The genome of Ectocarpus subulatus highlights unique...

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1

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;

2

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


3

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


4

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


5

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


6

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


7

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


8

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


9

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


10

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


11

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


12

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


13

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


14

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


15

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


16

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


17

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


18

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


19

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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