the genome of ectocarpus subulatus highlights unique...
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The genome of Ectocarpus subulatus1
highlights unique mechanisms for2
stresstoleranceinbrownalgae3
4
Simon M. Dittami1*, Erwan Corre
2, Loraine Brillet-Guéguen
1,2, Noé Pontoizeau
1,2, Meziane Aite
3,5
Komlan Avia1, Christophe Caron
2, Chung Hyun Cho
4, Jonas Collén
1, Alexandre Cormier
1, Ludovic6
Delage1,SylvieDoubleau
5,ClémenceFrioux
3,AngéliqueGobet
1, IreneGonzález-Navarrete
6,Agnès7
Groisillier1,CécileHervé
1,DidierJollivet
7,HettyKleinJan
1,CatherineLeblanc
1,AgnieszkaP.Lipinska
1,8
Xi Liu2, Dominique Marie
7, Gabriel V. Markov
1, André E. Minoche
6,8, Misharl Monsoor
2, Pierre9
Pericard2,Marie-Mathilde Perrineau
1, Akira F. Peters
9, Anne Siegel
3, Amandine Siméon
1, Camille10
Trottier3,HwanSuYoon
4,HeinzHimmelbauer
6,8,10,CatherineBoyen
1,ThierryTonon
1,1111
1SorbonneUniversité,CNRS,IntegrativeBiologyofMarineModels(LBI2M),StationBiologiquede12
Roscoff,29680Roscoff,France13
2CNRS,SorbonneUniversité,FR2424,ABiMSplatform,StationBiologiquedeRoscoff,29680,Roscoff,14
France15
3InstituteforResearchinITandRandomSystems-IRISA,UniversitédeRennes1,France16
4DepartmentofBiologicalSciences,SungkyunkwanUniversity,Suwon16419,Korea17
5IRD,UMRDIADE,911AvenueAgropolis,BP64501,34394Montpellier,France18
6 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr.19
Aiguader88,Barcelona,08003Spain20
7SorbonneUniversité,CNRS,AdaptationandDiversityintheMarineEnvironment(ADME),Station21
BiologiquedeRoscoff(SBR),29680Roscoff,France22
8MaxPlanckInstituteforMolecularGenetics,14195Berlin,Germany23
9BezhinRosko,40RuedesPêcheurs,29250Santec,France24
10DepartmentofBiotechnology,UniversityofNaturalResourcesandLifeSciences(BOKU),Vienna,25
1190Vienna,Austria26
11CentreforNovelAgriculturalProducts,DepartmentofBiology,UniversityofYork,Heslington,York,27
YO105DD,UnitedKingdom.28
29
*Correspondence:[email protected],phone+33298292362,fax+33298292324.30
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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Abstract31
32
Brownalgaearemulticellularphotosyntheticorganismsbelongingtothestramenopilelineage.They33
aresuccessfulcolonizersofmarinerockyshoresworld-wide.ThegenusEctocarpus,andespecially34
strainEc32,hasbeenestablishedasageneticandgenomicmodelforbrownalgae.Arelatedspecies,35
EctocarpussubulatusKützing,ischaracterizedbyitshightoleranceofabioticstress.Herewepresent36
the genome andmetabolic network of a haploidmale strain ofE. subulatus, establishing it as a37
comparative model to study the genomic bases of stress tolerance in Ectocarpus. Our analyses38
indicatethatE.subulatushasseparatedfromEctocarpussp.Ec32viaallopatricspeciation.Sincethis39
event,itsgenomehasbeenshapedbytheactivityofvirusesandlargeretrotransposons,whichin40
thecaseofchlorophyll-bindingproteins,mayberelatedtotheexpansionofthisgenefamily.We41
have identifiedanumberof furthergenes thatwesuspect tocontribute tostress tolerance inE.42
subulatus,includinganexpandedfamilyofheatshockproteins,thereductionofgenesinvolvedin43
the production of halogenated defense compounds, and the presence of fewer cell wall44
polysaccharide-modifyingenzymes.However,96%ofgenesthatdifferedbetweenthetwoexamined45
Ectocarpus species, aswell as 92% of genes under positive selection,were found to be lineage-46
specificandencodeproteinsofunknownfunction.Thisunderlinestheuniquenessofbrownalgae47
with respect to their stress tolerance mechanisms as well as the significance of establishing E.48
subulatusasacomparativemodelforfuturefunctionalstudies. 49
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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Introduction50
Brown algae (Phaeophyceae) are multicellular photosynthetic organisms that are successful51
colonizersofrockyshoresoftheworld’soceans,inparticularintemperateandpolarregions.Inmany52
placestheyconstitutethedominantvegetationintheintertidalzone,wheretheyhaveadaptedto53
multiple stressors includingstrongvariations in temperature, salinity, irradiation,andmechanical54
stress(waveaction)overthetidalcycle(DavisonandPearson,1996).Inthesubtidalenvironment,55
brownalgaeformlargekelpforeststhatharborhighlydiversecommunities.Theyarealsoharvested56
asfoodorforindustrialpurposes,suchastheextractionofalginates(McHugh,2003).Theworldwide57
annualharvestofbrownalgaehasreached10milliontonsby2014andisconstantlygrowing(FAO,58
2016).Brownalgaesharesomebasicphotosyntheticmachinerywithlandplants,buttheirplastids59
derivedfromasecondaryortertiaryendosymbiosiseventwitharedalga,andtheybelongtoan60
independent lineage of Eukaryotes, the Stramenopiles (Archibald, 2009). This phylogenetic61
background, together with their distinct habitat, contributes to the fact that brown algae have62
evolvednumerousuniquemetabolicpathways,lifecyclefeatures,andstresstolerancemechanisms.63
Toenablefunctionalstudiesofbrownalgae,strainEc32ofthesmallfilamentousalgaEctocarpussp.64
hasbeenestablishedasageneticandgenomicmodelorganism(Petersetal.,2004;Cocketal.,2010;65
Heeschetal.,2010).ThisstrainwasformerlydescribedasEctocarpussiliculosus,buthassincebeen66
showntobelongtoanindependentcladebymolecularmethods(Stache-Crainetal.,1997;Peterset67
al.,2015).Morerecentlytwoadditionalbrownalgalgenomes,thatofthekelpspeciesSaccharina68
japonica (Yeetal.,2015)andthatofCladosiphonokamuranus(Nishitsujietal.,2016),havebeen69
characterized.Comparisonsbetweenthesethreegenomeshaveallowedresearcherstoobtainafirst70
overviewoftheuniquegenomicfeaturesofbrownalgae,aswellasaglimpseofthegeneticdiversity71
withinthisgroup.However,giventheevolutionarydistancebetweenthesealgae,itisdifficulttolink72
genomicdifferences tophysiologicaldifferencesandpossibleadaptations to their lifestyle.Tobe73
abletogeneratemoreaccuratehypothesesontheroleofparticulargenesandgenomicfeaturesfor74
adaptivetraits,acommonstrategyistocomparecloselyrelatedstrainsandspeciesthatdifferonly75
ina fewgenomic features. ThegenusEctocarpus isparticularlywell suited for such comparative76
studiesbecauseitcomprisesawiderangeofmorphologicallysimilarbutgeneticallydistinctstrains77
andspeciesthathaveadaptedtodifferentmarineandbrackishwaterenvironments(Stache-Crain78
etal.,1997;Montecinosetal.,2017).Onespecieswithinthisgroup,EctocarpussubulatusKützing79
(Petersetal.,2015)hasseparatedfromEctocarpussp.Ec32approximately16millionyearsago(Mya;80
Dittamietal.,2012). Itcomprises isolateshighlyresistanttoelevatedtemperature(Bolton,1983)81
andlowsalinity.Astrainofthisspecieswasevenisolatedfromfreshwater(WestandKraft,1996),82
constitutingoneofthehandfulofknownmarine-freshwatertransitionsinbrownalgae(Dittamiet83
al.,2017).84
HerewepresentthedraftgenomeandmetabolicnetworkofastrainofE.subulatus,establishing85
thegenomicbasisforitsuseasacomparativemodeltostudystresstolerancemechanisms,andin86
particular of low salinity tolerance, in brown algae. Similar strategies have previously been87
successfullyemployedinterrestrialplants,where“extremophile”relativesofmodel-oreconomically88
relevant specieshavebeen sequenced toexplorenewstress tolerancemechanisms in thegreen89
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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lineage(Ohetal.,2012;DittamiandTonon,2012;Dassanayakeetal.,2011;Amtmann,2009;Maet90
al.,2013;Zengetal.,2015).Thestudyof theE.subulatusgenome,andsubsequentcomparative91
analysiswithotherbrownalgalgenomes,inparticularthatofEctocarpussp.Ec32,providesinsights92
into the dynamics of Ectocarpus genome evolution and divergence, and highlights important93
adaptive processes, such as a potentially retrotransposon driven expansion of the family of94
chlorophyll-binding proteins with subsequent diversification. Most importantly, our analyses95
underline that most of the observed differences between the examined species of Ectocarpus96
correspondtolineage-specificproteinswithyetunknownfunctions.97
Results98
SequencingandassemblyoftheE.subulatusgenome99
Atotalof34.7Gbofpaired-endreaddataandof28.8Gbofmatepairreads(correspondingto45100
millionnon-redundantmate-pairs)wereobtainedandusedtogenerateaninitialassemblywitha101
total length of 350 Mb, an N50 length of 159 kb, and 8% undefined bases (Ns). However, as102
sequencingwascarriedoutonDNAfromalgalmaterialthathadnotbeentreatedwithantibiotics,a103
substantialpartoftheassembledscaffoldswasofbacterialorigin.Removalofthesesequencesfrom104
thefinalassemblyresultedinthefinal227MbgenomeassemblywithanaverageGCcontentof54%105
(Table 1). After all cleaning and filtering steps, and considering only algal scaffolds, the average106
sequencingcoveragewas67Xforthepairendlibraryandthegenomiccoverage(numberofunique107
algalmatepairs*spansize/assemblysize)was6.9,14.4,and30.4Xforthe3kb,5kb,and10kb108
mate pair libraries, respectively. The bacterial sequences corresponded predominantly to109
Alphaproteobacteria (50%, with the dominant genera Roseobacter 8% and Hyphomonas 5%)110
followedbyGammaproteobacteria(18%)andFlavobacteria(13%).RNA-seqexperimentsyieldeda111
total of 4.2 Gb of sequence data for a culture of E. subulatus Bft15b cultivated in seawater.112
Furthermore,4.5Gband4.3GbwereobtainedfortwolibrariesofafreshwaterstrainofE.subulatus113
fromHopkinsRiverFallsaftergrowth in seawaterand indilutedmedium, respectively.Of these,114
96.6% (Bft15b strain in seawater), 87.6% (freshwater strain in seawater), and 85.3% (freshwater115
strainindilutedmedium)weresuccessfullymappedagainstthefinalgenomeassemblyoftheBft15b116
strain.117
Genepredictionandannotation118
GenepredictionwascarriedoutfollowingtheprotocolemployedforEctocarpussp.Ec32(Cocket119
al.,2010)usingEugene.Thenumberofpredictedproteinswas60%higherthanthatpredictedfor120
Ec32(Table1),butthisdifferencecanbeexplainedtoalargepartbythefactthatmono-exonicgenes121
(manyofwhichcorrespondingtotransposases)werenotremovedfromourpredictions,butwere122
manually removed fromtheEc32genome.This isalsocoherentwith the lowermeannumberof123
introns per gene observed in the Bft15b strain. For 10,395 (40 %) of these predicted proteins124
automaticannotationsweregeneratedbasedonBlastPsearchesagainsttheSwiss-Protdatabase;125
furthermore724proteinsweremanuallyannotated.Thecomplete setofpredictedproteinswas126
used to evaluate the completeness of the genome based on the presence of conserved core127
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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eukaryotegenesusingBUSCO(Simãoetal.,2015).ThisrevealedtheE.subulatusgenometobe86%128
completeusingthefullsetofconservedeukaryoticgenes,and91%whennotconsideringproteins129
alsoabsentfromallsequencedknownbrownalgae.130
Repeatedelements131
Using theREPETpipeline,wedetermined that, similar to results obtained for strain Ec32, theE.132
subulatus genome consistedof 30% repeatedelements, i.e. 10% less thanS. japonica. Themost133
abundantgroupsofrepeatedelementswerelargeretrotransposonderivatives(LARDs),followedby134
long terminal repeats (LTRs, predominantly Copia and Gypsy), and long and short interspersed135
nuclearelements (LINEs).Theoveralldistributionofsequence identity levelswithinsuperfamilies136
showedtwopeaks,oneatanidentitylevelof78-80%,andoneat96-100%(Figure1),indicatingtwo137
periodsofhightransposonactivityinthepast.Terminalrepeatretrotransposonsinminiature(TRIM)138
and LARDs, both non-autonomous groups of retrotransposons,were among themost conserved139
families (Figure 1B). In line with previous observations carried out in Ectocarpus sp. Ec32, no140
methylationwasdetectedintheE.subulatusgenomicDNA,anindicationthatmethylationwasmost141
likelynotamechanismtosilencetransposonsinthisspecies.142
Organellargenomes143
PlastidandmitochondrialgenomesfromE.subulatushave95.5%and91.5%sequenceidentitywith144
their Ectocarpus sp. Ec32 counterparts, respectively, in the conserved regions (Figure 2). The145
mitochondrial genome ofE. subulatus differed from that ofEctocarpus sp. Ec32 essentiallywith146
respecttothepresenceofthreeadditionalmaturasegenes,aswellasoneandtwointronswithin147
the16Sand23S rRNAgenes, respectively.A largestructuraldifferencewasobservedonly in the148
plastidgenomewhereone inversionofca. 50 kb in the small single copy (SSC) regionmayhave149
occurred.Furthermore,smalldifferencesingenecontentsoftheE.subulatusplastidwithrespectto150
Ectocarpussp.Ec32weredetectedaroundtwoinvertedrepeat(IR)regionsconcerningthefollowing151
genes:psbC(genetruncated),psbD(IRregionnexttogene),rpoB(largegap,frameshift),andtRNA-152
ArgandtRNA-Glu(duplicatedinthetRNAregion).PseudogenizationofgenesattheedgeofIRsis153
indeedacommonphenomenon(Leeetal.,2016).154
Globalcomparisonofpredictedproteomes155
GO-basedcomparisons156
OrthoFinderwasusedtodefineclustersofpredictedorthologsaswellasspecies-specificproteins.157
AsshowninFigure3,11,177predictedBft15bproteinshadnoorthologinEc32,whilethereverse158
was true for only 3,605 proteins of strain Ec32. Furthermore, among the clusters of genes, we159
observeddifferencesincopynumberforseveraloftheproteinsbetweenthetwospecies.Usinggene160
setenrichmentanalyses,weattempted toautomatically identify functional groupsof genes that161
wereover-representedeitheramongtheproteinsspecifictooneortheothergenome,orthatwere162
expandedinoneofthetwogenomes.Theresultsoftheseanalysespointtowardsseveralfunctional163
groupsofproteinsthatweresubjecttorecentvariationsbetweenE.subulatusandEctocarpussp.164
Ec32(Figure3).Categoriesidentifiedasover-representedamongthegenesuniquetoE.subulatus165
includeDNAintegration,chlorophyllbinding,andDNAbinding,butalsofalsepositivessuchasred166
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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light signaling, which arise from the presence of transposable elements in the genome (see167
Supporting InformationFileS1).However,nosignificantlyenrichedGOtermswere foundamong168
protein familiesexpanded in theE. subulatus genome. In contrast, several categorieswereover-169
representedamongthegenesandgenefamiliesspecifictoorexpandedintheEctocarpussp.Ec32170
strain,manyofwhichwererelatedeithertosignalingpathwaysortothemembraneandtransporters171
(Figure3), althoughdifferenceswith respect tomembraneand transporterswerenot confirmed172
aftermanualcuration.173
Domain-basedcomparisons174
Domain-basedcomparisonswerecarriedouttoavoidapossibleimpactofmoderateorpoor-quality175
annotationsonthegenomiccomparisons.Intotal,5,728differentInterProdomainsweredetected176
in both Ectocarpus genomes, with 133,448 and 133,052 instances in E. subulatus Bft15b and177
Ectocarpussp.Ec32strainsrespectively.ThemostcommondomainsinE.subulatuswereZincfinger,178
CCHC-type(IPR001878,3,861instances),andRibonucleaseH-like(IPR012337,3,742instances).Both179
werepresentlessthan200timesinEc32.ThemostcommondomainsinEctocarpussp.Ec32were180
theankyrinrepeatandankyrinrepeat-containingdomains(IPR002110,IPR020683:4,138and4,062181
occurrencesvsca.3,000inBft15b).Twohundredandninety-sixdomainswerespecifictoBft15b,182
while582werespecifictoEc32(seeSupportingInformationTableS2).183
Metabolicnetwork-basedcomparisons184
Intotal,theE.subulatusmetabolicnetworkreconstructioncomprised2,445genesassociatedwith185
2,074metabolic reactions and 2,173metabolites in 464 pathways, 259 ofwhichwere complete186
(Figure3).TheseresultsaresimilartodatapreviouslyobtainedforEctocarpussp.Ec32(Prigentet187
al., 2014; see http://gem-aureme.irisa.fr/ectogem for the most recent version; 1,977 reactions,188
2,132metabolites,2,281genes,459pathways,272completepathways).Comparisonsbetweenboth189
networks were carried out on a pathway level (Supporting Information Table S3), focusing on190
pathwayspresent(i.e.completetomorethan50%)inoneofthespecies,butwithnoreactionsin191
theother.This ledtotheidentificationof16pathwayspotentiallyspecifictoE.subulatusBft15b,192
and 11 specific to Ectocarpus sp. Ec32, which were further manually investigated. In all of the193
examined cases, the observed differences were due to protein annotation, but not due to the194
presence/absence of proteins associatedwith these pathways in both species. For instance, the195
pathways “spermine and spermidinedegradation III" (PWY-6441)was only found inE. subulatus196
becausethecorrespondinggeneshadbeenmanuallyannotatedinthisspecies,whilethiswasnot197
thecaseinEctocarpussp.Ec32.Ontheotherhand,threepathwaysrelatedtomethanogenesis(PWY-198
5247,PWY-5248,andPWY-5250)werefalselyincludedinthemetabolicnetworkofE.subulatusdue199
toanoverlypreciseautomaticGOannotationofthegeneBft140_7.Allinall,basedonournetwork200
comparisons,weconfirmednodifferencesregardingthepresenceorabsenceofknownmetabolic201
pathwaysinthetwoexaminedspeciesofEctocarpus.202
Genesunderpositiveselection203
In total, 7,147 pairs of orthologs were considered to search for genes under positive selection204
between the two examined strains of Ectocarpus, and we identified 83 gene pairs (1.2%) that205
exhibiteddN/dSratios>1(SupportingInformationTableS4).Thisproportionwaslowcomparedto206
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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the12%ofgenesunderpositiveselectionfoundinastudycomprisingalsokelpanddiatomspecies207
(Tengetal., 2017).Notehowever, thatouranalysis focusedon theglobaldN/dS ratiopergene,208
ratherthanthelocaldN/dSratiopercodonsite(implementedincodeml,PAML)usedbyTengetal.209
(2017).Thegenepairsunderpositiveselectionmayberelatedtotheadaptationtothedifferent210
environmental niches occupied by the strains investigated. These gene pairs were examined211
manually,butonlyoneofthem(Ec-11_002330,EsuBft305_15)couldbeassignedafunction,i.e.a212
putativemannosyl-oligosaccharide1,2-alpha-mannosidaseactivity,possiblyinvolvedinglycoprotein213
modification.Twelveadditionalpairscontainedknownproteindomains(twoZincfingerdomains,214
one TIP49 domain, oneDnaJ domain, oneNADH-ubiquinone oxidoreductase domain, one SWAP215
domain, and six ankyrin repeat domains). Ankyrin repeat domains were significantly over-216
represented among the genes under positive selection (p < 0.05, Fisher exact test), and the217
correspondinggenesweremanuallyexaminedbybestreciprocalblastsearchtoensurethatthey218
correspondedtotrueorthologs.Onlyonepairwaspartofaproteinfamilythathadundergonerecent219
expansion(Ec-27_003170,EsuBft1157_2),andinthiscasephylogeneticanalysisincludingtheother220
members of the family (EsuBft255_4, EsuBft2264_2, Ec-05_004510, Ec-08_002010) showed Ec-221
27_003170andEsuBft1157_2toformabranchwith100%bootstrapsupport(datanotshown).The222
remaining70pairsofproteinshadentirelyunknownfunctions,althoughfourgeneswerelocatedin223
thepseudoautosomalregionofthesexchromosomeofEctocarpussp.Ec32.Outofthe83genes224
underpositiveselection72werefoundonlyinbrownalgaeandanotherfouronlyinstramenopiles225
(e-value cutoff of 1e-10 against the nr database). They can thus be considered as taxonomically226
restricted genes. Furthermore, 75 of these genes were expressed in at least one of the two227
Ectocarpusspecies,andonly10ofthe83genesencodedshortproteinswithlessthan100amino228
acidresidues,suggestingthatthemajorityofthesegenesmaybefunctional.Noneofthemwere229
highlyvariable,asindicatedbythefactthatthedN/dSratioexhibitedaweaknegativecorrelation230
withtherateofsynonymousmutationsdS(PearsonCorrelationcoefficientr=-0.05,p<0.001;Figure231
4).ThissuggeststhatthesplitofEctocarpussp.Ec32andE.subulatuswastheresultofallopatric232
separationwithsubsequentspeciationduetogradualadaptationtothelocalenvironment.Indeed,233
in cases of sympatric or parapatric speciation, genes under positive selection are predominant234
among rapidly evolving genes (Swanson and Vacquier, 2002). There was no trend for positively235
selected genes tobe located in specific regionsof the genome (dispersion indexof genesunder236
positiveselectionclosetoarandomdistributionwithvaluesrangingbetween0.7and0.8depending237
onthewindowsize).238
Manualexaminationoflineage-specificandofexpandedgenesandgenefamilies239
ThefocusofourworkisonthegenesspecifictoandexpandedinE.subulatusandweonlygivea240
briefoverviewofthesituationregardingEctocarpussp.Ec32.ItisimportanttoconsiderthattheE.241
subulatusBft15bgenomeislikelytobelesscompletethantheEc32genome,whichhasbeencurated242
andimprovedforover10yearsnow(Cormieretal.,2017).Hence,regardinggenesthatarepresent243
in Ec32 but absent in Bft15b, it is difficult to distinguish between the effects of a potentially244
incompletegenomeassemblyandtruegenelossesinBft15b.Tofurtherreducethisbiasduringthe245
manual examination of lineage-specific genes, the list of genes to be examinedwas reduced by246
additionalrestrictions.First,onlygenesthatdidnothaveorthologsinS.japonicawereconsidered.247
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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This eliminated several predictedproteins thatmayhave appeared to be lineage-specific due to248
incomplete genome sequencing, but also proteins that have been recently lost in one of the249
Ectocarpus species. Secondly, the effect of possible differences in gene prediction, notably the250
manualremovalofmonoexonicgenemodelsinEctocarpussp.Ec32,wasminimizedbyincludingan251
additional validation step: onlyproteinswithout correspondingnucleotide sequences (tblastn, e-252
value<1e-10) intheotherEctocarpusgenomewereconsideredformanualexamination.Thirdly,253
onlyproteinswitha lengthofat least50aawere retained.This reduced thenumberof lineage-254
specificproteinstobeconsideredinstrainBft15bto1,629,andinstrainEc32to689(Supporting255
InformationTableS5).256
InE.subulatus,amongthe1,629lineage-specificgenes,1,436geneshadnohomologs(e-value<1e-257
5)intheUniProtdatabase:theyarethustrulylineage-specificandhaveunknownfunctions.Among258
the remaining 193 genes, 145 had hits (e-value < 1e-5) in Ectocarpus sp. Ec32. The majority259
corresponds tomulti-copy genes that had diverged prior to the separation of Ectocarpus and S.260
japonica,andforwhichtheEctocarpussp.Ec32andS.japonicaorthologswereprobablylost.The261
remaining48 genesweremanually examined (genetic context,GC content, EST coverage); 18of262
them corresponded to probable bacterial contaminations and the corresponding scaffolds were263
removed.Finally,theremaining30genesweremanuallyannotatedandclassified:13hadhomology264
onlywithuncharacterizedproteinsorwere toodissimilar fromcharacterizedproteins todeduce265
hypotheticalfunctions;anothereightprobablycorrespondedtoshortviralsequencesintegratedinto266
the algal genome (EsuBft1730_2, EsuBft4066_3, EsuBft4066_2, EsuBft284_15, EsuBft43_11,267
EsuBft551_12, EsuBft1883_2, EsuBft4066_4), and one (EsuBft543_9) was related to a268
retrotransposon.Twoadjacentgenes(EsuBft1157_4,EsuBft1157_5)werealsofoundindiatomsand269
may be related to the degradation of cellobiose and the transport of the corresponding sugars.270
Furthermore,twogenes,EsuBft1440_3andEsuBft1337_8,containedconservedmotifs(IPR023307271
andSSF56973) typically found in toxin families. Finally, twoadditionalproteins,EsuBft36_20and272
EsuBft440_20, consisted almost exclusively of short repeated sequences of unknown function273
(“ALEW”and“GAAASGVAGGAVVVNG”,respectively). 274
InEctocarpussp.Ec32,97proteinscorrespondedtotheE.siliculosusvirus-1insertedintotheEc32275
genome–no similar insertionwasdetected inE. subulatus. The largemajorityof proteins (511)276
correspondedtoproteinsofunknownfunctionwithoutmatchesinpublicdatabases.Theremaining277
81proteinsweregenerallypoorlyannotated,usuallyonlyviathepresenceofadomain.Examples278
are ankyrin repeat-containing domain proteins (12), Zinc finger domain proteins (6), proteins279
containingwallsensingcomponent(WSC)domains(3),proteinkinase-likeproteins(3),andNotch280
domainproteins(2)(seeSupportingInformationTableS5).281
Regarding expanded gene families, OrthoFinder indicated 232 clusters of orthologous genes282
(corresponding to4,064proteins)expanded in thegenomeofE. subulatus,and450expanded in283
Ectocarpus sp. Ec32 (corresponding to 1,685proteins; Supporting Information Table S5).Manual284
examinationoftheE.subulatusexpandedgeneclustersrevealed48ofthem(2,623proteins)tobe285
falsepositives,whichcanbeexplainedessentiallybysplitgenemodelsorgenemodelsassociated286
withtransposableelementspredictedintheE.subulatusbutnotintheEctocarpussp.Ec32genome.287
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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Theremaining184clusters(1,441proteins)correspondedtoproteinswithunknownfunction(139288
clusters,1,064proteins),98%ofwhichwerefoundonlyinbothEctocarpusgenomes.Furthermore,289
nine clusters (202 proteins) represented sequences related to transposons predicted in both290
genomes,andeightclusters(31proteins)weresimilartoknownviralsequences.Only28clusters291
(135proteins)couldberoughlyassignedtobiologicalfunctions(Table2).Theycomprisedproteins292
potentiallyinvolvedinmodificationofthecell-wallstructure(includingsulfation),intranscriptional293
regulationandtranslation,incell-cellcommunicationandsignaling,aswellasafewstressresponse294
proteins, notably a set of HSP20s, and several proteins of the light-harvesting complex (LHC)295
potentiallyinvolvedinnon-photochemicalquenching.296
AmongthemoststrikingexamplesofexpansioninEctocarpussp.Ec32,wefounddifferentfamilies297
ofserine-threonineproteinkinasedomainproteinspresentin16to25copiesinEc32comparedto298
only5or6(numbersofdifferentfamilies)inE.subulatus,Kinesinlightchain-likeproteins(34vs.13299
copies),twoclustersofNotchregioncontainingproteins(11and8vs.2and1copies),afamilyof300
unknownWSCdomaincontainingproteins(8copiesvs.1),putativeregulatorsofG-proteinsignaling301
(11vs.4copies),aswellasseveralexpandedclustersofunknownandofviralproteins.302
Targetedmanualannotationofspecificpathways303
Basedontheresultsofautomaticanalysisbutalsoonliteraturestudiesofgenesthatmaybeableto304
explainphysiologicaldifferencesbetweenE.subulatusandEctocarpussp.Ec32,severalgenefamilies305
andpathwaysweremanuallyexaminedandannotated.306
Cellwallmetabolism307
Cellwallsarekeycomponentsofbothplantsandalgaeand,asa firstbarrier to thesurrounding308
environment, important for many processes including development and the acclimation to309
environmental changes. Synthesis and degradation of cell wall oligo- and polysaccharides is310
facilitatedbycarbohydrate-activeenzymes(CAZymes)(http://www.cazy.org/;Cantareletal.2009).311
Thesecompriseseveralfamiliesincludingglycosidehydrolases(GHs)andpolysaccharidelyases(PLs),312
both involved in the cleavage of glycosidic linkages, glycosyltransferases (GTs), which create313
glycosidiclinkages,andadditionalenzymessuchasthecarbohydrateesterases(CEs)whichremove314
methyloracetylgroupsfromsubstitutedpolysaccharides.315
ThegenomeofthebrownalgaE.subulatusencodes37GHs(belongingto17GHfamilies),94GTs316
(belongingto28GTfamilies),ninesulfatases(familyS1-2),and13sulfotransferases,butlacksgenes317
homologoustoknownPLsandCEs (Figure5). Inparticular, theconsistent lackofknownalginate318
lyasesandcellulasesintheE.subulatusandtheotherbrownalgalgenomessuggeststhatother,yet319
unknowngenes,maybe responsible for cellwallmodificationsduringdevelopment.Overall, the320
genecontentofE.subulatusissimilartoEctocarpussp.Ec32andS.japonicaintermsofthenumber321
ofCAZYfamilies,butslightly lower intermsofabsolutegenenumber(Cock,etal.2010;Yeetal.322
2015;Figure5).EspeciallyS.japonicafeaturesanexpansionofcertainCAZYfamiliesprobablyrelated323
totheestablishmentofmorecomplextissuesinthiskelp(i.e.82GHsbelongingto17GHfamilies,324
131GTsbelongingto31GTfamilies).325
E. subulatus is frequently found inbrackish-andeven freshwaterenvironments (WestandKraft,326
1996)whereitscellwallexhibitslittleornosulfation(Torodeetal.,2015).Hence,wealsoassessed327
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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whetherE.subulatushadreducedthegenefamiliesresponsibleforthisprocess.Itsgenomeencodes328
onlyeightsulfatasesandsixsulfotransferasescomparedtotenandseven,respectively,inEctocarpus329
sp.Ec32.WealsodocumentedvariationsintheGTfamilies,somebeingpresentinoneortwoofthe330
brown algal genomes considered,while absent in other(s) (e.g. GH30, GT15, GT18, GT24, GT25,331
GT28,GT50,GT54,GT65,GT66,GT74,GT77).However,asgenenumbersforthesefamiliesarevery332
low(e.g.theGT24familyhasonememberinEctocarpussp.Ec32,twoinE.subulatus,andnoneinS.333
japonica),theresultsmustbetakenwithcaution.Finally,Ectocarpussp.Ec32haspreviouslybeen334
reportedtopossessnumerousproteinswithWSCdomains(Cocketal.,2010;Micheletal.,2010).335
These were initially found in yeasts (Verna et al., 1997) where they act as cell surface336
mechanosensors andactivate the intracellular cellwall integrity signaling cascade in response to337
hypo-osmoticshock(Gualtierietal.,2004).Inbrownalgae,theseWSCdomainsmayalsoregulate338
wall rigidity, through the control of the activity of appended enzymes, such asmannuronan C5-339
epimerases, which act on alginates (Hervé et al., 2016). Surprisingly, the total number of WSC340
domains is reduced in E. subulatus compared to Ectocarpus sp. Ec32 with around 320 vs. 444341
domains, respectively, based on InterProScan (Supporting Information Table S2). Additional342
informationregardingE.subulatusCAZYmescanbefoundinSupportingInformationFileS1.343
Centralandstoragecarbohydratemetabolism344
Acharacteristicfeatureofbrownalgaeisthattheystorecarbohydratesnotasglycogenorstarch,345
like most animals and plants, but as laminarin (Read et al., 1996). Brown algae also have the346
particularityofusingthephotoassimilateD-fructose6-phosphatetoproducethealcoholsugarD-347
mannitolinsteadofsucroselikelandplants.TheE.subulatusgenomecontainssimilarsetsofgenes348
forcarbonstoragecomparedtoEctocarpussp.Ec32:all thegenesencodingenzymes involved in349
sucrosemetabolism and starch biosynthesis are completely absentwhile all genes necessary for350
trehalosesynthesis,aswellas laminarinsynthesisandrecyclingwerefound.Also,threecopiesof351
M1PDHgeneswerefoundinbothEctocarpusspeciescomparedtotwoinS.japonica,probablydue352
toa recentduplicationofM1PDH1/M1PDH2 in theEctocarpales (Tononetal.,2017) (Supporting353
InformationFileS1).354
Sterolmetabolism355
Sterols are important modulators of membrane fluidity among eukaryotes, and provide the356
backbone for signaling molecules (Desmond and Gribaldo, 2009). Fucosterol, cholesterol, and357
ergosterolare themostabundant sterols inEctocarpussp.Ec32,where their relativeabundance358
variesaccordingtosexandtemperature(Mikamietal.,2018).Allthreemoleculesarethoughttobe359
synthesizedfromsqualenebyasuccessionof12to14steps,relyingonaroughlyconservedsetof360
twelveenzymes(DesmondandGribaldo,2009).TheE.subulatusandEctocarpussp.Ec32genomes361
eachencodehomologsof twelveof them(SQE,CAS,CYP51,FK,SMO,HSD3B,EBP,CPI1,DHCR7,362
SC5DL,and twoSMTs).The remaining two,adelta-24-reductase (DHCR24)andaC22desaturase363
(CYP710),wereprobablylostsecondarily.Inlandplants,theselatterenzymesareinvolvedinthetwo364
stepstransformingfucosterol intostigmasterol.Fucosterol is themainsterol inbrownalgae,and365
providesasubstrateforsaringosterol,abrown-algaspecificC24-hydroxylatedfucosterol-derivative366
withantibacterialactivity(Wächteretal.,2001).367
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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Algaldefense:metabolismofphenolicsandhalogens368
Polyphenolsareagroupofdefensecompoundsinbrownalgaethatarelikelytobeimportantboth369
forabiotic(Paviaetal.,1997)andbioticstresstolerance(GeiselmanandMcConnell,1981).Brown370
algaeproducespecificpolyphenolscalledphlorotannins,whichareanalogoustolandplanttannins.371
These products are polymers of phloroglucinol, which are synthesized via the activity of a372
phloroglucinolsynthase,atypeIIIpolyketidesynthasecharacterizedinEctocarpussp.Ec32(Meslet-373
Cladièreetal.,2013).Inanalogytotheflavonoidpathwayoflandplants,thefurthermetabolismof374
phlorotannins is thought to be driven by members of chalcone isomerase-like (CHIL), aryl375
sulfotransferase (AST), flavonoid glucosyltransferase (FGT), flavonoidO-methyltransferase (OMT),376
polyphenol oxidase (POX), and tyrosinase (TYR) families (Cocket al., 2010).While copynumbers377
betweenthetwoEctocarpusspeciesandS. japonicaare identical forPKS III,CHIL,FGT,OMTand378
POX,E.subulatusencodesfewerASTsandTYRs(Figure5).InthecaseofASTs,thismayberelatedto379
thelowerconcentrationofsulfateinlowsalinityenvironmentsfrequentlycolonizedbyE.subulatus.380
Asecondimportantandoriginaldefensemechanisminbrownalgaeistheproductionofhalogenated381
compoundsviatheactivityofhalogenatingenzymes,e.g.thevanadium-dependenthaloperoxidase382
(vHPO).While S. japonica has recently been reported to possess 17 potential bromoperoxidases383
(vBPO)and59putativeiodoperoxidases(vIPO)(Yeetal.,2015),Ectocarpussp.Ec32andE.subulatus384
possessonlyasinglevBPOeachandnovIPO,buthaveinturnslightlyexpandedahaloperoxidase385
familyclosertovHPOcharacterizedinseveralmarinebacteria(Fournieretal.,2014)(Figure5).One386
differencebetweenthetwoEctocarpusspeciesisthatE.subulatusBft15bpossessesonlythreevHPO387
genescomparedtothefivecopiesfoundinthegenomeofEc32.Inaddition,homologsofthyroid388
peroxidases (TPOs)may alsobe involved in halide transfer and stress response.Again, Ec32 and389
Bft15bshowareducedsetofthesegenescomparedtoS.japonica,andEc32containsmorecopies390
thanBft15b.Finally,asinglehaloalkanedehalogenase(HLD)wasfoundexclusivelyinEctocarpussp.391
Ec32.392
Transporters393
Transporters are key actors driving salinity tolerance in terrestrial plants (Volkov, 2015). We394
therefore carefully assessed potential differences in this group of proteins that may explain395
physiologicaldifferencesbetweenEc32andBft15bbasedonthefivemaincategoriesoftransporters396
described in the Transporter Classification Database (TCDB) (Saier et al., 2016): channels/pores,397
electrochemicalpotential-driventransporters,primaryactivetransporters,grouptranslocators,and398
transmembraneelectroncarriers.Atotalof292geneswereidentifiedinE.subulatus(Supporting399
InformationTableS1).Theyconsistmainlyof transportersbelonging to the three first categories400
listedabove.All27annotatedtransportersofthechannels/porescategorybelongtothealpha-type401
channel (1.A.) and are likely to be involved in movements of solutes by energy-independent402
processes.Onehundredandforty-fiveproteinswerefoundtocorrespondtothesecondcategory403
(electrochemical potential-driven transporters) containing transporters using a carrier-mediated404
process to catalyze uniport, antiport, or symport. The most represented superfamilies are APC405
(Amino Acid-Polyamine-Organocation, 24), DMT (Drug/Metabolite Transporter, 16), MFS (Major406
FacilitatorSuperfamily,32),andMC(MitochondrialCarrier,34).Primaryactivetransporters(third407
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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category) use a primary source of energy to drive the active transport of a solute against a408
concentrationgradient.Eightyproteinsrepresentingthiscategorywerefound intheE.subulatus409
genome, including 59 ABC transporters and 15 belonging to the P-type ATPase superfamily. No410
homologs of group translocators or transmembrane electron carriers were identified, but 14411
transporterswereclassifiedascategory9,whichispoorlycharacterized.A1:1ratiooforthologous412
genescoding forallof the transportersdescribedabovewasobservedbetweenbothEctocarpus413
genomes,exceptforEsuBft583_3,ananion-transportingATPase,whichisalsopresentindiatoms414
andS.japonica,butmayhavebeenrecentlylostinEctocarpussp.Ec32.415
Abioticstress-relatedgenes416
Reactive oxygen species (ROS) scavenging enzymes, including ascorbateperoxidases, superoxide417
dismutases, catalases, catalase peroxidases, glutathione reductases, (mono)dehydroascorbate418
reductases,andglutathioneperoxidasesareimportantfortheredoxequilibriumoforganisms(see419
DasandRoychoudhury2014 fora review).An increasedreactiveoxygenscavengingcapacityhas420
beencorrelatedwithstresstoleranceinbrownalgae(CollénandDavison,1999).Inthesamevein,421
chaperoneproteinsincludingheatshockproteins(HSPs),calnexin,calreticulin,T-complexproteins,422
andtubulin-foldingco-factorsareimportantforproteinre-foldingunderstress.Thetranscriptionof423
thesegenesisverydynamicandgenerallyincreasesinresponsetostressinbrownalgae(Roederet424
al.,2005;Motaetal.,2015). In total,104genesencodingmembersof theprotein families listed425
aboveweremanuallyannotatedintheE.subulatusBft15bgenome(SupportingInformationTable426
1).However,withtheexceptionofHSP20proteinswhichwerepresentinthreecopiesinBft15bvs.427
onecopyinEc32andhadalreadybeenidentifiedintheautomaticanalysis,nocleardifferencein428
genenumberwasobservedbetweenthetwoEctocarpusspecies.429
Differentfamiliesofchlorophyll-bindingproteins(CBPs),suchastheLI818/LHCXfamily,havebeen430
suspected to be involved in non-photochemical quenching (Peers et al., 2009). CBPs have been431
reportedtobeup-regulatedinresponsetoabioticstressinstramenopiles(e.g.ZhuandGreen2010;432
Dongetal.2016),includingEctocarpus(Dittamietal.,2009),probablyasawaytodealwithexcess433
lightenergywhenphotosynthesisisaffected.Theyhavealsopreviouslybeenshowntobeamong434
themost variable functional groups of genes between Ectocarpus sp. Ec32 and E. subulatus by435
comparativegenomehybridizationexperiments(Dittamietal.,2011).Wehaveaddedtheputative436
E. subulatusCBPs toapreviousphylogenyofEctocarpussp.Ec32CBPs (Dittamietal.,2010)and437
foundbothasmallgroupofLHCXCBPsaswellasalargergroupbelongingtotheLHCF/LHCRfamily438
that have probably undergone a recent expansion (Figure 6). Although some of the proteins439
appearedtobetruncated(markedwithasterisks),allofthemwereassociatedwithatleastsome440
RNA-seqreads,suggestingthattheymaybefunctional.AnumberofLHCRfamilyproteinswerealso441
flankedbyLTR-likesequencesaspredictedbytheLTR-harvestpipeline(Ellinghausetal.,2008).442
Discussion443
HerewepresentthedraftgenomeandmetabolicnetworkofE.subulatusstrainBft15b,abrownalga444
which,comparedtoEctocarpussp.Ec32, ischaracterizedbyhighabioticstresstolerance(Bolton,445
1983;Petersetal.,2015).Basedontime-calibratedmoleculartrees,bothspeciesseparatedroughly446
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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16Mya (Dittamiet al., 2012), i.e. slightly beforee.g. the split betweenArabidopsis thaliana and447
Thellungiella salsuginea 7-12Mya (Wuetal., 2012).According toouranalysis, the splitbetween448
Ectocarpus sp. Ec32 andE. subulatuswasprobablydue to allopatric separationwith subsequent449
adaptationofE.subulatustohighlyfluctuatingandlowsalinityhabitatsleadingtospeciation.450
GenomeevolutionofEctocarpusspeciesdrivenbytransposonsandviruses451
ComparedtotheextremophileplantmodelsT.salsugineaorArabidopsislyratawhichhavealmost452
doubledingenomesizewithrespecttoA.thaliana,theE.subulatusgenomeisonlyapproximately453
23%largerthanthatofEctocarpussp.Ec32.InT.salsugineaandA.lyrata,theobservedexpansion454
wasattributedmainlytotheactivityoftransposons(Wuetal.,2012;Huetal.,2011).Inthecaseof455
Ectocarpus,wealsoobservedtracesofrecenttransposonactivity,especiallyfromLTRtransposons,456
whichisinlinewiththeabsenceofDNAmethylation,andburstsintransposonactivityhaveindeed457
been identifiedasonepotentialdriverof localadaptationandspeciation inothermodelsystems458
such as salmon (de Boer et al., 2007). Furthermore, LTRs are known to mediate the459
retrotranspositionofindividualgenes,leadingtotheduplicationofthelatter(Tanetal.,2016).In460
theE.subulatusgenome,onlyafewcasesofgeneduplicationwereobservedsincetheseparation461
fromEctocarpussp.Ec32,andinmostofthemnoindicationoftheinvolvementofLTRswasfound.462
TheonlyexceptionwasarecentexpansionoftheLHCRfamily,inwhichproteinswereflankedbya463
pair of LTR-like sequences. These elements lacked both the group antigen (GAG) and reverse464
transcriptase(POL)proteins,whichimpliesthat,ifretro-transpositionwasthemechanismunderlying465
the expansion of this group of proteins, it would have depended on other active transposable466
elementstoprovidetheseactivities.467
ThesecondmajorfactorthatimpactedtheEctocarpusgenomeswereviruses.Viralinfectionsarea468
common phenomenon in Ectocarpales (Müller et al., 1998), and a well-studied example is the469
Ectocarpussiliculosusvirus-1(EsV-1)(Delaroqueetal.,2001).Itwasfoundtobepresentlatentlyin470
hostcellsofseveralstrainsofEctocarpussp.closelyrelatedtostrainEc32,andhasalsobeenfound471
integrated in thegenomeof the latter strain, although it isnotexpressed (Cocketal., 2010).As472
previouslyindicatedbycomparativegenomehybridizationexperiments(Dittamietal.,2011),theE.473
subulatusgenomedoesnotcontainacompleteEsV-1likeinsertion,althoughafewshorterEsV-1-474
like proteinswere found. Thus, the EsV-1 integration observed in Ectocarpus sp. Ec32 has likely475
occurredafterthesplitwithE.subulatus.This,togetherwiththepresenceofotherviralsequences476
specifictoE.subulatus,indicatesthat,inadditiontotransposableelements,viruseshaveshapedthe477
Ectocarpusgenomesoverthelast16millionyears.478
Few classical stress response genes but no transporters involved in479
adaptation480
Amainaimofthisstudywastoidentifygenefunctionsthatmaypotentiallyberesponsibleforthe481
highabioticstressandsalinitytoleranceofE.subulatus.Similarstudiesongenomicadaptationto482
changes in salinityor todrought in terrestrial plantshavepreviouslyhighlighted genes generally483
involved in stress tolerance to be expanded in “extremophile” organisms. Examples are the484
expansionofcatalase,glutathionereductase,andheatshockproteinfamiliesindesertpoplar(Ma485
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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etal.,2013),argininemetabolisminjujube(Liuetal.,2014),orgenesrelatedtocationtransport,486
abscisicacidsignaling,andwaxproductioninT.salsuginea(Wuetal.,2012).Inourstudy,wefound487
a few genomic differences thatmatch these expectations.E. subulatus possesses two additional488
HSP20 proteins and has an expanded family of CBPs probably involved in non-photochemical489
quenching,whichmaycontribute to itshighstress tolerance. Italsohasaslightly reducedsetof490
genesinvolvedintheproductionofhalogenateddefensecompoundswhichmayberelatedtoits491
habitatpreference:E.subulatusisfrequentlyfoundinbrackishandevenfreshwaterenvironments492
withlowavailabilityofhalogens.Italsospecializesinhighlyabioticstressfulhabitatsforbrownalgae493
andmaythusinvestlessenergyinhalogen-baseddefense.494
Another anticipated adaptation to life in varying salinities lies in modifications of the cell wall.495
Notably, the content of sulfated polysaccharides is expected to play a crucial role as these496
compounds are present in all marine plants and algae, but absent in their freshwater relatives497
(KloaregandQuatrano,1988;Popperetal.,2011).Thefactthatwefoundonlysmalldifferencesin498
thenumberofencodedsulfatasesandsulfotransferasesindicatesthattheabsenceofsulfatedcell-499
wall polysaccharides previously observed inE. subulatus in low salinities (Torodeet al., 2015) is500
probablyaregulatoryeffectorsimplyrelatedtotheavailabilityofsulfatedependingonthesalinity.501
This isalsocoherentwiththewidedistributionofE.subulatus,whichcomprisesmarine,brackish502
water,andfreshwaterenvironments.503
Finally,transportershavepreviouslybeendescribedasakeyelementinplantadaptationtodifferent504
salinities(seeRaoetal.,2016forareview).SimilarresultshavealsobeenobtainedforEctocarpusin505
astudyofquantitativetraitloci(QTLs)associatedwithsalinityandtemperaturetolerance(Aviaet506
al., 2017). In our study, however, we found no indication of genomic differences related to507
transporters between the two species. This observation corresponds to previous physiological508
experimentsindicatingthatEctocarpus,unlikemanyterrestrialplants,respondstostrongchanges509
in salinity as anosmoconformer rather thananosmoregulator, i.e. it allows the intracellular salt510
concentrationtoadjusttovaluesclosetotheexternalmediumratherthankeepingtheintracellular511
ioncompositionconstant(Dittamietal.,2009).512
Genesrelatedtocell-cellcommunicationareunderpositiveselection513
Inadditiontogenesthatmaybedirectlyinvolvedintheadaptationtotheenvironment,wefound514
several gene clusters containing domains potentially involved in cell-cell signaling that were515
expandedintheEctocarpussp.Ec32genome(Table2),notablyafamilyofankyrinrepeat-containing516
domainproteins(Mosavietal.,2004)wasmoreabundantinEc32.Furthermore,weidentifiedsix517
ankyrinrepeat-containingdomainproteinsamongthegenesunderpositiveselectionbetweenthe518
twospecies.Theexactfunctionoftheseproteins,however,isstillunknown.Theonlywell-annotated519
gene under positive selection, a mannosyl-oligosaccharide 1,2-alpha-mannosidase, is probably520
involved in the modification of glycoproteins which are also important for cell-cell interactions521
(Tulsianietal.,1982).AlthoughthesegenesarenotrapidlyevolvinginEctocarpus,theseobserved522
differencesmaybe,inpart,responsiblefortheexistingpre-zygoticreproductivebarrierbetweenthe523
twoexaminedspeciesofEctocarpus(Lipinskaetal.,2016).524
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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Genesofunknownfunctionandlineage-specificgenesarelikelytoplaya525
dominantroleinadaptation526
Despitethegenefunctionsidentifiedaspotentiallyinvolvedinadaptationandspeciationabove,itis527
importanttokeepinmindthatthevastmajorityofgenomicdifferencesbetweenthetwospeciesof528
Ectocarpuscorrespondstoproteinsofentirelyunknownfunctions.Amongthe83genepairsunder529
positive selection, 84% were also entirely unknown, and 92% represented genes taxonomically530
restrictedtobrownalgae.Inaddition,weidentified1,629lineage-specificgenes,ofwhich88%were531
entirelyunknown.Thesegeneswereforthemostpartexpressedandarethuslikelytocorrespond532
to true genes. For the lineage-specific genes, their absence from theEctocarpus sp. Ec32 andS.533
japonicagenomeswasalsoconfirmedonthenucleotidelevel.Alargepartofthemechanismsthat534
underlietheadaptationtodifferentecologicalnichesinEctocarpusmay,therefore,lieinthesegenes535
ofunknownfunction.Thiscanbeexplainedinpartbythefactthatstillonlyfewbrownalgalgenomes536
areavailableandthatcurrentlymostofourknowledgeonthefunctionsoftheirproteinsisbasedon537
studies in model plants, animals, yeast, or bacteria. Brown algae, however, are part of the538
stramenopilelineagethathasevolvedindependentlyfromtheformerforover1billionyears(Yoon539
etal.,2004).Theydifferfromlandplantseveninotherwisehighlyconservedaspects,forinstancein540
theirlifecycles,theircellwalls,andtheirprimarymetabolism(Charrieretal.,2008).Furthermore,541
substantial contributions of lineage-specific genes to the evolution of organisms and the542
developmentofinnovationshavealsobeendescribedforanimalmodels(seeTautzandDomazet-543
Lošo,2011forareview)andstudiesinbasalmetazoansfurthermoreindicatethattheyareessential544
forspecies-specificadaptiveprocesses(Khalturinetal.,2009).545
Despite the probable importance of unknown and lineage-specific genes for local adaptation,546
Ectocarpusmaystillheavilyrelyonclassicalstressresponsegenesforabioticstresstolerance.Many547
ofthegenefamiliesknowntoberelatedtostressresponseinlandplants(includingtransportersand548
genesinvolvedincellwallmodification)forwhichnosignificantdifferencesingenecontentswere549
observed, have previously been reported to be strongly regulated in response to environmental550
stress in Ectocarpus (Dittami et al., 2009; Dittami et al., 2012; Ritter et al., 2014). This high551
transcriptomicplasticity isprobablyoneof the features thatallowEctocarpus to thrive inawide552
range of environments and may form the basis for its capacity to further adapt to “extreme553
environments”suchasfreshwater(WestandKraft,1996).554
Conclusionandfuturework555
WehaveshownthatE.subulatushasseparatedfromEctocarpussp.Ec32probablyviaamechanism556
of allopatric speciation. Its genome has since been shapedmainly by the activity of viruses and557
transposons,particularlylargeretrotransposons.Overthisperiodoftime,E.subulatushasadapted558
toenvironmentswithhighabioticvariabilityincludingbrackishwaterandevenfreshwater.Wehave559
identified a number of genes that likely contribute to this adaptation, including HSPs, CBPs, a560
reduction of genes involved in halogenated defense compounds, or some changes in cell wall561
polysaccharidemodifyingenzymes.However,thevastmajorityofgenesthatdifferbetweenthetwo562
examinedEctocarpusspeciesorthathaverecentlybeenunderpositiveselectionarelineage-specific563
andencodeproteinsofunknownfunction.Thisunderlinesthefundamentaldifferencesthatexist564
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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betweenbrownalgaeandterrestrialplantsorotherlineagesofalgae.Studiesasthepresentone,565
i.e.withoutstrongaprioriassumptionsaboutthemechanismsinvolvedinadaptation,aretherefore566
essentialtostartelucidatingthespecificitiesofthislineageaswellasthevariousfunctionsofthe567
unknowngenes.Finally,E.subulatushasbecomeanimportantbrownalgalmodeltostudytherole568
of algal-bacterial interactions in response to environmental changes. This is due mainly to its569
dependenceonspecificbacterialtaxaforfreshwatertolerance(KleinJanetal.,2017;Dittamietal.,570
2016).Thepresentedalgalgenomeandmetabolicnetworkareindispensabletoolsinthiscontextas571
well,astheywillallowfortheseparationofalgalandbacterialresponsesincultureexperiments,and572
facilitate the implementation of global approaches based on the use of metabolic network573
reconstructions(Dittamietal.,2014;Levyetal.,2015).574
MaterialsandMethods575
Biological material. Haploid male parthenosporophytes of E. subulatus strain Bft15b (Culture576
CollectionofAlgaeandProtozoaCCAPaccession1310/34),isolatedin1978byDieterG.Müllerin577
Beaufort,NorthCarolina,USA,weregrownin14cm(ca.100ml)PetriDishesinProvasoli-enriched578
seawater (Starr and Zeikus, 1993) under a 14/10daylight cycle at 14°C. Approximately 1 g fresh579
weightofalgalculturewasdriedonapapertoweland immediatelyfrozenin liquidnitrogen.For580
RNA-seq experiments, in addition to Bft15b, a second strain, the diploid freshwater strain CCAP581
1310/196 isolated fromHopkins River Falls, Australia (West and Kraft, 1996),was included.One582
culturewasgrownasdescribedaboveforBft15b,andforasecondculture,seawaterwasdiluted20-583
foldwithdistilledwaterpriortotheadditionofProvasolinutrients(Dittamietal.,2012).584
Flow cytometry experiments to measure nuclear DNA contents were carried out as described585
(Bothwelletal.,2010),exceptthatyoungsporophytetissuewasusedinsteadofgametes.Samples586
of the genome-sequenced Ectocarpus sp. strain Ec32 (CCAP accession 1310/4 from San Juan de587
Marcona,Peru),wereruninparallelasasizereference.588
Nucleicacidextractionandsequencing.DNAandRNAwereextractedusingaphenol-chloroform-589
basedmethodaccordingtoLeBailetal. (2008).ForDNAsequencing,four Illumina librarieswere590
preparedandsequencedonaHiSeq2000:onepaired-endlibrary(IlluminaTruSeqDNAPCR-freeLT591
SamplePrepkit#15036187,sequencedwith2x100bpread length),andthreemate-pair libraries592
with span sizes of 3kb, 5kb, and 10kb respectively (Nextera Mate Pair Sample Preparation Kit;593
sequencedwith2x50bpreadlength).Onepoly-AenrichedRNA-seqlibrarywasgeneratedforeach594
ofthethreeaforementionedculturesaccordingtotheIlluminaTruSeqStrandedmRNASamplePrep595
kit#15031047protocolandsequencedwith2x50bpreadlength.596
Methylation.ThedegreeofDNAmethylationwasexaminedbyHPLConCsCl-gradientpurifiedDNA597
(LeBailetal.,2008)fromthreeindependentculturesperstrainaspreviouslydescribed(Rivaletal.,598
2013).599
Sequenceassembly.Redundancyofmatepairs(MPs)wasreducedbymappingMPstoapreliminary600
assembly,tomitigatethenegativeeffectofredundantchimericMPsduringscaffolding.CleanDNA601
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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readswereassembledusingSOAPDenovo2(Luoetal.,2012).Scaffoldingwasthencarriedoutusing602
SSPACEbasic2.0(Boetzeretal.,2011)(trimlengthupto5bases,min3linkstoscaffoldcontigs,min603
15readstocallabaseduringanextension)followedbyarunofGapCloser(partoftheSOAPDenovo604
package, default settings). Alternative assemblers (CLC and Velvet)were also tested but yielded605
significantlylowerfinalcontigandscaffoldlengths.RNA-seqreadswerecleanedusingTrimmomatic606
(defaultsettings),firstassembleddenovousingTrinity2.1.1(Grabherretal.,2011)andfilteredby607
coveragewithanFPKMcutoffof1.Later,asecondgenome-guidedassemblywasperformedwith608
Tophat2andwithCufflinks.609
Removal of bacterial sequences: As cultures were not treated with antibiotics prior to DNA610
extraction,bacterial scaffoldswere removed fromthe finalassemblyusing the taxoblastpipeline611
(DittamiandCorre,2017).Everyscaffoldwascutintofragmentsof500bp,andthesefragmentswere612
aligned(blastn,e-valuecutoff0.01)againsttheGenBanknon-redundantnucleotide(nt)database.613
Scaffoldsforwhichmorethan90%oftheir500bp-fragmentshadbacterialsequencesasbestblast614
hitswereremovedfromtheassembly(varyingthisthresholdbetween30and95%resultedinonly615
veryminordifferencesinthefinalassembly).“Bacterial”scaffoldsweresubmittedtotheMG-Rast616
servertoobtainanoverviewofthetaxapresentinthesample(Meyeretal.,2008).617
RepeatedelementsweresearchedfordenovousingTEdenovoandannotatedusingTEannotwith618
defaultparameters.BothtoolsarepartoftheREPETpipeline(Flutreetal.,2011),ofwhichversion619
2.5wasusedforourdataset.620
Assessmentof genomecompleteness:BUSCO2.0analyses (Simãoetal., 2015)were runon the621
servers of the IPlant Collaborative (Goff et al., 2011) with the general eukaryote database as a622
referenceanddefaultparameters.BUSCOinternallyusesAugustus(Stankeetal.,2004)topredict623
protein coding sequences. As the latter tool performed poorly on both Ectocarpus strains in624
preliminarytests,predictedproteinswereusedasinputinsteadofDNAsequences.625
Organellargenomes, i.e.plastidandmitochondrion,weremanuallyassembledbasedonscaffolds626
416and858respectively,usingthepublishedgenomeofEctocarpussp.Ec32asaguide(Delageet627
al.,2011;LeCorguilléetal.,2009;Cocketal.,2010).Inthecaseofthemitochondrialgenome,the628
correctnessofthemanualassemblywasverifiedbyPCRwheremanualandautomaticassemblies629
diverged. Both organellar genomeswere visualized usingOrganellarGenomeDRAW (Lohse et al.,630
2013)andalignedwiththeEctocarpussp.Ec32organellesusingMauve2.3.1(Darlingetal.,2004).631
Geneprediction.Putativeprotein-codingsequenceswereidentifiedusingEugene4.1c(Foissacet632
al., 2008). RNA-seq reads weremapped against the assembled genome using GenomeThreader633
1.6.5, and all available proteins from the Swiss-Prot database (Dec. 2014) as well as predicted634
proteinsfromtheEctocarpussp.Ec32genome(Cocketal.,2010)werealignedtothegenomeusing635
KLAST(NguyenandLavenier,2009).Bothaligneddenovo-assembledtranscriptsandproteinswere636
providedtoEugeneforgeneprediction,whichwasrunwiththeparametersetpreviouslyoptimized637
fortheEctocarpussp.Ec32genome(Cocketal.,2010).638
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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Functional annotation. Predicted proteins were compared to the Swiss-Prot database by BlastP639
search(e-valuecutoff1e-5),andtheresultsimportedtoBlast2GO(Götzetal.,2008),whichwasused640
to run InterPro domain searches and automatically annotate proteins with a description, GO641
numbers,andECcodes.ThegenomeandallautomaticannotationswereimportedintoApollo(Lee642
etal.,2013;Dunnetal.,2017)formanualcuration.643
Metabolic network reconstruction. The E. subulatus genome-scale metabolic model (GEM)644
reconstruction was carried out as previously described by Prigent et al. (2014) by merging an645
annotation-basedreconstructionobtainedwithPathwayTools(Karpetal.,2016)andanorthology-646
based reconstruction based on theArabidopsis thalianametabolic networkAraGEM (deOliveira647
Dal’Molinetal.,2010)usingPantograph(Loiraetal.,2015).Afinalstepofgap-fillingwasthencarried648
outusingtheMenecotool(Prigentetal.,2017).Theentirereconstructionpipelineisavailablevia649
the AuReMe workspace (Aite et al., 2018; http://aureme.genouest.org/). For pathway-based650
analyses,pathwaysthatcontainedonlyasinglereactionorthatwerelessthan50%completewere651
notconsidered.652
Genome comparisons. Functional comparisons of gene contents were based primarily on653
orthologousclustersofgenessharedwithversion2oftheEctocarpussp.Ec32genome(Cormieret654
al., 2017) as well as the Saccharina japonica (Areschoug) genome (Ye et al., 2015). They were655
determinedbytheOrthoFindersoftwareversion0.7.1(EmmsandKelly,2015).Foranypredicted656
proteins thatwere not part of amulti-species cluster,we verified the absence in the other two657
genomesalsobytblastnsearches.Proteinswithouthit(thresholde-valueof1e-10)wereconsidered658
lineage-specific proteins. Blast2GO 3.1 (Götz et al., 2008)was then used to identify significantly659
enrichedGOtermsamongthelineage-specificgenesortheexpandedgenefamilies(Fischer’sexact660
testwithFDRcorrectionFDR1weremanuallyexamined.Thedistributionofthesegenesacross671
thegenomewasexaminedbycalculatingvariancetomeanratiosbasedonwindowsizesof50to672
500genes.673
Phylogenetic analyses. Phylogenetic analyses were carried out for gene families of particular674
interest. For chlorophyll-binding proteins (CBPs), reference sequences were obtained from a675
previousstudy(Dittamietal.,2010),andalignedtogetherwithE.subulatusandS. japonicaCBPs676
using MAFFT (G-INS-i) (Katoh et al., 2002). Alignments were then manually curated, conserved677
.CC-BY-NC-ND 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/307165doi: bioRxiv preprint first posted online Apr. 25, 2018;
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positionsselectedinJalview(Waterhouseetal.,2009),andmaximumlikelihoodanalysescarriedout678
usingPhyML3.0(GuindonandGascuel,2003),theLGsubstitutionmodel,100bootstrapreplicates,679
and an estimation of the gamma distribution parameter. The resulting phylogenetic tree was680
visualized usingMEGA7 (Kumar et al., 2016). The same procedurewas also used in the case of681
selectedAnkyrinRepeatdomain-containingproteins.682
Data availability: Raw sequence data (genomic and transcriptomic reads) as well as assembled683
scaffolds and predicted proteins and annotations were submitted to the European Nucleotide684
Archive(ENA)underprojectaccessionnumberPRJEB25230usingtheEMBLmyGFF3script (Dainat685
andGourlé,2018).AJBrowse(Skinneretal.,2009)instancecomprisingthemostrecentannotations686
is available via the server of the Station Biologique de Roscoff (http://mmo.sb-687
roscoff.fr/jbrowseEsu/?data=data/public/ectocarpus/subulatus_bft). The reconstructed metabolic688
networkofE.subulatusisavailableathttp://gem-aureme.irisa.fr/sububftgem.Additionalresources689
and annotations including a blast server are available at http://application.sb-690
roscoff.fr/project/subulatus/index.html. The complete set of manual annotations is provided in691
SupportingInformationTableS1.692
Acknowledgements693
Wewould like to thankPhilippePotin,MarkCock, SusannaCoelho, FlorianMaumus, andOlivier694
Panaud for helpful discussions, as well as Gwendoline Andres for help setting up the Jbrowse695
instance.ThisworkwasfundedpartiallybyANRprojectIDEALG(ANR-10-BTBR-04)“Investissements696
d’Avenir, Biotechnologies-Bioressources”, the European Union’s Horizon 2020 research and697
innovationProgrammeundertheMarieSklodowska-Curiegrantagreementnumber624575(ALFF),698
andtheCNRSMomentumcall.SequencingwasperformedattheGenomicsUnitoftheCentrefor699
GenomicRegulation(CRG),Barcelona,Spain.700
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