Evidence-based green algal genomics reveals marine diversity and ancestral characteristics of ...

22 Pages·2016·2.84 MB·English

vanBarenetal.BMCGenomics (2016) 17:267
DOI10.1186/s12864-016-2585-6
RESEARCH ARTICLE Open Access
Evidence-based green algal genomics
reveals marine diversity and ancestral
characteristics of land plants
Marijke J. van Baren1, Charles Bachy1, Emily Nahas Reistetter1, Samuel O. Purvine2, Jane Grimwood3,4,
Sebastian Sudek1, Hang Yu1,6, Camille Poirier1, Thomas J. Deerinck7, Alan Kuo3, Igor V. Grigoriev3,
Chee-Hong Wong3, Richard D. Smith2, Stephen J. Callister2, Chia-Lin Wei3, Jeremy Schmutz3,4 and
Alexandra Z. Worden1,5*
Abstract
Background: Prasinophytes are widespread marine green algae that are related to plants. Cellularabundance of
theprasinophyte Micromonas has reportedly increased in theArctic due to climate-induced changes. Thus, studies
ofthese unicellular eukaryotes are importantfor marine ecology and for understanding Viridiplantae evolution and
diversification.
Results: Wegeneratedevidence-basedMicromonasgenemodelsusingproteomicsandRNA-Seqtoimprove
prasinophytegenomicresources.First,sequencesoffourchromosomesinthe22MbMicromonaspusilla(CCMP1545)
genomewerefinished.Comparisonwiththefinished21MbgenomeofMicromonascommoda(RCC299;named
herein)showstheyshare≤8,141of~10,000protein-encodinggenes,dependingontheanalysismethod.Unlike
RCC299andothersequencedeukaryotes,CCMP1545hastwoabundantrepetitiveintrontypesandahighpercent
(26%)GCsplicedonors.Micromonashasmoregenus-specificproteinfamilies(19%)thanothergenomesequenced
prasinophytes(11%).Comparativeanalysesusingpredictedproteomesfromotherprasinophytesrevealproteinslikely
relatedtoscaleformationandancestralphotosynthesis.Ourstudiesalsoindicatethatpeptidoglycan(PG)biosynthesis
enzymeshavebeenlostinmultipleindependenteventsinselectprasinophytesandplants.However,CCMP1545,polar
MicromonasCCMP2099andprasinophytesfromotherclassesretaintheentirePGpathway,likemossandglaucophyte
algae.Surprisingly,multiplevascularplantsalsohavethePGpathway,exceptthePenicillin-BindingProtein,andsharea
uniquebi-domainproteinpotentiallyassociatedwiththepathway.AlongsideMicromonasexperimentsusingantibiotics
thathaltbacterialPGbiosynthesis,thefindingshighlightunrecognizedphylogeneticcomplexityinPG-pathwayretention
andimplicatearoleinchloroplaststructureordivisioninseveralextantViridiplantaelineages.
Conclusions:Extensivedifferencesingenelossandarchitecturebetweenrelatedprasinophytesunderscoretheir
divergence.PGbiosynthesisgenesfromthecyanobacterialendosymbiontthatbecametheplastid,havebeen
selectivelyretainedinmultipleplantsandalgae,implyingabiologicalfunction.Ourstudiesproviderobustgenomic
resourcesforemergingmodelalgae,advancingknowledgeofmarinephytoplanktonandplantevolution.
Keywords:GreenCut,Archaeplastidaevolution,Viridiplantae,IntronerElements,RNAsequencing,Proteomics,
Evidence-basedgenemodels,Peptidoglycan,PPASP
*Correspondence:[email protected]
1MontereyBayAquariumResearchInstitute,7700SandholdtRd,Moss
Landing,CA95039,USA
5IntegratedMicrobialBiodiversityProgram,CanadianInstituteforAdvanced
Research,TorontoM5G1Z8,Canada
Fulllistofauthorinformationisavailableattheendofthearticle
©2016vanBarenetal.OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
vanBarenetal.BMCGenomics (2016) 17:267 Page2of22
Background Additionally, Bathycoccus and Ostreococcus are non-
Marine photosynthetic plankton are responsible for ap- motilewhileMicromonashasaflagellum(likemostprasi-
proximately half of global carbon fixation [1]. Prasino- nophytes) and is larger than the former two taxa.
phytes are a major lineage of unicellular green algae that Genomes have been sequenced for Micromonas species
can contribute significantly to marine primary produc- representing Clades D (Micromonas pusilla CCMP1545)
tion [2–4]. In the oceans, prasinophyte genera within and A (Micromonas RCC299) [6]. In addition, three
ClassIIareparticularlywidespread.Thesealgaeincludethe OstreococcusandoneBathycoccusspecieshavecompletely
picoplanktonic (<2 μm cell diameter) genera Bathycoccus, sequenced genomes [17–19], while targeted Bathycoccus
Micromonas and Ostreococcus which have small genomes metagenomes have been sequenced from coastal Chile
(12–22 Mb) and less gene family expansion than observed [13] and the tropical Atlantic Ocean [13, 20]. The Micro-
in other Viridiplantae groups (Fig. 1), specifically chloro- monas nuclear genomes are 22 Mb (CCMP1545) and
phytealgaeandstreptophytes[5].Like chlorophytes,prasi- 21 Mb (RCC299), while the genomes of Bathycoccus
nophytes provide insights into ancestral Viridiplantae gene prasinos (15 Mb) and various Ostreococcus (~13 Mb)
families. For example, key transcription factors formerly are smaller [6, 17–19]. Genomes of all three genera
considered innovations in vascular plants are present in contain two chromosomes with lower GC% than the
Micromonas although absent from model chlorophytes, overall average (e.g., 51 % versus the overall average
such as Chlamydomonas reinhardtii and non-vascular of 64–66 % in Micromonas). The larger low-GC region
plantslikePhyscomitrellapatens(moss)[6]. (LGC) is a proposed sex chromosome, while the other is
ThefirstdescribedeukaryoticpicoplankterwasChromu- much smaller and has few recognizable genes [6, 17, 19].
lina pusilla [7], later renamed Micromonas pusilla. Micro- TheRCC299genomesequenceisgapless,withtelomereto
monasformsatleast sevenphylogeneticallydistinctclades, telomere sequenced chromosomes [6]. In contrast the
six of which have cultured representatives [8–10]. These CCMP1545 genome was published as a high quality draft
clades appear to often co-exist in mid- to low- latitude genome(Sangersequenced)in21scaffoldsrepresenting19
systems [10, 11], with the exception of Micromonas Clade chromosomes.
E2 which is found in polar environments but not lower TofurtherdevelopgenomicresourcesforClassIIprasi-
latitude surface oceans [9]. Abundance of the latter has nophytes (the Mamiellophyceae), we finished sequences
reportedly increased in the Canadian Arctic in association from four CCMP1545 chromosomes and developed new
with climate induced changes [2]. Like Micromonas, the gene models for both CCMP1545 and RCC299 using
genus Bathycoccus is also found from tropical to polar evidence-based methods, including directional Illumina
systems, but is much less phylogenetically diverse [12, 13]. RNA-Seq and Liquid Chromatography Tandem Mass
Their sister genus Ostreococcus is found only in mid- and Spectrometry (LC-MS/MS) proteomics. Analyses of these
low-latitudewatersandhasseveralestablishedcladeswith datasets revealed characteristics of gene architecture,
distinctenvironmentaldistributions[14,15]. novel repetitive introns and deviations in splice donor se-
Morphologically the three genera have marked differ- quence.Wealsoanalyzedthepredictedproteomeofpolar
ences. All have a single chloroplast and lack visible cell CladeE2isolateCCMP2099andgeneratedgenomicinfor-
walls.UnlikeBathycoccusandotherknownprasinophytes, mation for a morebasalClassIIprasinophyte by growing
Micromonas and Ostreococcus do not have scales [16]. andsequencingthetranscriptomeofDolichomastixtenui-
lepis. Our comparative studies identified proteins that are
likely involved in scale formation and features of the land
Glaucophytes plant ancestor as well as essential components of photo-
100 Rhodophytes
100 Chlorophytes (e.g. Chlamydomonas reinhardtii) synthesis. Among these is the presence of a bacterial-like
100969388 100917105009D0MOoOMlMsiPicOcstirrirhtccsaoerBorertsmoooramoiecmnmtochooaonoooyccsdacncnocteiscoauacxru csc ssmstsc u eptcp ausanl.ouu s uuCcsmcs rioppiilCimlem lrl.oa MapRaon siCsPrdiCiain na2CCloeu 0MRs8s90CP991C 524959 (d) (c)(e) (b) (a) Prasinophytes pfsiereeoslpemhtciitdgiavohceglrillgyoyhsclstaonstththpeienactAohmmwrcuaphlytlaiepetmphlelaeatnisntthdaidaresaiptyebsneuodefpneetnwrrtgeoretoaevuivenponeltdu(sF.tiiinOogn.ulai1rnr)yes,atdubgiduess--t
100 Streptophytes tinct green algal groups, represented by Micromonas and
(a) Chlamydomonas(c) Bathycoccus (e) Micromonas(g) Physcomitrella(f) (g) (h) Chlamydomonas,forinvestigatingplantsystemsandprovide
(b) Dolichomastix (d) Ostreococcus(f) Chlorokybus (h) Helianthus
newinsightsintothedevelopmentofthegreenlineage.
Fig.1RelationshipsofphotosyntheticeukaryotesintheArchaeplastida.
ThecladogramwasinferredwiththecpREV+Gmodelusingmaximum
Results and discussion
likelihoodmethodsandaconcatenatedalignmentof16plastid-genome
encodedproteins.Bootstrapsupport>50%isshown.Plastidgenome Genomeimprovementandevidence-basedgenemodels
informationislackingforO.lucimarinusandRCC809,therefore,branching We finished four chromosomes in CCMP1545 which re-
withintheOstreococcuscladeisbasedonbranchinginthe18SrRNA
duced the total number of gaps in the genome sequence
genetree[9]andisrepresentedwithdashedlines
from 582 to 455. Specifically, scaffolds 2, 3, 18 and 19
vanBarenetal.BMCGenomics (2016) 17:267 Page3of22
were finished by closing all gaps, improving low quality set generated using multiple prediction algorithms [6]
regions andresolvingrepeat structures. These four chro- and to generate new models at previously unannotated
mosomes now represent 4,888,335 base pairs (bp) of loci. The new CCMP1545 protein-encoding gene model
finished sequence (Additional file 1: Table S1). Of the set contains 680 fewer genes than the initially published
other scaffolds in the initially published genome build Gene Catalog [6] (Table 1). The reduction was primarily
[6], scaffolds 20 (8,981 nt) and 21 (5,431 nt) are not duetomergingofadjacentgenemodelsaccordingtonew
considered to be full chromosomes, because the former transcriptional evidence and resulted in longer average
represents an unresolved repeat unit on scaffold 5 and gene length. In RCC299, average protein-encoding gene
the latter is rDNA. The newly finished Chromosome 19 size increased largely due to 5′ coding region extension
is the smallest (0.25 Mb) in the genome, and has 51 % (Table 1). Removal of unsupported RCC299 gene models,
GC.Chromosome2containsthe LGC(48%GC),which gene merging, and creation or addition of models based
spans 1.7 Mb of its total 2.2 Mb. The remainder of onnewevidenceresultedinasetthatcontained251more
Chromosome 2 is 63 % GC, similar to the overall gen- protein-encoding gene models than the prior Catalog [6].
ome average (66 %, Additional file 2: Figure S1). The Average exon size increased by 15 (CCMP1545) and
total chromosome count is 17 for RCC299 [6] and 19 23 % (RCC299) because of additional untranslated
for CCMP1545. We performed transmission electron region (UTR) sequence and because average coding
microscopy on CCMP1545 and RCC299 which showed length increased (Table 1). Overall, the new models
similar morphologies and structures, with the chloro- were 29 (CCMP1545) and 21 % (RCC299) longer than
plastoccupyingmorethan half the cell(Fig. 2a,b). the original models.
Two forms of biological evidence for protein-encoding Four independent tests were used to assess gene models
genes were generated for each Micromonas species. andvalidityofpredictedproteins.Specifically,(1)conserva-
Three hundred and twenty six million paired-end RNA- tion of predicted proteins was examined using blastp
Seq reads were generated for each Micromonas isolate searches (E-value ≤10−15) against NCBI’s non-redundant
alongside 758,467 (CCMP1545) and 236,683 (RCC299) protein set (nr); (2) transcription was verified using RNA-
peptide spectra generated by LC-MS/MS. These forms Seq data; (3) translation was verified using LC-MS/MS
of evidence were used to score, select and modify the support;and(4)predictedfunctionwascharacterizedusing
best gene model for each locus from an “allgenes” model Interproscan [21]. The vast majority (9,870) of CCMP1545
a b
Micromonas pusilla (CCMP1545) Micromonas commoda (RCC299)
Cp
Cp
Cp
Nu
Py Nu
Py
Fl
Fh
Fl
Th
500 nm 500 nm
c d
Transcribed LC/MS Peptides Transcribed LC/MS Peptides
Function 336 592 13 Conserved Function 117 513 24 0 1 Conserved
107 4 12 203
233 1077 12 77
21 27
2061
6475 0 1550
8 168
5 5492
30 698
203
81
Fig.2MicromonaspusillaandM.commodacellularstructuresandgenemodelsets.TransmissionelectronmicrographsofCCMP1545(a)and
RCC299(b)showthechloroplast(Cp)comprisingmorethanhalfthecell,thepyrenoid(Py),nucleus(Nu)andflagellum(Fl)withmicrotubules
visible(24nmdiameter).InRCC299thebeginningoftheflagellarhair(Fh)isalsovisibleasarethethylakoidmembranesinCCMP1545(Th).
EvidencefornewgenemodelsinCCMP1545(c)andRCC299(d)isshownbasedon:Function:Predictedproteinhadafunctionaldomainhitin
Interproscan;Transcribed:Predictedgenemodelwasexpressed(FPKM>2,000)intheRNA-Seqexperiment;LC-MSPeptides:Atleastonepeptide
matchingthepredictedproteinwasidentifiedintheLC-MS/MSanalysis;Conserved:BlastphitswerefoundinNCBI’snrdatabase(E-value≤10−15).
Thetotalnumberofgeneswithnoevidence:25(CCMP1545)and37(RCC299).NotethatforRCC299thelowernumberofgenemodels
supportedbyLC-MS/MSdata(2,306)thaninCCMP1545(8,432)isduetogenerationoffewerpeptides
vanBarenetal.BMCGenomics (2016) 17:267 Page4of22
Table1Comparisonoftheevidence-based(EB)proteincodinggenemodelsetspredictedhereforthenucleargenomesof
Micromonaspusilla(CCMP1545)andMicromonascommoda(RCC299)andtheoriginalmodelsets(“Catalog”)publishedin
Wordenetal.2009[6]
CCMP1545EBset CatalogCCMP1545 RCC299EBset CatalogRCC299
Proteincodinggenes(number) 9895 10575 10307 10056
Averagetranscriptlength(nt) 1799 1390 1808 1497
Averagecodinglength(nt) 1653 1317 1663 1419
Introns(number) 11229 9531 5418 5650
Averageintronlength(nt) 180 187 152 163
Exons(number) 21060 20106 15651 15706
Averageexonlength(nt) 840 731 1182 958
Splicedgenes(number) 5666 5311 3584 3688
Exonspermultiple-exongene 2.98 2.79 2.51 2.54
Totalintergenicbases(Mb) 2.3 n.c. 1.7 n.c.
Totalexonicbases(Mb) 17.6 n.c. 17.4 n.c.
Totalintronicbases(Mb) 0.20 n.c. 0.78 n.c.
GCsplicedonors(%) 25.60 5.90 1.50 0.70
Notethatforaveragetranscriptandcodinglengthsintronshavebeenremoved.FortheEBsetsgenecharacteristicswerecomputedon9826(CCMP1545)and
10233(RCC299)proteins,towhich69and74modelswerelateradded,respectively,duetonewRNA-seqsupport.Abbreviation:n.c.,notcomputed
protein-encoding gene models had supporting evidence, tail overlaps occur among LGC genes. Overlap between
while 25 did not (Fig. 2c). Ninety-eight percent of genes protein-coding genes in eukaryotes has been suggested as
were confirmed using RNA-Seq, and 96 % were supported a mechanism for reciprocal regulation [18, 22]. For single
by at least two types of evidence. Of the 21 predicted pro- cellorganismssuchasMicromonas,physicalseparationof
teinswithonlyInterproscanevidence,11wererelatedSel-1 cytoplasmicbiochemicalpathwaysisonlyfeasiblethrough
like repeat (SLR) proteins. For RCC299, 97 % of models temporalregulationandindeedrhythmicpatternsingene
were confirmed with RNA-Seq and 94 % were supported expression have been found in Ostreococcus [23] and
by at least two evidence types (Fig. 2d), while 37 gene CCMP1545 [24]. Further studies are needed to establish
models had no evidence. Twelve predicted proteins from whether reciprocal regulation of COPs provides a mech-
mixed families of unknown function (two with zinc finger anism for temporal partitioning of cellular processes and
predictions)hadonlyInterproscansupport. expressionalprogramsinunicellularorganisms.
Geneoverlap Architecturalandintronicnovelties
Forbothspecies,genedensityislowerthanaverageonthe CCMP1545andRCC299havetwocleardifferencesingene
smallestchromosomes(whichalsoexhibitlowerthanaver- architecture; both are related to intron characteristics. We
ageGC%;Additionalfile1:TableS1).Genedensityisalso identified numerous GC splice donors in CCMP1545
lower in the LGC, but unlike the smallest chromosomes, (25.6%)thatwerelargelyabsentininitial predictions. This
LGC genes are often organized in convergent overlapping islikelybecausemostpredictionprogramsrequireGT/AG
pairs (COPs). These have overlapping 3′ UTRs and rela- splice donor/acceptor pairs while the short read sequence
tivelylargeintergenicdistancestotheirrespectiveupstream aligner used here [25] accommodates both GT and GC
(5′) neighbors. In the CCMP1545 LGC, 316 of 591 genes splice donors. To our knowledge, only the marine hapto-
occurasCOPs,withanaverageintergenicdistanceof1,255 phyte alga Emiliania huxleyi has more GC splice donors
(± 837) nucleotides (nt) between the COP and non-COP (50%)[26]thanCCMP1545.UnlikeCCMP1545,the1.5%
neighbors. The averageintergenicdistanceontheother 18 GCsplicedonorsinRCC299(Table1)isnearlyidenticalto
chromosomes is 211±398 nt. Similarly, in the RCC299 other Viridiplantae, such as the streptophytes Arabidopsis
LGC, 242 of 738 genes occur in COPs, with intergenic thaliana(1.5%)andBrassicarapa(1.2%)[27–29].
distances of 898±810 nt that contrast with the average CCMP1545 also has twice as many introns as RCC299,
fortheother16chromosomesof167±238nt.COPnum- although both species contain similar numbers of
bers are likely underestimated because we required EST nucleus-encoded genes (Table 1). Many of the introns in
evidence (directionally cloned cDNAs, Sanger sequenced) CCMP1545 are Introner Elements (IE), a type of spliceo-
as validation of overlapping models. Visual inspection of somalintronthathasrecognizablebranchpoints,butalso
the RNA-Seq evidence indicates that many more tail to hashighsequenceidentitythroughoutthegenome(unlike
vanBarenetal.BMCGenomics (2016) 17:267 Page5of22
regular spliceosomal introns, RSIs) [6, 9, 30]. IE fragment onlyonemotif,thatmotifwaspresentinmultiple copies.
thegenemodelsproducedbysomepredictionalgorithms. Mixed-motif partials, with motifs from D-IE1 and D-
ToidentifyIEhere, predicted introns in CCMP1545were IE2, occurred seven times. The large number of partial
clustered to identify those with sequence similarity. A motifs inside introns indicates that both D-IE1 and D-
motif finder was used to identify sequence motifs; two IE2 are diverging from their source sequence and that
groupsofnon-overlappingmotifswerefound:afourmotif much of the specific primary sequence of the IE is not
group that identified IE1, IE3, and IE4 as reported in (6), necessary for splicing, supporting hypotheses put forth
and a three motif group that recognizes IE2 (Fig. 3a). in [9].
We will refer to these as D-IE1 and D-IE2 (exclusive WhilethevastmajorityofD-IEareintronicandoriented
to Clade D Micromonas [9]), respectively. in the same direction as the gene containing them, 34
The non-overlappingmotif sets had 0 to49 nucleotides intronic IEs appear to be on the opposite strand. Seven of
between those present in an intron (see Fig. 3a, Table 2) thesewerecompleteD-IE(sixD-IE1,oneD-IE2).Addition-
and were used to identify a total of 3,409 complete D-IE: ally,57completeand349partialIE(accordingtoourmotif
3,171 D-IE1 matched all of the motifs in the four motif analysis)appeartobeintergenic(Table2).SomeD-IEover-
group and 238 D-IE2 matched all the motifs in the three lapped coding (153) or noncoding (131) exons, most of
motifgroup.Inadditiontocompletematchestothe four which (204) were located on the opposite strand. The
motif (or three-motif) group, 3,131 partial D-IEs were majority(198outof284)wereD-IE1partialsthatonlycon-
identified.Amongthese,572matchedthreeofthefourD- tained motif 1, suggesting possible integration into the
IE1 motifs, 382 matched two D-IE1 motifs, and 1,501 CDS. Motif 1 does not encompass branch points, and is
matched only one D-IE1 motif (the first motif in 1,247 therefore unlikely to function as an independent intron.
cases;Fig.3a).TwofullD-IE1sandoneD-IE2matchedall These deviations may also represent lingering issues with
four (or three) motifs, but with an internal duplication, genemodelsorpotentiallyotheraspectsofMicromonasIE
possiblyduetoamergeoftwooriginallycompleteD-IE1s. dynamics and their proposed propagation at R-loops [9].
Partial D-IE2 elements were also found. These contained Overall, 5,850 D-IE (complete, partial, and mixed) are
only two (287) motifs or just one (382, of which 354 are located in 5,499 introns of the new model set, 90 % of
motif1).In18ofthe1,501caseswhereaD-IE1contained which are supported by spliced transcript data. Although
a
MicromonasClade D Introner Elements (represented by M. pusilla CCMP1545)
2
bits1 C 308 t ont
01 motif 1 50 1
E
2 2 2 D-I
s
bit1 0 to 0 to
49 nt 48 nt
0 0 0
1 motif 2 29 1 motif 3 29 1 motif 4 29
2 2 2
bits1 221 t ont 1 228 tnot D-IE2
0 0 0
1 motif 1 21 1 motif 2 33 1 motif 3 41
b Donor RSI D-IE c
MicromonasClade A Introner Element
Type 2 2
(represented by M. commoda RCC299)
GT bits1 bits1 2
E
0 exon intron 0 exon intron bits1 C-I
2 2 B
0 A
GC s s 1 sole motif 50
bit1 bit1
0 0
exon intron exon intron
Fig.3IntronerElements(IEs)andintronsplicedonorfeatures.(a)D-IE1andD-IE2containfourandthreemotifgroups,respectively.Minimum
andmaximumnucleotidestretchesinterveningbetweenmotifsareindicated.(b)CCMP1545containsahighpercentageofGCsplicedonorsin
IEcontainingintrons(43.5%)aswellasregularspliceosomalintrons(RSIs,9.7%).SplicedonormotifsareshownforGTandGCmotifsinbothRSIs
andIEs(motifsgeneratedwith500inputsequenceseach).(c)ThemotifoftheRCC299ABC-IE(basedon164ABC-IEcontainingintrons)
vanBarenetal.BMCGenomics (2016) 17:267 Page6of22
Table2IntronerElementfamiliesinMicromonasCladeD,asidentifiedinCCMP1545,andinMicromonasCladesA,B,andCas
identifiedinRCC299
IEfamily D-IE1(complete) D-IE1(partial) D-IE2(complete) D-IE2(partial) ABC-IE
Motifs 4 1–3 3 1–2 1
Count 3171 2455 238 669 164
Intronic 3117 2194 201 337 164
Intergenic 25 46 32 303 0
GCsplicedonor 46% 44% 7% 15% 0%
SequencesmatchingmotifsfrombothIE1andIE2wereomitted(7total)
351 of these introns contain two or three IE (summing to requiringthatatleast60%ofthelengthoftheshorterpro-
six or more motifs), most IE containing introns have a tein overlap with the longer pair member. However, this
singlecompleteD-IE1. approach could result in missed orthologs. Therefore, we
Presence of D-IEs (complete and partial) is connected to also used a lenient reciprocal best blastp hit approach (E-
higher percentages of GC splice donors, 79 % of which value ≤10−5), which identified 8,141 putative orthologs.
occurinD-IE1-containingintrons.Still,54%ofD-IE1have Thus, on average 19 % (reciprocal blastp) to 36 %
GTsplice donors, indicating that selection may act against (OrthoMCL) of predicted proteins can be considered
the GC splice donor. Only 15 % of introns containing presentinoneMicromonasbutnottheother,dependingon
complete D-IE2 have GC splice donors, while introns that themethodused.OnegenefamilyexpansioninCCMP1545
contain a full D-IE1 have GC splice donors 46 % of the relative to RCC99 involved SLR proteins. Eighty-four are
time. Interestingly, sequence proximal to D-IE splice do- present in CCMP1545, compared to nine in RCC99. SLR
nors is much more conserved than for RSIs, regardless of proteinsarewidespreadineukaryotesandbacteria,typically
GCorGTdonorstate(Fig.3b). have low similarity levels, and are involved in a wide range
WeappliedthesameanalysisapproachtoevaluateABC- of processes, including cellular stress, regulation of mitosis,
IEs in RCC299 [9, 30]. One hundred sixty four ABC-IEs and assembly of membrane-bound protein complexes [35].
were identified, less than the 221 reported elsewhere [30]. The significant expansion in M. pusilla (CCMP1545) pro-
Unlike D-IEs, a single highly conserved motif captured videsanexampleofexpansionthatmayrelatetobasicbio-
thesesequences,allofwhichwereintronic.TheIEsfurther logicaldifferencesbetweenthesetwoisolates.
differ from the abundant families in CCMP1545 because Collectively, the differences observed here and in prior
they are on average shorter (64±7 nt) than RSIs (152± studies [6, 8, 10] clearly support Clade A (RCC299) and
98 nt), more akin to Introner-Like Elements in fungi CladeD(CCMP1545)asbeingdifferentspecies.Therefore,
[31, 32]. Overall, our results demonstrate that D-IE are here we name RCC299 Micromonas commoda based on
an order of magnitude more abundant than repetitive molecular diagnoses and the protocols of the International
introns reported in genomes of other species, in par- Code of Nomenclature for Algae, Fungi and Plants. The
ticular fungi [31–33] and RCC299 [9, 30]. speciesnamereferstothefactthatRCC299iseasytogrow
inanaxenicstateinthelaboratory.Thisnamingwillavoid
Micromonasproteomecomparisonsanddesignationofa confusion in the literature [36, 37] where Clade A strains
newspecies such as RCC299 or its close relatives (e.g., Clade C isolate
Proteinfamiliesinthetwogenome-sequencedMicromonas Mp-Lac38)areincorrectlytermedMicromonaspusilla.
were compared using two approaches. Previous studies
have performed global analyses on niche differentiation Micromonas commoda van Baren, Bachy and Worden,
between the two Micromonas [6] and other Mamiellophy- sp.nov.–Fig.2b.
ceae[18,19]usingbestblastapproaches.Here,OrthoMCL
analysis [34] showed that a total of 6,265 predicted Morphological description — Naked cells, oblong. Motile
protein families were present in both CCMP1545 and with a single flagellum, conserved microtubule arrange-
RCC299, encompassing 6,402 and 6,484 proteins, re- ment and a flagellar hair of uncharacterized length. The
spectively (Fig. 4a). Forty additional families (collectively single chloroplast contains a starch granule and pyrenoid.
containing 156 proteins) and 3,337 ‘singleton’ proteins By Coulter Multisizer analysis average diameter (blind to
were present in CCMP1545 only. Eighty-six families orientation) is 1.43±0.16 μm and volume is 1.532±
(together containing 222 proteins) and 3,527 singletons 0.545μm3duringmid-exponentialgrowth(mu=1.09d−1)
werepresentinRCC299only.Weminimizedtheprobabil- under 90 μmol photons m2 sec−1 (photosynthetically ac-
ity of grouping proteins inaccurately (e.g., based only on tive radiation) at 21 °C in K-medium with an artificial sea
presence of a common domain) in this analysis by waterbase.
vanBarenetal.BMCGenomics (2016) 17:267 Page7of22
a b c
M. pusilla M. commoda
Dolichomastix Bathycoccus
Micromonas pusilla
(CCMP1545) Micromonass Ostreococcus
2246 358 33
42 67
(1777) (1812)
807 106 198
5237 2032 1158
40 6265 86
135 59
(156) (6402/ 6484) (222)
172 452
2986
157 226 40
(2306) 1769 318
Micromonas commoda
510
(RCC299) CCMP2099
Fig.4Core,shared,anduniqueprotein-encodinggenefamilies.Valuesindicatethetotalnumberofproteinfamilies(containingtwoormoreproteins)
andinparenthesesthegenecountcontainedwithinthosefamiliesfor(a)M.commodaRCC299andM.pusillaCCMP1545.Uniquesingletongenesarenot
representedinthispanel,andonlyonerepresentativewaskeptforidenticalparalogsinCCMP1545(30)andRCC299(21).bComparisonof
RCC299,CCMP1545,andtranscriptome-basedCCMP2099proteinsequences.Numbersinparenthesesaregenesforwhichnoparalogwas
identified.cComparisonofMicromonas(combinedCCMP1545,RCC299andCCMP2099),Dolichomastixtenuilepis,Bathycoccusprasinos,and
Ostreococcus(combinedO.lucimarinusandO.sp.RCC809).Notethatanindividualgenefamilyinouterlobescanbecomposedoftwoor
morepredictedproteinsfromonespecies
Molecular description — Sequences describe the type Habitat and ecology — Planktonic photosynthetic life-
specimen (RCC299, deposited at the National Center for style in marine photic zone waters. Habitat extent
Marine Algae and Microbiota (NCMA) as CCMP2709) known to date: coastal to open oceans; has not been ob-
and are available in GenBank under accession numbers servedinhighlatitudesystems(latitudes>60° NorS).
KU612123(ribosomalRNA operon) andXM_002507645
(β-tubulin). Etymology — The specific epithet commoda refers to the
‘ease and convenience’ of culturing and propagating this
Molecular diagnosis — Nucleotide character state “A” in species which grows well in artificial seawater when
positions 1343, 2455, 2761 and 2795 and “T” in position amended according to K or L1 [38] medium recipes and
2947 of ribosomal RNA operon sequence. These charac- inotherstandard marine algalmedia.
ters are also shared by all Clade A and Clade B Micro-
monas strains (sensu Slapeta et al. MBE 2006, Simmons Comparativegenomicsofmarinegreenalgae
et al. 2015) but not Micromonas pusilla (Clade D) or We also compared Micromonas with other Class II prasi-
Clades C and E (sensu Slapeta et al. MBE 2006, nophytes using predictedproteomes from eithergenomes
Simmons et al. 2015). Nucleotide character state “T” in orRNA-Seqtranscriptomeassemblies.First,wecompared
positions 120, 222, 1011 and 1233, and “A” in positions the proteomes predicted for M. pusilla CCMP1545 and
181and1429,and“C”inposition186ofβ-tubulincoding M. commoda RCC299toa protein set predicted from the
sequence. Multiple genes contain a repetitive intron se- CCMP2099 transcriptome [39, 40], which represents the
quencewiththe motif in Fig. 3C; this sequence is present polarMicromonasCladeE2[9].Afterremovingduplicates
in closely related lineages (Micromonas A/B/C lineage from the transcriptome assembly, a total of 9,494
sensu Slapeta et al. MBE 2006, Simmons et al. 2015) but CCMP2099 proteins were analyzed, ranging from 30 to
notinMicromonaspusilla. 7,612aminoacids(average587aa).Therelativeoverabun-
dance of short proteins indicates that the transcriptome-
Holotype — Strain CCMP2709 is the type specimen and basedgeneassemblieswereoftenincomplete.
is preserved in a metabolically inactive state at the OrthoMCL was used to create core, shared and unique
NCMA (https://ncma.bigelow.org/). CCMP2709 was de- protein families between CCMP1545, RCC299, and
posited at the NCMA by the Worden lab after rendering CCMP2099 (Fig. 4b). ‘Unique’ features in transcriptome-
the field isolate RCC299/NOUM17 clonal and axenic. based predicted proteomes can only be determined if the
RCC299 was collected on 10 February 1998 in open respective proteins are absent from genome-sequenced
ocean surface waters of the South Pacific at 22.3° S, taxa, but not the reverse: Absence from a transcriptome-
166.3° E and is available at the Roscoff Culture Collec- based proteome can reflect either absence from the gen-
tion (http://roscoff-culture-collection.org/). ome, or lack of transcription at the time of sampling. A
total of 5,237 families were shared between Micromonas
Validating illustration—Figure2b. CladesA,DandE2(Fig.4b).Anadditional2,246families
vanBarenetal.BMCGenomics (2016) 17:267 Page8of22
wereshared by CCMP1545 and RCC299,possibly reflect- orthologcoveragebetweenO.lucimarinusandCCMP1545
ing incomplete coverage of the CCMP2099 proteome. higher than between RCC299 and CCMP1545. While
CCMP2099 and RCC299 shared 452 gene families not furtherinvestigationisnecessary,theseresultssuggestthere
present in CCMP1545, and 172 gene families were exclu- may be less evolutionary divergence between core genes
sive to CCMP1545 and CCMP2099. This suggests shared by the most basal Micromonas (Clade D, repre-
CCMP2099issomewhatlessdivergedfromRCC299than sentedbyCCMP1545)[9]anditssistergroupOstreococcus
fromCCMP1545,atleastintermsofgenecontent. (andBathycoccus),thanwiththemorederivedMicromonas
CCMP2099 contained putative paralogs absent from CladesAandE2.
CCMP1545 and RCC299. One group contained three
genes encoding one or more discoidin domains (i.e., DS Scaledornaked
or F5/8 type C domains; Pfam 00754). The transcripts To understand differentiation between scaled and naked
from these are divergent, making it unlikely that they prasinophytes as well as other genus level differences,
represent alternative spliceoforms of a single gene. None four additional proteome sets were created and com-
of the proteins have predicted transmembrane domains pared. The first three sets comprised (i) all Micromonas
or signal peptides, but the protein predictions do not (CCMP1545,RCC299andCCMP2099combined),(ii)all
start with methionine and are likely to be 5′ incomplete. Ostreococcus (O. tauri, O. lucimarinus and O. RCC809)
Discoidin proteins are involved in binding of cell-surface and (iii) the predicted proteome from the B. prasinos
attached carbohydrates and are present in eukaryotes genome [19]. The fourth set contained just a predicted
andprokaryotes[41].Arecent publication proposed that proteome (transcriptome-based) of the more basal Class
CCMP2099 is capable of phagotrophy [42], in which II prasinophyte Dolichomastix tenuilepis (Fig. 1). Mem-
case it seems possible that these proteins may function in bers of the Dolichomastix genus are motile [3] and are
substrate recognition. Absence of these domains in the present in the Arctic and temperate oceans [43]. The
other Micromonascladesanalyzedwouldthenbe consist- four Class II prasinophyte genera shared 2,986 of 10,735
ent with the fact that only photoautotrophic growth has protein families (Fig. 4c). D. tenuilepis caused the largest
beenobservedfortheCladeAandDisolates. reduction in core numbers and, when excluded, the
The number of “unique” genes and families in each Class II prasinophyte core is just 9 % smaller than the
Micromonas clade is extensive and raises questions on Micromonas core set (Fig. 4b). The D. tenuilepis protein
similarity levels between orthologs shared between two set consisted of 16,884 unique proteins, of which 25 %
or more clades. On average, amino acid identity between were between 30 and 100 amino acids long (Additional
Micromonas orthologs was low, for example when 8,141 file 2: Figure S2). In contrast, only 2 % of CCMP1545
reciprocal best blast hits (orthologs) were aligned be- and RCC299 protein predictions are <100 amino acids,
tween CCMP1545 and RCC299, on average only 60 % of with the smallest being 39 and 33 amino acids, respect-
amino acid residues were identical over the length of the ively. This suggests issues withthe predicted D. tenuilepis
alignment(Table3andAdditionalfile1:TableS2).Aver- proteome arising from libraryconstruction, RNA sequen-
age coverage was 78 %, based on the fraction of aligned cing or assembly methods [39]. Alternatively, incomplete
regions in the shorter sequence. Results were similar for proteinpredictionsmighthavecausedissuesconnectedto
orthologs shared between CCMP2099 and RCC299 and OrthoMCLcriteriaonproteinoverlap.Hence,aconserva-
those shared between CCMP2099 and CCMP1545. In tiveestimateoftheClassIIprasinophytecoreexcludesD.
contrast, the 6,449 OrthoMCL-identified orthologs in O. tenuilepis,resultingin4,755sharedfamilies(44%).
lucimarinus and Ostreococcus sp. RCC809 averaged 73 % BothBathycoccusandDolichomastixformscales[44,45]
identity and 93 % coverage. Comparison of O. lucimari- as do nearly all prasinophytes described to date [46, 47],
nus and CCMP1545 orthologs (5,534 proteins) showed making their absence in Micromonas and Ostreococcus
54 % identity with 83 % average overlap. This makes unusual. The biosynthetic pathway for scale formation is
Table3Orthologsimilaritiesamongprasinophyteswithsequencedgenomes
Comparedorganisms CCMP1545 CCMP1545 RCC299 CCMP1545 O.lucimarinus
(pairs) RCC299 CCMP2099 CCMP2099 O.lucimarinus O.RCC809
ID(%) 60 57 57 54 73
Coverage(%) 78 74 77 83 93
Genefraction(%) 83 62 65 76 88
Orthologcount 8141 5859 6160 5534 6449
Thegenefractionrepresentstheorthologcountdividedbythetotalnumberofpredictedproteinsinthesmallermemberofthepair(withrespecttogenecount)
vanBarenetal.BMCGenomics (2016) 17:267 Page9of22
unknown[48],butfourgenefamilieshavebeenreportedas clades[53],B.prasinos(0.3%),andD.tenuilepis(3%).For
expanded in B. prasinos, compared to other genome- thelattertwo,inclusionofgenomesortranscriptomesfrom
sequenced Class II prasinophytes: sialyltransferases, siali- other members of the genus might expand the observed
dases (neuraminidases), ankyrin-repeat proteins, and zinc values. These results highlight the greater gene diversifica-
finger proteins [19]. Here, of 106 gene families shared tion within the Micromonasgenus compared to Ostreococ-
between B. prasinos and D. tenuilepis that were not found cus and likely more extensive genome reduction prior to
in the other sequenced genera, 31 were sialyltransferases divergencewithintheOstreococcusgenus.
(Pfam 00777), and 11 were neuraminidases (IPR011040). Each genomesequencedClassIIprasinophytegenushas
The sialyltransferase families contained 34 B. prasinos and low redundancy within protein families. Among the
32 D. tenuilepis proteins and the total number in these families identified here, only 4 % of those that contain
organisms was even higher, 78 and 71, respectively. Sialyl- CCMP1545 proteins have more than one CCMP1545 pro-
transferases were otherwise found only in M. commoda tein (Additional file 1: Table S3). The same is true for
andO.RCC809(oneeach),makingthesegenesreasonable RCC299,B.prasinos,O.tauri,andO.lucimarinuswhileO.
candidatesforinvestigationofscaleformation. RCC809 shows even less expansion (3 % of families). Low
About half of the 23 B. prasinos and 24 D. tenuilepis genefamilyexpansionmakestheseorganismsstrongcandi-
neuraminidases belonged to families shared between the datesforfutureexperimentalworkonproteinfunction.
two species, but none were shared with naked prasino-
phytes. No neuraminidases were found in RCC299, O. TheViridiplantaeancestor
tauri, and O. RCC809, one neuraminidase was present in Presence/absence patterns between prasinophyte protein
CCMP1545. O. lucimarinus contained four other neur- families provide insights into the evolution of theViridi-
aminidases. Blastp of the B. prasinos proteins against the plantae as a whole. This is true at the level of individual
NCBI nr database revealed just single neuraminidase protein families, biosynthesis pathways [40] and the
proteins in the Trebouxiophyceae Chlorella variabilis, ancestral suite of photosynthesis-related machinery. One
Coccomyxa subellipsoidea C-169, Auxenochlorella proto- such protein is phytochrome, a master regulator in
thecoides, and Helicosporidium sp. ATCC 50920. Hence, plants that is present in M. pusilla and D. tenuilepis, but
these proteins provide a second example of enrichment not in M. commoda, the M. sp. CCMP2099 transcrip-
thatispotentiallyrelatedtoscaleformation. tome, Ostreococcus, Bathycoccus or chlorophyte algae.
Another protein present only in B. prasinos and D. The prasinophyte phytochromes share conserved signal-
tenuilepiswasaGolgi-targetedxyloglucanfucosyltransfer- ing mechanisms with land plants but detect shorter
ase.Xyloglucanisahemicellulosethatmakesup~20%of wavelengths [24]. Here, the ‘unique’ overlap of families
the primary cell wall of vascular plants [49]. Other containing Micromonas representatives and other Class
enzymes for xyloglucan synthesis such as xyloglucan IIprasinophyteswasgreatestwithDolichomastix(Fig.4c;
endo-transglycosylase/hydrolase(XTH)andβ1→4-glucan Additional file 1: Table S4). Our analysis identified
synthasearepresentincharophytealgae[50],butnotinB. multiple enzymes involved in peptidoglycan biosynthesis
prasinos or the D. tenuilepis transcriptome. A. thaliana (Fig. 5a) in protein families shared by D. tenuilepis and
fucosyltransferase 1 (AtFUT1) has been shown to fucosy- two ofthreeMicromonasspecies (Fig. 5b).
late xyloglucan and at least two of the remaining nine Peptidoglycan (PG) formation involves ten core en-
AtFUT proteins appear to have some function in cell wall zymes, seven of which participate in the conversion of
formation [51, 52], suggestive of a possible role of FUT in UDP-N-acetyl-D-glucosamine (GlcNAc) to GlcNac-N-
wall or scale formation in these prasinophytes. In contrast acetylmuramyl-pentapeptide-pyrophosphoryl-undecapre-
to prior genome-based studies on scale formation [19], we nol [54] (Fig. 5a). In bacteria, including cyanobacteria,
did not find enrichment of ankyrin repeats or zinc fingers this compound istransferredto theperiplasm byMURG
in the families shared only between the scaled taxa. Many and MRAY, and multiple linear strands are then cross-
zincfinger and ankyrin repeat genes were found inMicro- linked by penicillin-binding proteins (PBPs) to form
monas (408 and 132 in CCMP1545, 425 and 129 in the 3-dimensional structure of the cell wall PG layer
RCC299, respectively) and Ostreococcus (O. tauri: 230 and [55, 56]. Glaucophyte algae, which also belong to the
69; O. lucimarinus: 60 and 75; and O. RCC809: 213 and Archaeplastida (Fig. 1), maintain the PG-wall of the
57). The majority of these were in families present in all cyanobacterial endosymbiont around their chloroplast
theprasinophytesanalyzed. [57]. However, PG has not been observed in plastids
WhentheClassIIprasinophyteproteomeswereanalyzed of other Archaeplastida groups and is presumed lost,
together,19%(2,032)ofthe10,735proteinfamiliesidenti- resulting in modifications of the mechanisms for
fied were exclusive to Micromonas (Fig. 4c). This is higher chloroplast division or wall formation that are not
thanthefractionofproteinsuniquetothethreeOstreococ- understood [57, 58]. In the vascular plant A. thaliana,
cus (11 %), which represent three of the four Ostreococcus only four PG pathway genes remain: MURE, MRAY,
vanBarenetal.BMCGenomics (2016) 17:267 Page10of22
MURG,andDDL[58,59](Fig.5b).Indeed,amongstrepto- are also present in the D. tenuilepis transcriptome. In
phytes,thecompletesetofenzymeshasonlybeenreported contrast, M. commoda has only MURE, MRAY, MURG
in P. patens [58, 60] and Selaginella moellendorffii (spike and PBP, and the three Ostreococcus as well as Bathycoccus
moss), a non-seed species thatbelongs to the oldest extant only contain DDL and MURE (Additional file 1: Table S5).
vascular plant division [61]. A PG-layer has not been ob- Twooftheseenzymes(MURGandPBP)werereportedpre-
servedinchloroplastsofthesetaxa. viously in RCC299 (M. commoda) [59]. Chlorophyte algae
We identified complete PG pathways in M. pusilla, M. alsohaveonlyafewPG-pathwaygenesandshowdifferences
sp. CCMP2099, and prasinophytes from Class III and betweenCoccomyxasubellipsoideaversusC.reinhardtiiand
VII (Fig. 5b and Additional file 1: Table S5). Most genes Volvox carteri (Fig. 5b, Additional file 1: Table S5). A PG
a UDP b MurAMurBMurCMurDMurEDdlMurFMraYMurGPBPPpasp
MurA Gloeochaete wittrockiana Glaucophytes
Galdieria sulphuraria Rhodophytes
enolpyruvate Porphyra purpurea
UDP Chondrus crispus
MurB Volvox carteri Chlorophytes
Chlamydomonas reinhardtii
Coccomyxa subellipsoidea
UDP Picocystis salinarum Class VII s
MurC,MurD,MurE Nephroselmis pyriformis Class III yte
Mpl Dolichomastix tenuilepis Class II h
p
Bathycoccus prasinos o
Ddl UDP OMsictrreoomcooncacsu ss pta. uCrCi MP2099 asin
MurF Micromonas commoda RCC299 Pr
Micromonas pusilla CCMP1545
Physcomitrella patens s
Sphagnum fallax e
UDP yt
Selaginella moellendorffii s h
PP MraY OPBarraynczichauy smpao tvdiviiraugmat udmistachyon Grassophyte mbryop
P Setaria italica he E
P UDP Sorghum bicolor ac
MurG Zea mays Tr
UDP Linum usitatissimum s s
Populus trichocarpa bid cot
Manihot esculenta a di
p ants RCiuccinuumsi sc osmatmivuusnis F Eu
la P pl Fragaria vesca
stid P eed PMraulnuus sd poemresisctaica
membrane cular s MGPhleyadcsiicneaeog lmuos at rvxuunlgcaartuislata
s
PBP Va Solanum lycopersicum
Solanum tuberosum
Mimulusguttatus
Vitis vinifera
Eucalyptus grandis s
d
peptidoglycan Citrus sinensis vi
Gossypium raimondii al
P Theobroma cacao M
P undecaprenolpyrophosphate Carica papaya
Brassica rapa
D-Glu m-DAP or L-Lys L-Ala Arabidopsis thaliana
Capsella rubella
D-Ala GlcNAc MurNAc
gene present not in transcriptome not in genome
Fig.5Peptidoglycanbiosynthesis.aProteinsinvolvedinthepeptidoglycanbiosyntheticpathwayandfinalcrosslinkingsteps(after[63,110]).
bPeptidoglycanpathwayproteinsintheArchaeplastida,showingMURE,DDL,MRAYandMURGarepresentthroughouttheViridiplantae.ThefullPG
pathwaycomplementwiththeexceptionofPBPoccursinseveralmajorplantlineages.Thefinalcolumnindicatesthegeneforplantpeptidoglycan
associatedstreptophyteprotein(PPASP)identifiedherein,aputativeassignmentbasedonitsexclusivepresenceinplantswiththecompleteornearly
completePG-pathway