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ReportRed and Green Algal Monophylyand Extensive Gene Sharing Foundin a Rich Repertoire of Red Algal Genes
Cheong Xin Chan,
Eun Chan Yang,
2,5
Titas Banerjee,
1
Hwan Su Yoon,
2,
* Patrick T. Martone,
3
Jose´  M. Estevez,
4
and Debashish Bhattacharya
*
1
Department of Ecology, Evolution, and Natural Resourcesand Institute of Marine and Coastal Sciences, RutgersUniversity, New Brunswick, NJ 08901, USA 
2
Bigelow Laboratory for Ocean Sciences, West BoothbayHarbor, ME 04575, USA 
3
Department of Botany, University of British Columbia, 6270University Boulevard, Vancouver, BC V6T 1Z4, Canada
4
Instituto de Fisiologı´a, Biologı´a Molecular y Neurociencias(IFIBYNE UBA-CONICET), Facultad de Ciencias Exactas yNaturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina
SummaryThe Plantae comprising red, green (including land plants),and glaucophyte algae are postulated to have a singlecommon ancestor that is the founding lineage of photosyn-thetic eukaryotes [1, 2]. However, recent multiproteinphylogenies provide little [3, 4] or no [5, 6] support for this hypothesis. This may reflect limited complete genome dataavailable for red algae, currently only the highly reducedgenome of
 Cyanidioschyzon merolae 
 [7], a reticulate geneancestry [5], or variable gene divergence rates that misleadphylogenetic inference [8]. Here, using novel genome datafrom the mesophilic
 Porphyridium cruentum 
 and
 Calliar- throntuberculosu
,weanalyze60,000novelredalgalgenesto test the monophyly of red + green (RG) algae and theirextent of gene sharing with other lineages. Using a gene-by-gene approach, we find an emerging signal of RGmonophyly (supported by
 w
50% of the examined proteinphylogenies) that increases with the number of distinctphyla and terminal taxa in the analysis. A total of 1,808phylogenies show evidence of gene sharing betweenPlantaeandotherlineages.Wedemonstratethatarichmeso-philic red algal gene repertoire is crucial for testing contro-versial issues in eukaryote evolution and for understandingthe complex patterns of gene inheritance in protists.Results and DiscussionAssessing Red and Green Algal Monophyly Basedon Exclusive Gene Sharing
Here,with36,167expressedsequence-tagged(EST)unigenesfrom
 Porphyridium cruentum
 and 23,961 predicted proteinsfrom
 Calliarthron tuberculosum
, we report analyses of >60,000 novel genes from mesophilic red algae. Of the 36,167
P. cruentum
 unigenes (6.7-fold greater than the gene number [5,331] from
 Cyanidioschyzon merolae
 [7]), 13,632 encodeproteins with significant BLASTp hits (e value
 %
 10
2
10
 ) tosequences in our local database, in which we included the23,961 predicted proteins from
 C. tuberculosum
 (see TableS1 available online). Of these hits, 9,822 proteins (72.1%,includingmany
P.cruentum
paralogs)werepresentin
C.tuber-culosum
 and/or other red algae, 6,392 (46.9%) were sharedwith
C.merolae
,and1,609werefoundonlyinredalgae.Atotalof 1,409 proteins had hits only to red algae and one other phylum. Using this repertoire, we adopted a simplified recip-rocal BLAST best-hits approach to study the pattern of exclu-sive gene sharing between red algae and other phyla (seeExperimentalProcedures ).Wefoundthat644proteinsshowedevidence of exclusive gene sharing with red algae. Of these,145 (23%) were found only in red + green algae (hereafter,RG) and 139 (22%) only in red + Alveolata Figure 1 A).Incomparison,wefoundonly34(5%)proteinsinred+Glauco-phyta,likelyasaresultofthelimitedavailabilityofglaucophytedata in the database. As we restricted this search by requiringa larger number of hits per query (x) from both phyla, theproportion of RG proteins increased relative to other taxa.Forinstance, thenumber ofred +Alveolataand red+Metazoaproteins was reduced from 139
/
1
/
0 and from 55
/
3
/
0 when x
R
2 (644 proteins), x
R
10 (96 proteins), and x
R
20(22 proteins), respectively Figures 1 A–1C). This BLASTpanalysis is based on the implicit assumption that significantsimilarity among a group of sequences indicates a putativehomologous relationship (i.e., a shared common ancestry).This approach could potentially be misled by convergence atthe amino acid level that results in high similarity among non-homologous sequences (i.e., homoplasy [9, 10]). Alternatively,because RG are primarily photoautotrophs, exclusive genesharing could be explained by these lineages having retainedacommonsetofancestralgenesthatwerelostinothereukary-otes. With these potential issues in mind, we suggest thatexclusive gene sharing (as defined by significant reciprocalBLASTp hits) provisionally favors the RG grouping.
Gene Sharing between RG and Other Lineages
Using a phylogenomic approach, we generated maximum-likelihood (ML) trees for each of the 13,632
 P. cruentum
proteins with significant hits to the local database. One of the major confounding issues in phylogenomic analysis isinadequate and/or biased taxon sampling. To reduce suchbiases in our inference of gene phylogeny, we restricted our analysis to trees that contain
R
3 phyla (per tree) and analyzedthese phylogenies based on the minimum number of terminaltaxa per tree (n), ranging from 4 to 40 ( Figure 1D). The expec-tation was that the impact of inadequate taxon samplingon our interpretation of the data would be minimal in treeswith large n. Applying these restrictions, n
 R
 4 returned1,367 trees that contained red algae positioned withina strongly supported (bootstrap
R
90%) monophyletic cladeFigure 1D); the majority of these trees (1,129 of 1,367; 83%)had n
 R
 10. Among the 1,367 trees, 329 showed exclusiveRG monophyly, of which 53 trees defined RG + glaucophytes(i.e., were putatively Plantae-specific). The number of treesthat recovered the RG remained similar between cases of n
R
4 and n
R
10, with only 71 trees having n between 4 and9. As n increased, the proportion of RG groupings remained
*Correspondence: hsyoon@bigelow.org (H.S.Y.), bhattacharya@aesop. rutgers.edu (D.B.)
5
These authors contributed equally to this work
 
similar across all categories, although the number of treessupporting this clade gradually decreased. These estimatesreflect our current database and will change as more genomedata become available. Figure 2 A shows the phylogeny of a putative Plantae-specific gene (of unknown function) thatappears to have undergone an ancient gene duplication inthe Plantae ancestor followed by subsequent duplications(particularly among land plants).
ACBD
Figure 1. Analysis of Predicted Proteins from the Red Alga
 Porphyridiumcruentum
(A–C) The distribution of phyla with exclusive BLASTp hits to
 P. cruentum
proteins where the number of hits per query (x) is as follows, (A) x
R
2, (B)x
R
10, and (C) x
R
20. The colors indicate the different phyla that shareproteins exclusively with
 P. cruentum
.(D) The percentage of maximum-likelihood (ML) protein trees (raw numbersshown in thebarsfor the fivemost frequentlyfoundgroupings)that supportthe monophyly of red algae with other eukaryote phyla (bootstrap
R
90%).The impact of increasing the number of terminal taxa in each tree (n) onthese proportions is shown for the progression from 4
/
10
/
20
/
30
/
40. The total number of trees for each category is shown on top of eachbar. Thecategory‘‘Red-Green(RG)exclusive’referstotreesin whichthesetwophylaformanexclusiveclade,whereas‘‘Red-Green(RG)shared’referstotrees inwhichred-greenmonophylyis well supported but other phyla arefound within this clade (i.e., due to gene sharing). See also Figure S1.
Polysphondylium pallidum gi281208101Dictyostelium discoideum AX4 gi66819463Dictyostelium purpureum jgi44467 
99
Lachancea thermotolerans CBS 6340 gi255714072 Phaeosphaeria nodorum SN15 gi169617722 Gibberella zeae PH-1 gi46127145 Schizosaccharomyces pombe gi19113907 
609999
Viridiplantae-Chlorella vulgaris jgi25303Viridiplantae-Chlorella NC64A jgi32681
Rhodophyta-Porphyridium cruentum Contig11360 4Rhodophyta-Porphyridium cruentum GCDJ7DB01DXL12 5 
Viridiplantae-Chlamydomonas reinhardtii gi159480824Viridiplantae-Volvox carteri jgi67793
100
100
Viridiplantae-Micromonas CCMP1545 jgi6463Viridiplantae-Micromonas sp. RCC299 gi255086013Viridiplantae-Micromonas RCC299 jgi103549
100
Viridiplantae-Ostreococcus RCC809 jgi42833Viridiplantae-Ostreococcus tauri jgi22643Viridiplantae-Ostreococcus lucimarinus CCE9901 gi145356458 Viridiplantae-Ostreococcus lucimarinus jgi42284
10071
100100
Viridiplantae-Physcomitrella patens subsp. patens gi167999572 Viridiplantae-Physcomitrella patens jgi112456 Viridiplantae-Zea mays gi226508476 Viridiplantae-Oryza sativa Japonica Group gi115445815 Viridiplantae-Ricinus communis gi255540591Viridiplantae-Arabidopsis lyrata jgi496486 Viridiplantae-Arabidopsis thaliana gi79546079
100
Viridiplantae-Populus trichocarpa gi224135861
6769
741009672
Viridiplantae-Populus trichocarpa gi224129280 Viridiplantae-Ricinus communis gi255540033Viridiplantae-Arabidopsis lyrata jgi470113Viridiplantae-Arabidopsis thaliana gi15223439Viridiplantae-Zea mays gi226496273
98
Viridiplantae-Sorghum bicolor gi242062504Glaucophyta-Cyanophora paradoxa Contig23 4
Rhodophyta-Cyanidioschyzon merolae CMT608C Rhodophyta-Galdieria sulphuraria Gs42470.1
Fragilariopsis cylindrus jgi206207 Thalassiosira pseudonana gi219125311Phaeodactylum tricornutum gi219125311
60
88100699110096
10095
990.2 substitutions/site
STRAMENOPILESPLANTAEFUNGIAMOEBOZOA
B
Rhodophyta-Porphyra haitanensis 61663183 2 Rhodophyta-Calliarthron tuberculosum g6851t1Rhodophyta-Cyanidioschyzon merolae CMX001C Rhodophyta-Porphyridium cruentum Contig9125 2 
9166
Glaucophyta-Glaucocystis nostochinearum GNL00002567 1Viridiplantae-Physcomitrella patens jgi20837 Viridiplantae-Zea mays gi212274441Viridiplantae-Arabidopsis lyrata jgi483095 Viridiplantae-Arabidopsis thaliana gi22331818 
91
90
Viridiplantae-Chlorella vulgaris jgi81458 Viridiplantae-Volvox carteri jgi79235 Viridiplantae-Chlamydomonas reinhardtii gi159490938 
100
Viridiplantae-Vitis vinifera gi225450009Viridiplantae-Oryza sativa Japonica Group gi115442333Viridiplantae-Sorghum bicolor gi242059969Viridiplantae-Zea mays gi226533441
100
Viridiplantae-Oryza sativa Japonica Group gi115463661Viridiplantae-Zea mays gi226529786 Viridiplantae-Sorghum bicolor gi242087801
100
98
Viridiplantae-Ricinus communis gi255544908 Viridiplantae-Populus trichocarpa gi224059548 Viridiplantae-Populus trichocarpa gi224104171Viridiplantae-Arabidopsis lyrata jgi909228 Viridiplantae-Arabidopsis thaliana gi18416870 
91
100
Viridiplantae-Vitis vinifera gi225435391
93
93
69
82
Viridiplantae-Populus trichocarpa gi224059600 Viridiplantae-Ricinus communis gi255553448 
62
63
Viridiplantae-Physcomitrella patens subsp. patens gi168034841Viridiplantae-Physcomitrella patens subsp. patens gi168028204
9267100991001009097
Rhodophyta-Cyanidioschyzon merolae CMM247C 
Glaucophyta-Cyanophora paradoxa Contig638 4Viridiplantae-Ostreococcus lucimarinus CCE9901 gi145349444Viridiplantae-Micromonas sp. RCC299 gi255076153Viridiplantae-Micromonas pusilla CCMP1545 gi303278506 
92
Viridiplantae-Chlorella vulgaris jgi84560 
100
Viridiplantae-Ostreococcus tauri gi116055893Viridiplantae-Chlorella NC64A jgi137672 Viridiplantae-Chlorella vulgaris jgi72495 Viridiplantae-Volvox carteri jgi106345 Viridiplantae-Chlamydomonas reinhardtii gi159473677 
619896
Viridiplantae-Arabidopsis thaliana gi15242979Viridiplantae-Arabidopsis lyrata jgi326573Viridiplantae-Populus trichocarpa gi224103009Viridiplantae-Vitis vinifera gi225436365 Viridiplantae-Arabidopsis lyrata jgi482793Viridiplantae-Arabidopsis thaliana gi79324637 Viridiplantae-Vitis vinifera gi225440504Viridiplantae-Oryza sativa Japonica Group gi115471329Viridiplantae-Sorghum bicolor gi242043424Viridiplantae-Ricinus communis gi255577977 
100
100
Viridiplantae-Sorghum bicolor gi242073740 Viridiplantae-Zea mays gi226491452 Viridiplantae-Oryza sativa Japonica Group gi115459498 
97
68
97
Viridiplantae-Physcomitrella patens subsp patens gi168035809
927210093100
Viridiplantae-Chlorella vulgaris jgi77486 Viridiplantae-Chlorella NC64A jgi23974Viridiplantae-Chlorella NC64A jgi20786 Viridiplantae-Chlamydomonas reinhardtii gi159472571
Rhodophyta-Calliarthron tuberculosum g9362t1Rhodophyta-Cyanidioschyzon merolae CMT574C 
100
81100
Rhodophyta-Cyanidioschyzon merolae CMT191C 
87
0.5 substitutions/site
A
Figure 2. Plantae Evolution and Gene Sharing(A) Phylogeny of a gene of unknown function that is putatively specific toPlantae.(B) Phylogeny of a gene encoding a putative phosphoglyceride transfer protein, SEC14, with a well-supported monophyly (bootstrap 95%) of plants, red algae, the glaucophytes, and diatoms and a monophyly (boot-strap 100%) between
 Porphyridium cruentum
 and green algae (includingother plants). RAxML [30] bootstrap support values
 R
 60% based on100 nonparametric replicates are shown at the nodes. Red algae are shownin boldface and glaucophytes in gray. The unit of branch length is thenumber of substitutions per site. See also Figure S2.
 Assessing Red and Green Algal Monophyly
329
 
In these analyses, we also examined instances of RGmonophyly in which other taxa interrupted this clade, e.g.,Stramenopiles, presumably resulting from endosymbiotic/ horizontal gene transfer (E/HGT). We referred to suchinstances as ‘‘RG shared’’ ( Figure 1D), whereby there wasa strongly supported monophyly (i.e., bootstrap
 R
 90%) of RG algae with other non-Plantae lineages. We applied thecondition that RG shared clades include
R
75% of all terminaltaxa in a tree, and within this clade, a majority (>50%) of thetips defined red and green algae. Using this definition, wefoundanadditional413treesthatsupportRGmonophylyFig-ure 1D). Therefore, at n
R
4, a total of 742 (54%) of 1,367 treesreturned by our pipeline supported the RG union (bootstrap
R
90%). At a less stringent bootstrap threshold of 
R
70%, 997(46%) of 2,167 trees showed support for RG monophyly ( Fig-ure S1 ). An example of a phylogeny showing nonexclusivegene sharing with Plantae lineages is shown in Figure 2B for a putative phosphoglyceride transfer protein, SEC14. Thephylogeny shows a well-supported monophyly (bootstrap95%) of plants, red algae ( 
Galdieria sulphuraria
 and
 C. mero- lae
 ), the glaucophyte
 Cyanophora paradoxa
, and diatoms.The diatom gene likely arose via secondary endosymbioticgene transfer from a red algal donor [11]. In addition, a diver-gent red algal-derived gene copy is present in
 P. cruentum
that groups with green algae and other gene copies found inplants (bootstrap 100%). Although complete genome datafrom glaucophytes and other red algae are required to delin-eate the extent of gene duplication and convergence betweenthese two lineages, this phylogeny illustrates two key proper-tiesofprotistgeneandgenomeevolutionthatposechallengesto the inference of lineage relationships: ancient gene duplica-tion (e.g., multiple copies in plants) and loss (i.e., putatively of a gene copy in green algae, e.g.,
 Ostreococcus
 spp.), andnonlineal gene sharing involving algal lineages.The next most frequently found positions of red algaein these trees were as sister to Stramenopiles (168, 12%), Alveolata (91, 7%), Excavata (68, 5%), and Cryptophyta (66,5%). Increasing the minimum number of terminal taxa per tree (n) from 4 through 40 (while maintaining
 R
3 phyla) didnot affect the relative proportion of trees that supportRG monophyly, but the number of cases with other well-supported phylogenetic affiliations (e.g., red + Metazoa,red + Fungi) fell sharply ( Figure 1D). When we relaxed thebootstrap threshold to
 R
70%, the patterns reported heregenerally remained unchanged Figure S1 ) but allowed theidentification of single-protein markers that may prove usefulfor delineating the eukaryote tree of life (e.g., V-type ATPaseI 116 kDa subunit family; Figure S2; see also [12]). We found 1,808 trees that showed strong support (at boot-strap
 R
90%) for the monophyly of RG with other ‘‘foreign’’taxa. Figure 3 shows the number of these trees that containdifferent foreign phyla within the well-supported RG clade.The sources of the foreign genes are depicted in a schematicrepresentation of the putative tree of life. The most commonpartners of gene sharing with RG (i.e., barring significantphylogenetic artifacts in our approach) are Stramenopiles(e.g., the diatoms; 1,264 proteins), bacteria other than Cyano-bacteria (1,108), Haptophyta (839), Cyanobacteria (827), Alveolata (622), and Metazoa (473). The majority of theseproteins (1,322 of 1,808) are shared between RG and two or more other phyla, demonstrating the complex evolutionaryhistory of the algal genes. We recognize that our results arebiased by the unbalanced contribution of available genomedata from microbial eukaryotes in our database (e.g., diatomsare gene rich, cryptophytes are gene poor). In addition, thedetection of gene transfer using phylogenetic approachesis susceptible to a number of technical limitations such asmodularity [13, 14] and amelioration [15, 16] of the transferred genes, which result in underestimation of the extent of HGT ingene-by-gene surveys. Nevertheless, our findings indicatethat single-gene or multigene analysis of Plantae should takeinto account extensive gene sharing vis-a `-vis other eukaryotelineages (e.g., nongreen affiliation in nearly one-half of 
P. cruentum
 proteins shown in Figure 1D).Lastly, we examined whether the observed signal of RGmonophyly was contributed primarily or solely by nuclear-encoded plastid-targeted proteins (i.e., whether they reflectthe evolution of the organelle rather than the host cell). To dothis, we analyzed all RG-exclusive and RG-shared proteinsFigure 1D) with n ranging from 4 through 40. Using anintegrated pipeline that incorporates three independenttarget-prediction approaches, we found that circa 40% of the proteins that support RG monophyly at bootstrap
R
90%may be plastid targeted (253 of 742, 34.1% at n
R
4; 119 of 283, 42.1% at n
R
40; see Supplemental Experimental Proce-dures ). Although bioinformatic predictions of organelle target-ing are clearly provisional, these results suggest that inaddition to the expected significant contribution to plastidfunction by proteins that unite the RG (i.e., the vast majorityof these taxa are photoautotrophs), over one-half of themmay not be destined for the plastid.
Enrichment of Red Algal Genes EnhancesOur Understanding of Eukaryote Evolution
To investigate the impact of increasing the number of genesavailable from mesophilic red algae in comparison to use of the genes of 
 C. merolae
 alone, we applied the reciprocalBLASTp best-hits approach using
 C. merolae
 proteins as thequery against our database. In this case, however, weexcluded
 P. cruentum
 and
 C. tuberculosum
 from the data-base. With this approach, we found 127 proteins that showedexclusive gene sharing with red algae, of which 39 (31%)
 Archaea
153
Other Bacteria
1108 
Cyanobacteria
827 
Stramenopiles
1264
 Alveolata
622 401
Haptophyta
839
Cryptophyta
379378 
Euglenozoa
295 
Fungi
400 
Metazoa
473
     B    a    c     t    e    r     i    a
PROKARYOTES
RhodophytaViridiplantaeGlaucophyta
PlantaeRhizariaExcavataAmoebozoaOpisthokonta
     E     U     K     A     R     Y     O     T     E     S
20040060080010001200Color key
Other Excavates
275 
Figure3. SchematicRepresentationofthePutativeTreeofLifeShowingtheExtent of RG Gene Sharing with Other Eukaryote and Prokaryote PhylaThe branch shown as a dashed line represents ambiguous relationshipsamong the lineages to the right. The color key indicates the number of treesfound for each ‘‘foreign’(non-RG) phylum.
Current Biology
 Vol 21 No 4330

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