UBC Faculty Research and Publications

Complex distribution of EFL and EF-1α proteins in the green algal lineage Noble, Geoffrey P; Rogers, Matthew B; Keeling, Patrick J May 23, 2007

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


52383-12862_2007_Article_376.pdf [ 296.44kB ]
JSON: 52383-1.0220561.json
JSON-LD: 52383-1.0220561-ld.json
RDF/XML (Pretty): 52383-1.0220561-rdf.xml
RDF/JSON: 52383-1.0220561-rdf.json
Turtle: 52383-1.0220561-turtle.txt
N-Triples: 52383-1.0220561-rdf-ntriples.txt
Original Record: 52383-1.0220561-source.json
Full Text

Full Text

ralssBioMed CentBMC Evolutionary BiologyOpen AcceResearch articleComplex distribution of EFL and EF-1α proteins in the green algal lineageGeoffrey P Noble, Matthew B Rogers 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: Geoffrey P Noble - gnoble@interchange.ubc.ca; Matthew B Rogers - mbrogers@interchange.ubc.ca; Patrick J Keeling* - pkeeling@interchange.ubc.ca* Corresponding author    AbstractBackground: EFL (or elongation factor-like) is a member of the translation superfamily of GTPaseproteins. It is restricted to eukaryotes, where it is found in a punctate distribution that is almostmutually exclusive with elongation factor-1 alpha (EF-1α). EF-1α is a core translation factorpreviously thought to be essential in eukaryotes, so its relationship to EFL has prompted thesuggestion that EFL has spread by horizontal or lateral gene transfer (HGT or LGT) and replacedEF-1α multiple times. Among green algae, trebouxiophyceans and chlorophyceans have EFL, butthe ulvophycean Acetabularia and the sister group to green algae, land plants, have EF-1α. Thisdistribution singles out green algae as a particularly promising group to understand the origin ofEFL and the effects of its presence on EF-1α.Results: We have sampled all major lineages of green algae for both EFL and EF-1α. EFL isunexpectedly broad in its distribution, being found in all green algal lineages (chlorophyceans,trebouxiophyceans, ulvophyceans, prasinophyceans, and mesostigmatophyceans), exceptcharophyceans and the genus Acetabularia. The presence of EFL in the genus Mesostigma and EF-1αin Acetabularia are of particular interest, since the opposite is true of all their closest relatives. Thephylogeny of EFL is poorly resolved, but the Acetabularia EF-1α is clearly related to homologuesfrom land plants and charophyceans, demonstrating that EF-1α was present in the commonancestor of the green lineage.Conclusion: The distribution of EFL and EF-1α in the green lineage is not consistent with thephylogeny of the organisms, indicating a complex history of both genes. Overall, we suggest thatafter the introduction of EFL (in the ancestor of green algae or earlier), both genes co-existed ingreen algal genomes for some time before one or the other was lost on multiple occasions.BackgroundHorizontal or lateral gene transfer (HGT or LGT) is thenon-sexual movement of genetic information betweenimportance of such events is debated, there is little debatethat it plays some role [1-4]. The importance of HGT inthe evolution of eukaryotic genomes is not as well appre-Published: 23 May 2007BMC Evolutionary Biology 2007, 7:82 doi:10.1186/1471-2148-7-82Received: 5 February 2007Accepted: 23 May 2007This article is available from: http://www.biomedcentral.com/1471-2148/7/82© 2007 Noble 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 9(page number not for citation purposes)two species. This process has been amply documented toaffect prokaryotic genomes: although the frequency andciated, in part because comparative nuclear genomics haslagged behind that of prokaryotes. Nevertheless, aBMC Evolutionary Biology 2007, 7:82 http://www.biomedcentral.com/1471-2148/7/82number of genome-wide surveys have now shown theprocess has had a broad impact in several species [5-9],and a number of single gene transfers have also beeninvestigated and found to have interesting implicationsfor ecology, protein function, or genome evolution [10-12].One potential gene transfer with interesting functionalimplications is EFL, or elongation factor-like protein [13].EFL is member of a large GTPase superfamily containingtranslation initiation, elongation, and termination fac-tors. EFL is specifically related to a subset of proteinsfound exclusively in eukaryotes, including elongation fac-tor-1 alpha (EF-1α), eukaryotic translation release factor-3, and HSP70-binding protein HBS1. EFL is itself onlyknown from eukaryotes, but not all eukaryotes have it. Ithas presently been reported from eight eukaryotic lineages[13-15], nearly all of which have close relatives which lackit (many of which have completely sequenced genomessupporting the absence of EFL). Moreover, most or all thelineages where EFL has been reported to date appear tolack EF-1α (again, this includes both organisms with com-pletely sequenced genomes and some with large scale ESTsequencing surveys). Altogether there is generally a mutu-ally exclusive distribution of EFL and EF-1α, and both arescattered around the tree of eukaryotes. This is of interestfor several reasons. First, EF-1α was formerly consideredessential because of the important and universal role itplays in translation, but this is clearly not the case sincesome EFL containing organisms clearly don't use EF-1α.Second, the distribution suggests EFL might substitute orpartially substitute for EF-1α function. Third, the punctatedistribution of EFL suggests it may be moving betweeneukaryotes [13]. As more lineages with EFL are found[14,15], the alternative that EFL is ancestral to all the lin-eages that possess it, and that either EFL or EF-1α has beenlost many times in different lineages becomes more defen-sible. However, the number of such events is very largesince the lineages possessing EFL are scattered across thetree of eukaryotes, and the number of lineages with EFLremains far fewer than those with EF-1α. Based on the cur-rent distribution we favour the movement of EFL betweeneukaryotes, but this must be reassessed as the distributionbecomes better known.Within each group where EFL has been found, the distri-bution of the protein is sometimes simple but uninforma-tive; for example, all dinoflagellates appear to useEFL[14]. Conversely, it's distribution among other groupsis more complex but difficult to interpret; for example,some fungi, ichthyosporeans, and choanoflagellates haveEFL while others do not and the relationships betweenthese is unclear [13,15-17]. A group where the distribu-instead have EF-1α, whereas the well-sampled chloro-phyceans and trebouxiophyceans have EFL and lack EF-1α. The single ulvophycean eukaryotic elongation factor,from Acetabularia acetabulum, was found to use EF-1α butnot EFL, altogether leading to the conclusion that thegreen algal EFL originated in a common ancestor of chlo-rophyceans and trebouxiophyceans {Keeling, 2004#652}. This makes green algae a good model for the studyof the fine scale distribution of EFL for two reasons. First,EFL seems to have arisen within the group rather thenprior to its diversification, so the age of this event could inprinciple be predicted. Second, while the phylogeny ofgreen algae is not perfectly known, it is better understoodthan most other eukaryotic groups [18], so the distribu-tion can be interpreted according to a reasonable phylog-eny. A better idea of this distribution can help us answerseveral questions. In particular, if EFL is introduced into alineage by HGT, what happens to EF-1α? Do they bothpersist for some time, and does lineage sorting take place?Only a fine-scale survey of a group where EFL exists insome but not all members could address such questions,and these questions are important because they are oneway to determine the possible functional relationshipbetween EFL and EF-1α.Here we have sampled EFL and EF-1α from representa-tives of all major missing lineages of green algae. Theviridiplantae are divided into two main groups, strepto-phytes and chlorophytes [18]. The streptophytes includeland plants, charophyceans, and the enigmatic scaly greenflagellate Mesostigma. The chlorophytes include four sub-divisions, prasinophyceans, ulvophyceans, trebouxio-phyceans and chlorophyceans (we will use the suffix '-ceans' so as to distinguish the larger group chlorophytesfrom the subgroup chlorophyceans). The expectation,based on the relatively simple distribution originallydescribed, is that EF-1α but not EFL should be found in allgreen lineages except chlorophyceans and trebouxiophyc-eans. In contrast, however, we have found that EFL isabundant in chlorophytes and EF-1α comparatively rare.Indeed, among chlorophyte green algae, Acetabularia isthe only lineage in which we could find EF-1α: EFL wasfound in all other chlorophyte lineages, and in the strep-tophyte Mesostigma. Phylogenetic reconstruction showsthe Acetabularia EF-1α is related to homologues from cha-rophytes and land plants, and therefore ancestral (and theonly known chlorophyte EF-1α to be retained). Thisimplies that EFL and EF-1α were likely both present in theancestor of streptophytes and chlorophytes, and that line-age sorting led to the current distribution of EFL and EF-1α. This has implications for the functional co-existenceof the two proteins, and also suggests that EFL was morefrequently retained than was EF-1α.Page 2 of 9(page number not for citation purposes)tion appeared to be most precisely explained was thegreen algae: here the well-sampled plants lack EFL andBMC Evolutionary Biology 2007, 7:82 http://www.biomedcentral.com/1471-2148/7/82Results & discussionCharacterisation of new EFL and EF-1α genes from green algaePreviously, EFL was found in a number of species of chlo-rophyceans and trebouxiophyceans, but not in any landplants or the ulvophycean A. acetabulum. This lead to theconclusion that it originated in a common ancestor ofchlorophyceans and trebouxiophyceans [13], which inturn leads to the prediction that all other green algaeshould only contain EF-1α. In contrast, however, we havefound that most green algal lineages possess EFL. Genesencoding EFL were characterized from three prasinophyc-eans (M. pusilla, T. tetrathele, and O. tauri), two ulvophyc-eans (U. fenestrata and U. intestinalis), an additionalchlorophycean (Chlorococcum sp.), and the only mesostig-matophycean (M. viride). From none of these lineageswere we able to detect the presence of a gene for EF-1α.Moreover, EF-1α was not present in the complete genomeof O. tauri or in EST surveys of M. viride [19,20]. An ESTsurvey of a third ulvophycean, U. linza [21], was alsosearched and in agreement with our results contained ashort fragment of EFL but no detectable EF-1α. Con-versely, EF-1α was characterized from two charophyceans(C. australis and Spirogyra sp.), and we were not able todetect the presence of EFL in either of these species. EFLfragments were identified in EST or genomic surveyprojects from four land plants, Pinus taeda, Oryza sativa,Lactuca saligna, and Triticum aestivum. With the exceptionof the first two, however, these do not form a group inphylogenetic analyses (Suppl. Figure 1) as one wouldexpect if they were ancestrally land plant sequences. More-over, most are closely related to either a subset of greenalgae or fungi, and are poorly represented in the surveyswhere they are found. In addition, in cases where substan-tial genomic data are available from the same organism,the EFL is only found in EST sequences and not in thegenomic data. The best cases are O. sativa and T. aestivum,where Blast searches against their respective genome data-bases revealed no EFL sequence, and specifically no matchto the EST purportedly from that organism. On balance,we conclude that these are contaminant sequences untilevidence that they are truly encoded in the plant genomesis available (which, according to the phylogeny, wouldsuggest several recent acquisitions in certain plants).In general, organisms from the streptophyte lineagemostly possess EF-1α while organisms from the chloro-phyte lineage mostly possess EFL. However, M. viride andA. acetabulum are important exceptions because, based ontheir currently accepted taxonomic positions, one wouldpredict that they should use EF-1α and EFL respectively,whereas EST sampling from both reveals the opposite. Itis possible that both genes are present in these genomes,cially in M. viride, where there is a large sample of 10,395ESTs [19]. It is also possible that these species are misclas-sified; that M. viride is really a chlorophyte and A. acetab-ulum is really a streptophyte. The phylogenetic position ofboth genera, and in particular M. viride, have been ana-lysed many times using a variety of data [18,19,22-27],making this somewhat unlikely. Nevertheless, we carriedout a phylogeny based on nine concatenated protein-cod-ing genes from the EST surveys to re-confirm their phylo-genetic affinities to streptophytes or chlorophytes. Thephylogeny (Figure 1) is consistent with previous analysesthat place M. viride at the base of the streptophytes and A.acetabulum within the chlorophytes. This rules out thepossibility that the possession of EF-1α and EFL by A.acetabulum and M. viride, respectively, reflects their rela-tionship to other groups that possess the same gene. Inshort, the distribution of EFL and EF-1α is not strictly con-sistent with the phylogenetic relationships of the organ-isms where the proteins are found.Phylogenies of EFL and EF-1αThe complex distribution of EFL and EF-1α could havearisen from multiple transfers of EFL to green algae, rever-sions by re-introduction of EF-1α, or an extended periodof time where both genes co-existed in the same ancestrallineage followed by differential loss in the descendants ofthis lineage. Each of these hypotheses is significant for dif-ferent reasons, and each leads to predictions for the phyl-ogeny of EFL and EF-1α. If EFL invaded green algaemultiple times, the green algal EFLs will not form a singlewell-supported clade. If EF-1α was re-introduced to one ormore groups, then the green algal and plant EF-1α geneswill not form a single well-supported clade. Lastly, if line-age sorting has taken place, then the green lineage shouldform a single clade in both genes.The first possibility cannot be confidently accepted orrejected because the phylogeny of green algal EFL genes isnot sufficiently well resolved. Figure 2 shows a phylogenyof all full-length EFL genes with the exception of thehighly divergent gene from the chlorarachniophyteBigelowiella natans; this gene has been included in analysespreviously [13], and we similarly found no indication thatit was related to green algal EFL (Suppl. Figure 1). Thegreen algal EFL sequences fall into three groups. One largeand weakly supported clade includes representatives of allfour major chlorophyte subgroups (noteworthy amongwhich are the ulvophyceans since Acetabularia is currentlybelieved to be related to this group). A second strongly-supported clade includes marine picoplanktonic prasino-phyceans (noteworthy among which is a previously uni-dentified environmental sequence from the Sargasso Seathat is specifically related to M. pusilla, consistent with thePage 3 of 9(page number not for citation purposes)although this would still suggest the expected gene is notexpressed as highly as the one characterized here, espe-source of this material and the size of cells collected:Suppl. Figure 1). Lastly, M. viride branches independentlyBMC Evolutionary Biology 2007, 7:82 http://www.biomedcentral.com/1471-2148/7/82of other green algae without any support for its position,which is unfortunate since it would be expected to possessEF-1α rather than EFL. None of these three groups ofgreen algae is related to any other group or to one anotherwith strong support in any analysis.The phylogeny of EF-1α is much more informative (Figure3). In contrast to EFL, the positions of all members of thegreen lineage, A. acetabulum, charophyceans, and landplants, are resolved with strong support, and the relation-ships of the EF-1α genes match those expected for theorganisms. The green clade is well supported overall (91–98%), with A. acetabulum sister to all streptophytes with94–100% support. The charophyceans are paraphyleticwith Spirogyra sister to land plants, although this branch-ing order is not supported by bootstrap analyses. Theposition of A. acetabulum is of particular importance,ulvophyceans, do not appear to possess EF-1α. In previ-ous analyses based on a truncated EST the position of theA. acetabulum EF-1α was not resolved with strong support,but it did not branch with plants [13]. However, havingnow sequenced an additional 561 bp of the 5' end (about50% of the alignable sequence), it is clear that the A.acetabulum EF-1α is indeed sister to other members of thegreen lineage. This suggests very strongly that A. acetabu-lum has not 'reacquired' EF-1α secondarily (e.g., throughHGT), and by extension that its EF-1α gene is the singleknown representative of the ancestral chlorophyte EF-1α.The evolution and distribution of EFL in the green algal lineageThe range of green algae that possess EFL is much broaderthan expected based on previous data, and more impor-tantly its distribution is not easily reconciled with the evo-Bayesian phylogeny of concatenated proteins from green algae and plants with maximum likelihood branch lengthsFigure 1Bayesian phylogeny of concatenated proteins from green algae and plants with maximum likelihood branch lengths. Numbers at nodes correspond to bootstrap support from protein maximum likelihood methods ProML (top) and PhyML (bottom). Major groups are labeled to the right. Note that the relationships between M. viride and streptophytes and A. acetabulum and chlorophytes are both recovered and supported by bootstrap. The 'Trebouxiophyte' is a composite of sequences from Chlorella, Prototheca, and Helicosporidium (for which genes come from each taxon, see methods).Chlamydomonas reinhardtiiTrebouxiophyteAcetabularia acetabulumOstreococcus tauriOryza sativaTriticum aestivumNicotiana tabacumArabidopsis thalianaPhyscomitrella patensMesostigma virideCyanidioschyzon merolaeCyanophora paradoxa92995984731008287 6010097100669781990.05ChlorophyceanUlvophyceanGlaucophyteMesostigmaPrasinophyceanRhodophyteTrebouxiophyceanLand PlantsPage 4 of 9(page number not for citation purposes)because the presence of EF-1α is unexpected in this ulvo-phycean, given chlorophytes as a whole, including otherlutionary relationships among the organisms that possessit. On the whole, streptophytes possess EF-1α, with theBMC Evolutionary Biology 2007, 7:82 http://www.biomedcentral.com/1471-2148/7/82exception of M. viride, whereas chlorophytes possess EFL,with the exception of A. acetabulum. Figure 4 shows a sche-matic phylogeny of the green lineage with the distributionof EFL and EF-1α. The phylogeny of EF-1α demonstratesthat EF-1α existed in the ancestor of the green lineage, andthat the land plants and charophyceans as well as thechlorophyte A. acetabulum have all retained this ancestralgene.of the green lineage. This would require three or perhapsmore origins of EFL (this is unclear because the phylogenyof basal chlorophytes is not resolved), which would sug-gest that EFL was very mobile indeed. While this is consist-ent with the present data (because the EFL tree does notreject such a scenario), it seems unlikely that one groupwould acquire such a gene many times.Another possibility is that EFL and EF-1α were bothBayesian phylogeny of EFL proteins with maximum likelihood branch lengthsFigure 2Bayesian phylogeny of EFL proteins with maximum likelihood branch lengths. Numbers at nodes correspond to bootstrap support from protein maximum likelihood methods ProML (left) and PhyML (right). Major groups are labeled to the right. Note that the relationships of many of the green algal groups are not well supported, and that they fall into three poorly separated groups.0.05Micromonas pusillaPavlova lutheriiEmiliania huxleyiIsochrysis galbanaChlamydomonas reinhardtiiScenedesmus obliquusTetraselmis tetratheleUlva fenestrataUlva intestinalisChlorococcum sp.Helicosporidium sp. ex Simulium jonesiiBlastocladiella emersoniiSpizellomyces punctatusRhodomonas salinaGuillardia thetaMesostigma virideHeterocapsa triquetraKarlodinium micrumOxyrrhis marinaPerkinsus marinus 22399Perkinsus marinus 22735Perkinsus marinus 6891Proterospongia-like sp. ATCC50818Sphaeroforma arcticaAllomyces macrogynusChlorophyceansUlvophyceansTrebouxiophyceanFungiCryptophytesMesostigmaHaptophytesPrasinophyceansDinoflagellatesPerkinsusChoanozoaMonosiga brevicollisOstreococcus tauri87/92100/100100/100-/50100/100100/10098/10091/94100/10077/9199/10095/9590/8862/9266/75100/100Page 5 of 9(page number not for citation purposes)It is possible that the distribution of EFL is due to multipleindependent invasions of the gene to different subgroupspresent in the common ancestor of streptophytes andchlorophytes and co-existed for some extended period ofBMC Evolutionary Biology 2007, 7:82 http://www.biomedcentral.com/1471-2148/7/82Page 6 of 9(page number not for citation purposes)Bayesian phylogeny of EF-1α proteins with maximum likelihood branch lengthsFigure 3Bayesian phylogeny of EF-1α proteins with maximum likelihood branch lengths. Numbers at nodes correspond to bootstrap support from protein maximum likelihood methods ProML (left) and PhyML (right). Major groups are labeled to the right. Note that A. acetabulum is sister to the streptophytes and that the charophyceans are sister to plants, in agreement with previous molecular phylogenies of the viridiplantae.+H[DPLWDLQIODWD*LDUGLDODPEOLD7ULFKRPRQDVWHQD[1DHJOHULDDQGHUVRQL6WUHEORPDVWL[VWUL['LQHQ\PSKDH[LOLV%ODVWRF\VWLVKRPLQLV7HORWURFKLGLXPKHQQHJX\LL(QWDPRHEDKLVWRO\WLFD7HWUDK\PHQDS\ULIRUPLV&ROSRGDLQIODWD3DUDPHFLXPWHWUDXUHOLD3RUSK\UDSXUSXUHD&\DQLGLRVFK\]RQPHURODH0DVWLJDPRHEDEDODPXWKL3ODQRSURWRVWHOLXPDXUDQWLXP'LFW\RVWHOLXPGLVFRLGHXP3K\VDUXPSRO\FHSKDOXP1XFOHDULDVLPSOH[8VWLODJRPD\GLV5KL]RSKO\FWLVVS6DFFKDURP\FHVFHUHYLVLDH1HXURVSRUDFUDVVD,FKWK\RSKRQXVLUUHJXODULV'URVRSKLODPHODQRJDVWHU;HQRSXVODHYLV+RPRVDSLHQV0RQRVLJDRYDWD0\WLOXVJDOORSURYLQFLDOLV5HFOLQRPRQDVDPHULFDQD&\DQRSKRUDSDUDGR[D(XJOHQDJUDFLOLV$FUDVLVURVHD/HLVKPDQLDEUD]LOLHQVLV7U\SDQRVRPDEUXFHL0DOORPRQDVUDVLOLV3KDHRGDFW\OXPWULFRUQXWXP3K\WRSKWKRUDLQIHVWDQV&U\SWRVSRULGLXPSDUYXP3ODVPRGLXPIDOFLSDUXP7KHLOHULDDQQXODWD$FHWDEXODULDDFHWDEXOXP&KDUDDXVWUDOLV6SLURJ\UDVS3LFHDDELHV*O\FLQHPD[9LFLDIDED=HDPD\V+RUGHXPYXOJDUH7ULWLFXPDHVWLYXP$UDELGRSVLVWKDOLDQD1LFRWLDQDWDEDFXP8OYRSK\FHDQ$SLFRPSOH[DQV+HWHURNRQWV*ODXFRSK\WH-DNRELG$QLPDOVDQG&KRDQRIODJHOODWHV,FKWK\RVSRUHDQ)XQJL1XFOHDULD0\FHWR]RD$PRHER]RDQ5KRGRSK\WHV&LOLDWHV$PRHER]RDQ&LOLDWH+HWHURNRQW2[\PRQDGV3DUDEDVDOLG+HWHURORERVHDQ'LSORPRQDGV.LQHWRSODVWLGV+HWHURORERVHDQ(XJOHQLG&KDURSK\WHV/DQG3ODQWVBMC Evolutionary Biology 2007, 7:82 http://www.biomedcentral.com/1471-2148/7/82time. Assuming that both genes do not still co-exist in anyone green algal genome, this would mean EFL was lostonce in the ancestor of charophyceans and land plants,and once in an ancestor of A. acetabulum, whereas EF-1αwas lost once in an ancestor of M. viride and several timesin chlorophyte lineages. Because the phylogeny of chloro-phytes is still uncertain it is impossible to state how manytimes EF-1α would have to have been lost, but it is inter-esting that EF-1α loss would likely have been more fre-quent than EFL loss. It is also possible that both genespersist in some green algae, although we know this is notthe case in several plants, O. tauri, and C. reinhardtii, andhave good reason to suspect it is not true in the manygreen algae for which there are large EST surveys. A longperiod of co-existence followed by differential loss of oneso. More specifically, EFL has been argued to share somefunctional overlap with EF-1α in translation [13]. How-ever, EF-1α is a multifunctional protein, so even if EFL iscapable of taking over its role in translation elongation, itstands to reason that EF-1α would have to persist until itsother roles in the cell were also obsolete or at least non-essential. Accordingly, a period of co-existence is probablynot only possible, but necessary, and during such a timeeither protein could conceivably be lost. This has theinteresting implication that plants, although they nowonly have EF-1α, once had EFL as well. It also bolstersexpectations that organisms with both proteins shouldexist. The zygomycete fungus Spizellomyces punctatus isreported to have both genes [17], and we have detectedboth in the diatom Thalassiosira pseudonana (G. Gile andPJK, unpublished data), but so far co-existence of the twoproteins is curiously rare.ConclusionWe have shown that the apparently mutually exclusivedistribution of the GTPase genes EFL and EF-1α in thegreen lineage is not consistent with the phylogeny of theorganisms in which the genes are found. Specifically,members of the streptophyte lineage tend to encode EF-1α and not EFL, except for the basal genus Mesostigmawhere the opposite is found. Conversely, members of thechlorophytes tend to encode EFL and not EF-1α, exceptfor Acetabularia where the opposite is found. The phylog-eny of EFL does not resolve the origin of the Mesostigmagene, but the phylogeny of EF-1α clearly shows the Acetab-ularia gene to be a relict, ancestral chlorophyte EF-1αrather than an independent re-acquisition. Altogether, wesuggest that the ancestor of the green lineage encoded EF-1α, but at some point EFL was introduced and the twogenes co-existed in green algal genomes for some timebefore one or the other was lost on multiple occasions.MethodsStrainsMultiple whole plants of Ulva fenestrata and Ulva intestina-lis were collected from the intertidal zone at SpanishBanks, Vancouver, BC Canada. Micromonas pusilla (strainNEPCC 29), Chlorococcum sp. (strain NEPCC 478), Tet-raselmis tetrathele (strain NEPCC 500) were grown in nat-ural seawater medium [28] at 16° with a 16:8 light:darkcycle. Chara australis (strain FWAC 7144) and Spirogyra sp.(strain FWAC 125) were grown in soilwater medium[29]at 16° with a 16:8 light:dark cycle. A cDNA libraryfrom Acetabularia acetabulum (strain Aa0005) was gener-ously provided by D. Mandoli.Characterisation of EFL and EF-1α genesMaterial from all cultures was harvested by centrifugationSchematic of evolutionary relationships within the green algal lineage showing distribution of EFL and EF-1α and possible ev nts explaining the distributionFigur  4Schematic of evolutionary relationships within the green algal lineage showing distribution of EFL and EF-1α and possible events explaining the distribution. A phylogeny of the Plantae, with the known presence of either EFL or EF-1α in extant lineages plotted at the tips of each branch (EFL is a square containing "L" and EF-1α is an octagon containing an "α"). All known rhodophytes and glau-cophytes contain EF-1α, as do most other eukaryotic line-ages, so the ancestor in inferred to have contained EF-1α as well (indicated by a gray octagon at the base of the tree). Also plotted is one possible explanation for the current dis-tribution of the two genes in the green lineage, where EFL was gained once in the ancestor of the green viridiplantae (indicated by the green box), and subsequently either EFL or EF-1α lost in several lineages each (indicated by red boxes with indicated which gene would have been lost). Other models to explain this distribution that include multiple ori-gins of EFL are also possible, but are not shown for simplicity. The tree of the green lineage is based on Figure 1 and other analyses [18].Land PlantsCharophyceansMesostigmaPrasinophyceansUlvophyceansAcetabulariaTrebouxiophyceansChlorophyceansStreptophytesChlorophytesRhodophytesGlaucophytesLLLLLααααα+L- L- Lα- α- α- α- α- αPage 7 of 9(page number not for citation purposes)or the other gene may seem unlikely at first glance, but ifone considers the function of these proteins it may not beand total RNA and DNA was isolated using protocolsdescribed previously [30]. RNA and DNA was used as a tem-BMC Evolutionary Biology 2007, 7:82 http://www.biomedcentral.com/1471-2148/7/82plate for RT-PCR and PCR reactions, respectively, usingprimers for each of EFL and EF-1α. EFL primers were CTGTC-GATCGTCATHTGYGGNCAYGTNGA and CTTGATRT-TNAGNCCNACRTTRTCNCC, EF-1α primers wereAACATCGTCGTGATHGGNCAYGTNGA and CTTGATCAC-NCCNACNGCNACNGT or CAACATCGTCGTCATCGGN-CAYGTNGA and GCCGCGCACGTTGAANCCNACRTTRTC.Products of the expected size (or larger in the case of genomicDNA amplifications) were cloned and multiple clones com-pletely sequenced. In all cases the RT-PCR approach wasmore successful, leading us to conclude EFL and EF-1α mayencode a number of introns. The A. acetabulum EFL was par-tially characterized as part of an EST survey [13], and toimprove the resolution of its phylogenetic position we char-acterized the 5' portion of the gene by amplification from acDNA library using the specific primer TTCCGACCG-GCACGGTTCCAATTCCG and the 5' degenerate EF-1αprimer AACATCGTCGTGATHGGNCAYGTNGA. During thecourse of this work the complete nuclear genome of the pra-sinophycean Ostreococcus tauri and an EST survey from themesostigmatophycean Mesostigma viride (strain NIES 476)both became available [19,20]. EFL and EF-1α were bothsought from this sequence data, from which only EFLsequences could be found. New sequences were deposited inGenBank under accession numbers EF551321-EF551331.Phylogenetic analysesNew EFL and EF-1α sequences were added to existingamino acid alignments [14]. The two proteins were ana-lysed separately since their relationship to one anotherhas been examined previously, and separate analysesallow the inclusion of more unambiguously alignablecharacters, 425 and 407 for EFL and EF-1α, respectively.An alignment of nine concatenated proteins from algaeand plants was also constructed using actin, alpha-tubu-lin, beta-tubulin, RbcS, Rps10, Rps13, Rpl3, Rpl11, andRpl13, for a total of 1,206 unambiguously alignableamino acid characters (with no missing data). The tre-bouxiophytes are represented by a composite ofsequences from three species: actin, Rps10, and Rpl11 arefrom Helicosporidium sp., beta-tubulin, Rpl3, Rpl13 andRps13 are from Prototheca wickerhamii, and alpha-tubulin,RbcS, and TufA are from Chlorella vulgaris. Trees wereinferred using distance, maximum likelihood and Baye-sian methods. Bayesian trees were inferred using Mr.Bayes 3.1 [31] employing the WAG substitution modelwith site-to-site rate variation modeled on a gamma distri-bution with 8 variable rate categories and one category ofinvariable sites, three heated chains and one cold one, and1,000,000 generations with sampling every 1,000 genera-tions. Log likelihoods were plotted and showed a rapidplateau after only five samples, so a burnin of 40 trees wasremoved before constructing the consensus (constructingtopology were calculated using ProML 3.6 [32] with JTT, 8gamma rate categories and one category of invariablesites. ProML trees were inferred using the same settings,and 100 bootstraps were inferred with the gamma shapeparameter alpha and proportion of invariant sites esti-mated using Tree-Puzzle 5.2 [33]. ML trees and 1,000bootstrap trees were also inferred using PhyML 2.4.4 [34]with the WAG model with 8 gamma rate categories andone category of invariable sites (when p-inv was not zero),and parameters estimated from the data. Distances werealso calculated using Tree-Puzzle with the same settings(and parameters estimated from the data), and trees con-structed using WEIGHBOUR 1.0.1a [35]. 100 bootstrapreplicates were carried out using puzzleboot [36], whichgave similar results as maximum likelihood (not shown).Authors' contributionsG.P. Noble and P.J. Keeling characterized all new genesequences. Phylogenetic analyses were performed by M.B.Rogers, G.P. Noble and P.J. Keeling. P.J. Keeling conceivedof the study and wrote the manuscript.Additional materialAcknowledgementsThis work was supported by a grant (227301) from the Natural Sciences and Engineering Research Council of Canada (NSERC). We thank D. Man-doli for providing the A. acetabulum cDNA library and C. Bowler and I. Grig-oriev for unpublished data from P. tricornutum. GPN was supported by a student award from NSERC and PJK is a Fellow of the Canadian Institutes for Advanced Research and a Senior Scholar of the Michael Smith Founda-tion for Health Research.References1. Lawrence JG, Hendrickson H: Lateral gene transfer: when willadolescence end?  Mol Microbiol 2003, 50:739-749.2. Doolittle WF: Lateral genomics.  Trends Cell Biol 1999, 9:M5-8.3. Gogarten JP, Doolittle WF, Lawrence JG: Prokaryotic evolution inlight of gene transfer.  Mol Biol Evol 2002, 19:2226-2238.4. Kurland CG, Canback B, Berg OG: Horizontal gene transfer: acritical view.  Proc Natl Acad Sci USA 2003, 100:9658-9662.5. Andersson JO: Lateral gene transfer in eukaryotes.  Cell Mol LifeSci 2005, 62:1182-1197.6. Bergthorsson U, Adams KL, Thomason B, Palmer JD: Widespreadhorizontal transfer of mitochondrial genes in floweringplants.  Nature 2003, 424:197-201.7. Archibald JM, Rogers MB, Toop M, Ishida K, Keeling PJ: Lateral geneAdditional file 1Bayesian phylogeny of all known EFL genes and fragments with maximum likelihood branch lengths. Numbers at nodes correspond to bootstrap sup-port from protein maximum likelihood methods ProML (top) and PhyML (bottom). Major groups are labeled to the right.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-7-82-S1.eps]Page 8 of 9(page number not for citation purposes)a consensus of all trees resulted in the same topology).Maximum likelihood branch lengths for the consensustransfer and the evolution of plastid-targeted proteins in thesecondary plastid-containing alga Bigelowiella natans.  ProcNatl Acad Sci USA 2003, 100:7678-7683.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 Evolutionary Biology 2007, 7:82 http://www.biomedcentral.com/1471-2148/7/828. Loftus B, Anderson I, Davies R, Alsmark UC, Samuelson J, Amedeo P,Roncaglia P, Berriman M, Hirt RP, Mann BJ, Nozaki T, Suh B, Pop M,Duchene M, Ackers J, Tannich E, Leippe M, Hofer M, Bruchhaus I,Willhoeft U, Bhattacharya A, Chillingworth T, Churcher C, Hance Z,Harris B, Harris D, Jagels K, Moule S, Mungall K, Ormond D, SquaresR, Whitehead S, Quail MA, Rabbinowitsch E, Norbertczak H, Price C,Wang Z, Guillen N, Gilchrist C, Stroup SE, Bhattacharya S, Lohia A,Foster PG, Sicheritz-Ponten T, Weber C, Singh U, Mukherjee C, El-Sayed NM, Petri WA Jr., Clark CG, Embley TM, Barrell B, Fraser CM,Hall N: The genome of the protist parasite Entamoeba histo-lytica.  Nature 2005, 433:865-868.9. Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bar-tholomeu DC, Lennard NJ, Caler E, Hamlin NE, Haas B, Bohme U,Hannick L, Aslett MA, Shallom J, Marcello L, Hou L, Wickstead B, Als-mark UC, Arrowsmith C, Atkin RJ, Barron AJ, Bringaud F, Brooks K,Carrington M, Cherevach I, Chillingworth TJ, Churcher C, Clark LN,Corton CH, Cronin A, Davies RM, Doggett J, Djikeng A, FeldblyumT, Field MC, Fraser A, Goodhead I, Hance Z, Harper D, Harris BR,Hauser H, Hostetler J, Ivens A, Jagels K, Johnson D, Johnson J, JonesK, Kerhornou AX, Koo H, Larke N, Landfear S, Larkin C, Leech V,Line A, Lord A, Macleod A, Mooney PJ, Moule S, Martin DM, MorganGW, Mungall K, Norbertczak H, Ormond D, Pai G, Peacock CS,Peterson J, Quail MA, Rabbinowitsch E, Rajandream MA, Reitter C,Salzberg SL, Sanders M, Schobel S, Sharp S, Simmonds M, Simpson AJ,Tallon L, Turner CM, Tait A, Tivey AR, Van Aken S, Walker D, Wan-less D, Wang S, White B, White O, Whitehead S, Woodward J,Wortman J, Adams MD, Embley TM, Gull K, Ullu E, Barry JD, FairlambAH, Opperdoes F, Barrell BG, Donelson JE, Hall N, Fraser CM,Melville SE, El-Sayed NM: The genome of the African trypano-some Trypanosoma brucei.  Science 2005, 309:416-422.10. Jenkins C, Samudrala R, Anderson I, Hedlund BP, Petroni G,Michailova N, Pinel N, Overbeek R, Rosati G, Staley JT: Genes forthe cytoskeletal protein tubulin in the bacterial genus Pros-thecobacter.  Proc Natl Acad Sci USA 2002, 99:17049-17054.11. Huang J, Mullapudi N, Lancto CA, Scott M, Abrahamsen MS, KissingerJC: Phylogenomic evidence supports past endosymbiosis,intracellular and horizontal gene transfer in Cryptosporid-ium parvum.  Genome Biol 2004, 5:R88.12. Waller RF, Slamovits CH, Keeling PJ: Lateral gene transfer of amultigene region from cyanobacteria to dinoflagellatesresulting in a novel plastid-targeted fusion protein.  Mol BiolEvol 2006, 23:1437-1443.13. Keeling PJ, Inagaki Y: A class of eukaryotic GTPase with a punc-tate distribution suggesting multiple functional replace-ments of translation elongation factor 1alpha.  Proc Natl AcadSci USA 2004, 101:15380-15385.14. Gile GH, Patron NJ, Keeling PJ: EFL GTPase in cryptomonadsand the distribution of EFL and EF-1alpha in chromalveo-lates.  Protist 2006, 157:435-444.15. Ruiz-Trillo I, Lane CE, Archibald JM, Roger AJ: Insights into theevolutionary origin and genome architecture of the unicellu-lar opisthokonts Capsaspora owczarzaki and Sphaeroformaarctica.  J Eukaryot Microbiol 2006, 53:379-384.16. Ragan MA, Murphy CA, Rand TG: Are Ichthyosporea animals orfungi? Bayesian phylogenetic analysis of elongation factor1alpha of Ichthyophonus irregularis.  Mol Phylogenet Evol 2003,29:550-562.17. James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V, Cox CJ,Celio G, Gueidan C, Fraker E, Miadlikowska J, Lumbsch HT, RauhutA, Reeb V, Arnold AE, Amtoft A, Stajich JE, Hosaka K, Sung GH, John-son D, O'Rourke B, Crockett M, Binder M, Curtis JM, Slot JC, WangZ, Wilson AW, Schussler A, Longcore JE, O'Donnell K, Mozley-Stan-dridge S, Porter D, Letcher PM, Powell MJ, Taylor JW, White MM,Griffith GW, Davies DR, Humber RA, Morton JB, Sugiyama J, Ross-man AY, Rogers JD, Pfister DH, Hewitt D, Hansen K, Hambleton S,Shoemaker RA, Kohlmeyer J, Volkmann-Kohlmeyer B, Spotts RA,Serdani M, Crous PW, Hughes KW, Matsuura K, Langer E, Langer G,Untereiner WA, Lucking R, Budel B, Geiser DM, Aptroot A, Died-erich P, Schmitt I, Schultz M, Yahr R, Hibbett DS, Lutzoni F, McLaugh-lin DJ, Spatafora JW, Vilgalys R: Reconstructing the earlyevolution of Fungi using a six-gene phylogeny.  Nature 2006,443:818-822.18. Lewis LA, McCourt RM: Green algae and the origin of landplants.  Am J Bot 2004, 91:1535-1556.phyta): implications for the evolution of green plants(Viridiplantae).  BMC Plant Biol 2006, 6:2.20. Derelle E, Ferraz C, Rombauts S, Rouze P, Worden AZ, Robbens S,Partensky F, Degroeve S, Echeynie S, Cooke R, Saeys Y, Wuyts J, Jab-bari K, Bowler C, Panaud O, Piegu B, Ball SG, Ral JP, Bouget FY, Piga-neau G, De Baets B, Picard A, Delseny M, Demaille J, Van de Peer Y,Moreau H: Genome analysis of the smallest free-living eukary-ote Ostreococcus tauri unveils many unique features.  ProcNatl Acad Sci USA 2006, 103:11647-11652.21. Stanley MS, Perry RM, Callow JA: Analysis of expressed sequencetags from the green algal Ulva linza (Chlorophyta).  J Phycol2005, 41:1219-1226.22. Lemieux C, Otis C, Turmel M: Ancestral chloroplast genome inMesostigma viride reveals an early branch of green plantevolution.  Nature 2000, 403:649-652.23. Lemieux C, Otis C, Turmel M: A clade uniting the green algaeMesostigma viride and Chlorokybus atmophyticus repre-sents the deepest branch of the Streptophyta in chloroplastgenome-based phylogenies.  BMC Biol 2007, 5:2.24. Rodriguez-Ezpeleta N, Philippe H, Brinkmann H, Becker B, MelkonianM: Phylogenetic analyses of nuclear, mitochondrial and plas-tid multi-gene datasets support the placement of Mes-ostigma in the Streptophyta.  Mol Biol Evol 2006.25. Petersen J, Teich R, Becker B, Cerff R, Brinkmann H: The GapA/Bgene duplication marks the origin of Streptophyta (charo-phytes and land plants).  Mol Biol Evol 2006, 23:1109-1118.26. Nedelcu AM, Borza T, Lee RW: A land plant-specific multigenefamily in the unicellular Mesostigma argues for its close rela-tionship to Streptophyta.  Mol Biol Evol 2006, 23:1011-1015.27. Turmel M, Otis C, Lemieux C: The complete mitochondrialDNA sequence of Mesostigma viride identifies this greenalga as the earliest green plant divergence and predicts ahighly compact mitochondrial genome in the ancestor of allgreen plants.  Mol Biol Evol 2002, 19:24-38.28. Harrison PJ, Waters RE, Taylor FJR: A broad spectrum artificialmedium for coastal and open ocean phytoplankton.  J Phycol1980, 16:28-35.29. Pringsheim EG: The biphasic or soil-water culture method forgrowing algae and flagellata.  J Ecol 1946, 33:193-204.30. Keeling PJ, Leander BS: Characterisation of a non-canonicalgenetic code in the oxymonad Streblomastix strix.  J Mol Biol2003, 326:1337-1349.31. Ronquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogeneticinference under mixed models.  Bioinformatics 2003,19:1572-1574.32. Felsenstein J: PHYLIP (Phylogeny Inference Package).  3.5thedition. Seattle, J. Felsenstein, University of Washington; 1993. 33. Schmidt HA, Strimmer K, Vingron M, von Haeseler A: TREE-PUZ-ZLE: maximum likelihood phylogenetic analysis using quar-tets and parallel computing.  Bioinformatics 2002, 18:502-504.34. Guindon S, Gascuel O: A simple, fast, and accurate algorithmto estimate large phylogenies by maximum likelihood.  SystBiol 2003, 52:696-704.35. Bruno WJ, Socci ND, Halpern AL: Weighted neighbor joining: alikelihood-based approach to distance-based phylogenyreconstruction.  Mol Biol Evol 2000, 17:189-197.36. www.tree-puzzle.de: .  .yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 9 of 9(page number not for citation purposes)19. Simon A, Glockner G, Felder M, Melkonian M, Becker B: EST anal-ysis of the scaly green flagellate Mesostigma viride (Strepto-


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items