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1 Secondary metabolic symbiosis in shipworms (Teredinidae) Authors: Marvin A. Altamia a,b *, Zhenjian Lin c *, Amaro E. Trindade-Silva d,e , Iris Diana Uy b,f , J. Reuben Shipway g , Diego Veras Wilke e , Gisela P. Concepcion b,f , Daniel L. Distel a , Eric W. Schmidt c **, Margo G. Haygood c ** a Ocean Genome Legacy Center, Department of Marine and Environmental Science, Northeastern University, Nahant, MA, USA b The Marine Science Institute, University of the Philippines Diliman, Quezon City 1101, Philippines c Department of Medicinal Chemistry, University of Utah d Bioinformatic and Microbial Ecology Laboratory - BIOME, Federal University of Bahia, Salvador, Bahia, Brazil e Drug Research and Development Center, Department of Physiology and Pharmacology, Federal University of Ceara, 60430275, Ceara, Brazil f Philippine Genome Center, University of the Philippines Diliman, Quezon City 1101, Philippines g Institute of Marine Science, School of Biological Sciences, University of Portsmouth, UK *equal contribution authors **co-corresponding authors Significance Shipworms play critical roles in recycling wood in the sea and in shaping mangrove habitats. Symbiotic bacteria supply the enzymes that they need for nutrition and wood degradation. Here, we show that the same nutritional symbionts also have an immense capacity to produce a multitude of diverse and likely novel bioactive secondary metabolites. The compounds likely support the ability of shipworms to degrade wood in marine environments and include a compound under investigation for its therapeutic potential. Because many of the symbionts can be cultivated, they provide a model for understanding how secondary metabolism impacts microbial symbiosis in animals. Abstract Shipworms, assisted by intracellular γ-proteobacteria in their gills, are the principal degraders of wood in the sea. Shipworm symbionts have been cultivated in the laboratory. The genomes of these symbionts, in addition to being replete with lytic enzymes capable of degrading wood and/or enzymes of thioautotrophic metabolism, are among the bacterial genomes richest in secondary metabolite genes. These cultivated symbionts represent the dominant species in the gills in diverse shipworm species. We investigated how the isolate secondary metabolites might impact the host animals: which bacterial pathways are present, how widely distributed they are, and how they vary. Focusing on 14 wood-eating shipworm specimens, we found between one to three major bacterial species in each gill, with each species comprising a complex mixture of closely related strains. The mixture allows the shipworm host to access a much more . CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted October 31, 2019. . https://doi.org/10.1101/826933 doi: bioRxiv preprint

Secondary metabolic symbiosis in shipworms (Teredinidae) · The compounds likely support the ability of shipworms to degrade wood in marine environments and include a compound under

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Page 1: Secondary metabolic symbiosis in shipworms (Teredinidae) · The compounds likely support the ability of shipworms to degrade wood in marine environments and include a compound under

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Secondarymetabolicsymbiosisinshipworms(Teredinidae)Authors:MarvinA.Altamiaa,b*,ZhenjianLinc*,AmaroE.Trindade-Silvad,e,IrisDianaUyb,f,J.ReubenShipwayg,DiegoVerasWilkee,GiselaP.Concepcionb,f,DanielL.Distela,EricW.Schmidtc**,MargoG.Haygoodc**aOceanGenomeLegacyCenter,DepartmentofMarineandEnvironmentalScience,

NortheasternUniversity,Nahant,MA,USAbTheMarineScienceInstitute,UniversityofthePhilippinesDiliman,QuezonCity1101,

PhilippinescDepartmentofMedicinalChemistry,UniversityofUtahdBioinformaticandMicrobialEcologyLaboratory-BIOME,FederalUniversityofBahia,

Salvador,Bahia,BrazileDrugResearchandDevelopmentCenter,DepartmentofPhysiologyandPharmacology,Federal

UniversityofCeara,60430275,Ceara,BrazilfPhilippineGenomeCenter,UniversityofthePhilippinesDiliman,QuezonCity1101,PhilippinesgInstituteofMarineScience,SchoolofBiologicalSciences,UniversityofPortsmouth,UK*equalcontributionauthors**co-correspondingauthorsSignificanceShipwormsplaycriticalrolesinrecyclingwoodintheseaandinshapingmangrovehabitats.Symbioticbacteriasupplytheenzymesthattheyneedfornutritionandwooddegradation.Here,weshowthatthesamenutritionalsymbiontsalsohaveanimmensecapacitytoproduceamultitudeofdiverseandlikelynovelbioactivesecondarymetabolites.Thecompoundslikelysupporttheabilityofshipwormstodegradewoodinmarineenvironmentsandincludeacompoundunderinvestigationforitstherapeuticpotential.Becausemanyofthesymbiontscanbecultivated,theyprovideamodelforunderstandinghowsecondarymetabolismimpactsmicrobialsymbiosisinanimals.AbstractShipworms,assistedbyintracellularγ-proteobacteriaintheirgills,aretheprincipaldegradersofwoodinthesea.Shipwormsymbiontshavebeencultivatedinthelaboratory.Thegenomesofthesesymbionts,inadditiontobeingrepletewithlyticenzymescapableofdegradingwoodand/orenzymesofthioautotrophicmetabolism,areamongthebacterialgenomesrichestinsecondarymetabolitegenes.Thesecultivatedsymbiontsrepresentthedominantspeciesinthegillsindiverseshipwormspecies.Weinvestigatedhowtheisolatesecondarymetabolitesmightimpactthehostanimals:whichbacterialpathwaysarepresent,howwidelydistributedtheyare,andhowtheyvary.Focusingon14wood-eatingshipwormspecimens,wefoundbetweenonetothreemajorbacterialspeciesineachgill,witheachspeciescomprisingacomplexmixtureofcloselyrelatedstrains.Themixtureallowstheshipwormhosttoaccessamuchmore

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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complexmetabolismthancanbeaffordedbyasinglesymbiontstrain.Weanalyzedsequencesfrom22shipwormgillmetagenomesfromsevenshipwormspeciesandcomparedthemwiththegenomesof23cultivatedbacterialisolatesfromshipwormgills.Limitingouranalysestowell-characterizedbiosyntheticgenefamilies,wefoundmorethan400polyketide,nonribosomalpeptide,andrelatedbiosyntheticgeneclusters,onlyahandfulofwhichresembleknownones,comprisingover100geneclusterfamilies(GCFs).OnlyfourGCFsarefoundinallspecimensinvestigated.SeveralGCFsexhibitedahostspecies-specificdistribution,butmostoccurredstochastically.Shipwormsandtheirsymbiontsthusprovideamodelsystemforunderstandingtheroleofsecondarymetabolisminsymbioses.

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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IntroductionShipworms(FamilyTeredinidae)arebivalvemollusksfoundthroughouttheworld’soceans(1,2).Manyshipwormseatwood,assistedbycellulasesfromintracellularsymbioticγ-proteobacteriathatinhabittheirgills(Figure1)(3-6).Othersusesulfidemetabolismalsorelyingongill-dwellingγ-proteobacteriaforsulfuroxidation(7).Shipwormgillsymbiontsofseveraldifferentspeciesarethusessentialtoshipwormnutritionandsurvival.Oneofthemostremarkablefeaturesoftheshipwormsystemisthatwooddigestiondoesnottakeplacewherethebacteriaarelocated,sothatthebacterialcellulaseproductsaretransferredfromthegilltoanearlysterilececum(8),wherewooddigestionoccurs(Figure1)(9).Thisenablesthehostshipwormstodirectlyconsumeglucoseandothersugarsderivedfromwoodlignocelluloseandhemicellulose,ratherthanthelessenergeticfermentationproductsofcellulolyticgutmicrobesasfoundinothersymbioses.Shipwormsymbiontsarealsoessentialfornitrogenfixationinthelow-nitrogenwoodenvironment(10).Thus,shipwormshaveevolvedstructuresandmechanismsenablingbacterialmetabolismtosupportanimalhostnutrition.Whileinmanynutritionalsymbiosesthebacteriaaredifficulttocultivate,shipwormgillsymbioticγ-proteobacteriahavebeenbroughtintostableculture(5,11,12).Thisledtothediscoverythatthesebacteriaareexceptionalsourcesofsecondarymetabolites(13).Ofbacteriawithsequencedgenomes,thegillsymbiontsTeredinibacterturneraeT7901andrelatedstrainsareamongtherichestsourcesofbiosyntheticgeneclusters(BGCs),comparableincontenttofamousproducersofcommercialimportancesuchasStreptomycesspp.(12-15).Thisimpliesthatshipwormsmightbeagoodsourceofnewcompoundsfordrugdiscovery.Ofequalimportance,thesymbioticbacteriaarecrucialtosurvivalofhostshipworms,andbioactivesecondarymetabolitesmightplayaroleinshapingthosesymbioses.ThegenomesequenceofT.turneraeT7901revealedninecomplexpolyketidesynthase(PKS)andnonribosomalpeptidesynthetase(NRPS)BGCs(13),andmorecomprehensiveanalysisidentifiesupto14potentialBGCs.Oneofthesewasshowntoproduceanovelcatecholatesiderophore,turnerbactin,whichiscrucialinobtainingironandtothesurvivalofthesymbiontinnature(16).AsecondBGCsynthesizestheboratedpolyketidetartrolonsD/E,whichareantibioticandpotentlyantiparasiticcompounds(17).Bothweredetectedintheextractsofshipworms,implyingapotentialroleinproducingtheremarkablenearsterilityobservedinthececum(8).Thesedatasuggestedspecificrolesforsecondarymetabolisminmaintainingshipwormfunction.T.turneraeT7901isjustoneofmultiplestrainsandspeciesofγ-proteobacterialivingintracellularlyinvariousshipwormgills(3,11),andthustheseanalysesjustbegintodescribeshipwormsecondarymetabolism.Manyshipwormspeciesaregeneralists,consumingwoodfromavarietyofsources(1,18).Otherwood-eaters,suchasDicyathifermannii,Bactronophorusthoracites,andNeoteredoreynei,arespecialiststhatliveinthesubmergedbranches,trunksandrhizomesofmangroves(19,20).There,theyplayanimportantroleinecologicalprocessesinmangroveecosystems,i.e.transferringlargeamountofcarbonfixedbymangrovestothemarineenvironment(18).Severalshipwormspecies,suchasKuphus

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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polythalamius,liveinothersubstrates.K.polythalamiusoftenisfoundinsedimenthabitats(aswellasinwood)whereitsgillsymbiontsarecrucialtosulfideoxidationandcarryoutcarbonfixation(7).K.polythalamiuslackssignificantamountsofcellulolyticsymbiontssuchasT.turnerae,andinsteadcontainsThiosociusteredinicola,whichoxidizessulfideandgeneratesenergyforthehost(21).Othershipwormsarefoundinsolidrockandinseagrass(22,23).Thus,gillsymbiontsvary,butinallcasesthesymbiontsappeartobeessentialtothesurvivalofshipworms.WhilethepotentialofT.turneraeasanunexploredproducerofsecondarymetaboliteshasbeendescribed(13,15),thecapacityofothershipwormsymbiontsisstilllargelyunknown.Moreover,severalpiecesofdataindicatethattheBGCsfoundincultivatedisolatesmightalsobefoundinshipwormgills,buttheirpresence,distributionandvariabilityinnatureareunknown.PreviousdataincludethedetectionoftartrolonsandturnerbactinsandtheirBGCsinshipworms(16,17);aninvestigationoffourisolategenomesandonemetagenomethatobservedsharedpathways(24);alsoanexploratoryinvestigationofthemetagenomeofN.reyneigillsanddigestivetractledtothedetectionofknownT.turneraeBGCsaswellasnovelclusters(25).Thesefindingsleftmajorquestionsabouttheorigin,abundance,variability,distribution,andpotentialrolesofshipwormsecondarymetabolites.Here,weuseacomparativemetagenomicsapproachtoanswerthesequestions.ResultsandDiscussionGenomicdatafromshipwormsandtheirsymbioticbacteria.CellulolyticT.turneraeisrelativelyrichinsecondarymetabolismincomparisontosulfide-oxidizingT.teredinicola.Therefore,forthisanalysisweselectedsixspeciesofwood-eatingshipworms,comparingthesetoaseventhsulfide-oxidizingspecies,K.polythalamius.Wecomparedgillmetagenomesfrom22specimenscomprisingsevenanimalspecieswiththegenomesof23cultivatedbacteriaisolatedfromshipworms(TableS1).Oftheanimalsobtained,weanalyzedthreespecimenseachofBactronophorusthoracites,Kuphusspp.,Neoteredoreynei,andTeredosp.,twospecimensofBankiasp.,andfivespecimensofBankiasetacea.Theseanimalsweredividedintothreegeographicalregions(Figure1):thePhilippines(B.thoracitesandD.manniifromInfanta,Quezon;Kuphusspp.fromMindanaoandMabini);Brazil(N.reyneifromRiodeJaneiro,Teredosp.,andBankiasp.fromCeará);andtheUnitedStates(B.setacea).Thepurposeofsamplingthisrangewastodeterminewhetherthereareanygeographicaldifferencesingillsymbionts.Mostoftheshipwormswereobtainedfrommangrovewood,withtheexceptionofB.setaceafromunidentifiedfoundwood,andKuphusspp.frombothfoundwoodandmud.Thegillmetagenomesoftwospecimensofmud-dwellingK.polythalamiusfromMindanaowerepreviouslysequenced(7).Here,wesequencedandanalyzedgillmetagenomesfromathirdwood-burrowingKuphussp.specimenfromMabini.Sincethetaxonomyofthisspecimenhasyettobefullyresolved,wehavenotprovidedaspeciesnameforthisspecimen.Nonetheless,itsmetagenomewasverysimilartothatofthemuddwellingK.polythalamius(seebelow).B.

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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setaceawassequencedbytheJGIwithlowcoverage.TheremainingspecimensfromthePhilippinesweresequencedatUtahwithhighestcoveragedata(HiSeq),whileBrazilianspecimensweresequencedinBrazilandareintermediateincoverage(MiSeq)(seeTableS1formetagenomestatistics).Becauseoftheirlowercoverage,B.setaceasequenceswereusedonlyinasubsetoftheanalysesdescribedbelow. γ-ProteobacteriaofOrdersCellvibrionales(Teredinibacterandallies)andChromatiales(Thiosociusandallies)aredescribedsymbiontsthatliveintracellularlyinthegillsofshipworms.Weselected23cellulolyticandsulfur-oxidizingisolatescultivatedfromshipwormtissuesamples.Someofthestrainswerepreviouslyisolated,whileothersoriginatedinourrecentsamplecollections(TableS2).Forcomparison,wealsoincludedAgarilyticarhodophyticola017(26)(Ga0198945),afree-livingbacteriumthatiscloselyrelatedtoshipwormstrainsandthathasasequencedgenome.ThestrainsweresequencedattheJGI.Sixofthesecirculargenomeswereclosed,whileremainingassemblieshadbetween2-141scaffolds(seeTableS2forstraindataandaccessionnumbers).Twoofthesegenomes,T.turneraeT7901andT.teredinicola2141T,werepreviouslydescribed(13,21).Examinationof16SrRNAgenesequencesofthecultivatedstrainsallowedustoidentifytheisolateswithgenomesmostsimilartothoseidentifiedinmetagenomesequences(Figures2AandS1;TableS2).Ofthese,3arecellulolyticsymbionts,while2consistofsulfide-oxidizingsymbionts.Crucially,11ofthestrains,belongtoasinglespecies,T.turnerae.Eachshipwormgillsampleisdominatedby1-3majorbacterialspecies,withalargeunderlyingstrainvariation.Metagenomesequencingwasusedtodeterminewhichbacterialspeciesinhabiteachshipwormgill.Afterinitialanalysis,wefoundthatmostofthemetagenomicDNAfrombacteriacouldbemappedtogenomesequencesfromindividualstrainsinourculturecollection.Readcountswereusedtoquantifytheabundanceofeachspecieswithineachgillsample.Thisenabledustoaccuratelyquantifythemajorsymbiontspecieswithhighconfidence.Forthisanalysis,weremovedB.setacea,whichhadlowreadcoverage,andfocusedonthesixspecieswithgoodcoverage(Fig.2B).Eachshipwormgillmetagenomeisdominatedbyonetothreebacterialspecies,whicharerepresentedbycultivatedisolatesthatweobtainedfromthosesamespecies.Threeshipwormspeciesaredominatedbyasinglebacterialspecies,whilethreeothershavemixedsymbiontcommunities.Aspreviouslyreported,Kuphusspp.isdominatedbythesulfur-oxidizerT.teredinicola,andN.reyneiisdominatedbythecellulolyticbacteriumT.turnerae((7,25).B.thoracitiesisdominatedbystrain2753L.Incontrast,BrazilianshipwormsBankiasp.andTeredosp.containmostlyT.turnerae,buttheyalsoharborspeciescloselyrelatedtoCellvibrionalesstrain1162T.D.manniihasthemostdiversecommunity,containingamixtureofT.turneraeandCellvibrionalesstrain2753L,andasignificantfractionofthesulfideoxidizingsymbiontChromatialesstrain2719K.Inthemetagenomesofallshipwormgills,thereisalsoacomplexmixtureofminor,variablebacterialstrains(~1-15%oftotalreads,Fig.2Bshowningray).Thus,theshipwormgillisarelativelysimplesystem,dominatedbyafewspeciesofintracellularsymbioticbacteriathatarecrucialtohostnutritionandsurvival.

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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Underlyingthissimplicity,foreachbacterialspeciespresentinasample,therearemultiplestrainvariants.WemeasuredtheprevalenceofthesevariantsusingapreviouslyreportedmethodinwhichwelookatsinglenucleotidepolymorphismsinconservedgenessuchasDNAgyraseBattheindividualreadlevel(FigureS2)(7).TheresultsareverysimilartoourpreviousreportofbacterialstrainvariationinthegillmetagenomesofK.polythalamius.Thepresenceofmultiple,closelyrelatedstrainvariantsaddstothecomplexityofsecondarymetabolitepathwaysfoundineachhostorganism,sinceineachanimalgilltherearemanymorebiosyntheticpathwaysthanarefoundinindividualsequencedisolates(seebelow).Thesymbiontmixturesarerepresentativeoftheanimallifestyles.K.polythalamiusappearstothriveentirelyonsulfideoxidation(7),asrequiredinitssedimenthabitat,whiletheothershipwormscontainvariouscellulolyticbacteriaresponsibleforwooddegradation.D.manniilikelyhasamorecomplexlifestyle,sinceitcontainsthesulfur-oxidizingbacteriumstrain2719KandthecellulolyticspeciesT.turneraeandstrain2753L.Morecompletedescriptionsofthesesymbioseswillbepublishedinarticlesfocusedonindividualshipwormspecies,whilehereourfocusisonanalysisandcomparisonofsecondarymetabolism.Shipwormsymbiontsandgillmetagenomesareunusuallyrichincomplexsecondarymetabolitepathways.TheprogramantiSMASH(27)wasusedtocombthegenomesforsecondarymetaboliteBGCs.WerefinedtheantiSMASHoutputtofocusonBGCsthatarewellcharacterizedtoencodesecondarymetabolites:polyketidesynthases(PKSs),nonribosomalpeptidesynthetases(NRPSs),siderophores,terpenes,homoserinelactones,andthiopeptides.Usingthesecriteria,weidentified168BGCsfrom23cultivatedisolatesand401BGCsfrom22shipwormgillmetagenomes(Fig.3).Becausethegenomesofcultivatedisolateswerewellassembled,wecoulddiscernandanalyzeentireBGCs.Bycontrast,theanimalmetagenomescontainedsomelargebutmanysmallercontigs(N50sshowninTableS1),inwhichBGCswereoftenfragmented.TheseBGCsnearlyuniversallyoriginatefromOrderCellvibrionales,withveryfewBGCsfoundinthesulfideoxidizingstrainsChromatiales.Thus,thecellulolyticshipwormsymbiontsarerichsourcesofdiverseBGCs.WefoundonlyfiveBGCsthatweresimilartopreviouslyidentifiedclustersfromoutsideofshipworms,basedupon>70%ofgenesconservedinantiSMASH.TheremainderappearedtobeunknownoruncharacterizedBGCs.Thisresultreinforcesthatshipwormsymbioticbacteriaarepromisingsourcesofnewbioactivemolecules.ItfurthersupportsapreviousanalysiscomparinggenomesacrossdomainBacteria,whichrevealedthatT.turneraerepresentsanotablyrich,yetnearlyuntapped,sourceofnewsecondarymetabolitegenes(15).Tofacilitatecomparisonbetweenmetagenomes,wegroupedBGCsintogeneclusterfamilies(GCFs).Thisisamethodthatcomparesgroupsofgenesthatareallinvolvedinaparticularpathway,ratherthancomparingindividualgenes(28,29).Wesetanidentitythresholddefinedin“Methods,”leadingtotheidentificationof122GCFscomprisingall569discreteBGCsinthegenomesandmetagenomescombined(Fig.4andTableS3).GiventhattheseGCFsoriginatedin

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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asmallhandfulofbacterialspecies,thisisalargenumberthatreinforcestheroleofstrainvariationingeneratingchemicaldiversityinshipwormgills.AcaveatisthatweignoredantiSMASHhitsfrompoorlycharacterizedoruncharacterizedbiosyntheticpathwayfamilies,sothat122GCFsrepresentsaveryconservativeestimateofthechemicaldiversitypresentinshipwormgills.ByparingdownthegenestobeanalyzedtoasetofwellcharacterizedBGCs,comparisonbecamepossible,butwealsopotentiallymissedsomeinterestingpathways.Asoneexample,weanalyzedthegenomeofChromatialesstrain2719Kanddiscoveredageneclusterfortabtoxin(30,31)orrelatedcompound(Fig.5).ThisclusterdoesnotcontaincommonPKS/NRPSelementsandthuswasexcludedfromthecomparativeanalysis(forexample,itisnotadefinedGCFasshowninFigures4,6,or7).Akeybiosyntheticgeneinthetabtoxin-likeclusterwaspseudogenousinstrain2719K,buttheD.manniigillmetagenomecontainedanapparentlyfunctionalpathway.Tabtoxinisanimportantβ-lactamthatisusedbyPseudomonasinplantpathogenesis(32).Sincetabtoxinpreventsglutaminesynthesisandleadstotheaccumulationoftoxicammoniainplants(33),itistemptingtospeculateonhowtabtoxinmightimpactthissymbiosis,perhapsimprovingaccesstonitrogen.Anothercaveatisthat,althoughweidentifiedalargenumberofGCFsfromasmallspecimenset,eventhisisanunderestimate.B.setaceametagenomesfromOceanGenomeLegacywereincludedinouranalysisforcomparison,buttheyarevastlyundersampledincomparisontotheotherspeciesduetolowsequencecoverage,andthuswearemissingmostoftheirBGCsintheanalysis.CultivatedisolateGCFsareabundantcomponentsofthesourcegillmetagenomes.WecomparedBGCsandGCFsbetweenshipwormsandbacteria.Of401BGCsidentifiedinthemetagenomes,305ofthemalsohadcloserelativesincultivatedisolates,indicatingthat~75%ofBGCsinthemetagenomesarecoveredinoursequencedculturecollection(Fig.3).Conversely,of168isolateBGCs,148(90%)ofthemarefoundinthemetagenomes.Thus,sequencingfurthercultivatedisolatesinourstraincollectionsislikelytoyieldadditionalnovelBGCs.Only8GCFsarewidelydistributedin10ormoreisolates,andthesearemostlypathwaysthatareuniversalornearlyuniversalinT.turnerae,whichisoverrepresentedinourdataset(Figs.6and7).BycontrasttoisolategenomesinwhichwefoundmanyGCFsthatoccurinonlyasinglegenome,inthemetagenomesmostGCFSarefoundinmultiplespecimens.Only24outof107totalGCFsarefoundonlyonceinmetagenomes(Fig.4).Sincedifferentshipwormspeciesinourspecimencollectioncontaindifferentgroupsofbacteria,thisresultreinforcestheneedtosamplemoreindividualspecimensacrossthediversityofshipwormspeciestooptimizethediscoveryofbioactivesecondarymetabolites. ToobtainamorerefinedviewofBGCdistribution,wefirstusedtheMultiGeneBlast(28)outputtoconstructasimilaritynetwork(Fig.6).ThenetworkprovidedaneasilyinterpretablediagramofhowGCFsaredistributedinbacteria.However,twonotableproblemswereobservedinthisdiagram.First,fromexperiencewehavefoundthetartrolonBGCinnearlyallT.turnerae

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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strains.However,thisBGCwasobservedinonlyafewoftheT.turnerae-hostingshipwormsviaMultiGeneBlast.Thisiscausedbyatechnicalprobleminassemblythatweoftenseewithlargetrans-ATpathwaysfromcomplexsamples.Second,becausethismethodreliesuponcomparinglargercontigs,wecouldnotincludethelow-coverageB.setaceasequencesfromJGIinthisanalysis.Toremedytheseproblems,weusedasecondmethodthatobtainedGCFsfromcultivatedisolatesandusedthoseGCFsintBLASTnsearchesofmetagenomecontigs(Fig.7).Thisprovidedanorthogonalviewofsecondarymetabolisminshipworms,revealingthepresenceofthetartrolonpathway,aswellasotherpathwaysthatdonotassemblewellinmetagenomesbecauseofcharacteristicssuchasrepetitiveDNAsequences.ItalsoenabledustocompareB.setaceawiththeotherspecies.AweaknessofthissecondmethodisthattheclosenessofrelationshipsbetweenBGCsisnotreadilydiscernedintermsofsequencesimilarityandgenespresent.Thus,thesetwomethodsprovidedifferentinsightsintoBGCsinshipwormgills.Usingthosedata,focusingonthesixshipwormspecieswithgoodsequencingcoverage,wecouldobservecleartrendsofwidelysharedGCFs,GCFsthatwerespecifictoshipwormandsymbiontspecies,andstochasticallyoccurringGCFs.WidelysharedGCFs.Fourpathways(GCF_2,GCF_3,GFC_5,andGCF_8)wereprevalentinwood-eatingshipworms,regardlessofsamplelocation(Figs.6and7).TheseGCFswereencodedinthegenomesofT.turneraeandthoseofseveralotherCellvibrionalesisolates(especiallythepathway-rich2753L),explainingtheirwidespreaddistribution.GCF_2encodesaNRPS/trans-acyltransferase(trans-AT)PKSpathway,thechemicalproductsofwhichareunknown.ItisfoundinallshipwormspecimensinthisstudyandinallT.turneraestrains.ItisalsopresentinCellvibrionalesstrain2753L.ThisexplainsitspresenceinB.thoracitesdespitetheabsenceofT.turneraeinthisspecies.GCF_2issynonymouswith“region3”describedintheannotationoftheT.turneraeT7901genome(13).ThemostprominentlyoccurringpathwayinshipwormgillmetagenomesisGCF_3.Itwasidentifiedinallgillmetagenomeswithcellulolyticsymbionts,includingthemetagenomesofspecimenB.setaceaBSG2.ItoccursinallT.turneraestrains,aswellasinCellvibrionalesstrains2753LandBs08.Itwasfirstannotatedas“region1”intheT.turneraeT7901genomeandencodesanelaboratehybridtrans-ATPKS-NRPSpathway(13).UnlikeallotherGCFsidentifiedinshipwormmetagenomesandisolates,GCF_3couldbesubdividedintoatleastthreediscretecategories,eachofwhichincludeddifferentgenecontent(Fig.8).Thefirstcategory,identifiedinT.turneraeT7901,encodesaPKSandasingleNRPS,inadditiontoseveralpotentialmodifyingenzymes.StrainBs08containsasimilarpathway,exceptwithanadditionaltwoNRPSgenesthatpresumablyleadtoadditionalaminoacidsontheresultingproduct.Cellvibrionales2753Lencodedthethirdpathwaytype,whichwassimilartothatfoundinT7901exceptwithdifferentflankinggenecontent.ThepresenceofasingleGCFthatencodessimilarbutnon-identicalproductssuggestsadynamicpathwayevolutionwithinshipworms.

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GCF_5encodesacombinationofterpenecyclaseandpredictedarylpolyenebiosyntheticgenes,whichwereunrecognizedintheinitialBGCscreeningofT.turneraeT7901genome,sincethearylpolyenepathwaysarerecentdiscoveries.TheGCF_5biosyntheticproductisunknown,althoughthecyclaseandsurroundingregionshaveallofthegenesnecessarytomakeandexporthopanoids.InadditiontooccurringinallT.turneraestrains,GCF_5ispresentinCellvibrionalesstrains1120Wand2753L.Thepathwaywasdetectedinallwood-eatingspecimensexceptTeredosp.TBF07(Fig.8).GCF_8isexemplifiedbythepreviouslydescribedturnerbactinBGC,fromT.turneraeT7901.Turnerbactinisacatecholatesiderophore,crucialtoironacquisitioninT.turnerae(16).TheBGCforturnerbactinwaspreviouslyidentifiedanddescribedas“region7”intheT.turneraeT7901genome.GCF_8ispresentinallT.turneraegenomessequencedhere.OtherCellvibrionalesstrains,including2753LfromB.thoracitesandBs08fromB.setacea(neitherofwhichcontainsT.turnerae),alsoencodeturnerbactin-likesiderophoresynthesis.OnespecimenofB.thoracitescontainedGCF_8.Beyondbacterialironacquisition,siderophoresarealsoimportantinstraincompetitionandpotentiallyinhostanimalphysiology(34,35),possiblyexplainingthewidespreaddistributionofGCF_8.FromtheclusteringpatterninFigure6,itislikelythatGCF_8comprisesatleastthreedifferent,butrelatedtypesofgeneclusters.Thus,GCF_8likelyrepresentscatecholatesiderophores,butnotnecessarilyturnerbactin.ImportantGCFswidelydistributedinT.turnerae-containingshipworms.InadditiontothefourGCFsdescribedabovethathaveawidedistribution,GCFs1,4,and11werefoundinallT.turnerae-containingshipworms.GCF_1isatrans-ATPKS-NRPSpathwaythatappearstobesplitintotwoclustersinsomeshipwormisolates,includingT.turneraeT7901,inwhichitwaspreviouslyannotatedas“region4”and“region5”.GCF_4isthepreviouslydescribed“region8”PKS-NRPSfromT.turneraeT7901.Mostnotably,GCF_11encodestartrolonbiosynthesis(17).TartrolonisanantibioticandpotentantiparasiticagentisolatedfromculturebrothsofT.turneraeT7901(17,36,37).Ithasalsobeenidentifiedinthececumoftheshipworm.Itwasproposedthatthebacteriasynthesizetartroloninthegill,anditistransferredtothececumwhereitmayplayaroleinkeepingthedigestivetractfreeofbacteria(17).GCFsspecifictoshipwormscontainingstrain2753L-likesymbonts.D.manniiandB.thoracitescontainabundant2753L-likestrains.LikeT.turneraeT7901,the2753LisolategenomeencodesGCFs2,3,and5.However,2753LcontainsseveralGCFsnotfoundinT.turnerae,includingGCFs6,10,12,13,14,16,30,and31(listedinorderoftheirrelativefrequencyofoccurrenceinsamples).AlloftheseGCFsarealsofoundinD.manniiandB.thoracitesgillmetagenomes.ThesearePKSandNRPSclustersthatlackcloserelativesaccordingtoantiSMASHannotationandthushaveapotentialtosynthesizenovelsecondarymetaboliteclasses.GCFsspecifictostrain1162T-likesymbiontsinshipwormsfromBrazil.AllthreeBrazilianshipwormspeciescontainsymbiontgenomessimilartoourcultivatedisolate,1162T.Thisislikelyduetothespeciessampledandnottogeographicalvariation,sinceweobtained1162TfromaPhilippinesspecimenofLyrodussp.InBankiasp.andTeredosp.,wherestrain1162T-likesymbiontsareamajorcomponent,thereareseveraluniqueGCFsthatoriginateinthestrain

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2753L-likesymbionts(Fig.S4B).Becausethestrain2753L-likesymbiontfromBrazilmetagenomesisrelativelydistantlyrelatedtothecultivatedisolateandhaslittleoverlapintermsofBGCs,wecouldnotidentifymostofthosefull-lengthBGCsinthecultivatedisolates.Thus,ifthesetrendsholdupthroughfurthersampling,strain1162T-likesymbiontsmayexhibitthehighlevelofchemicaldiversitysimilartowhatisfoundinstrain2753L-likegenomes.Inaddition,whencomparingthemetagenomesofBankiasp.andTeredosp.,itisclearthatthe1162T-likesymbiontsarenotidenticalinthesetwoshipwormspecies,andthattheymaylikelyharbordifferentGCFs.GCFsspecifictosulfuroxidizingshipwormsymbionts.KuphusanditssymbiontscontainedrelativelyfewBGCs,butstrikinglytwoNRPS-containingGCFshavebeenuniversallyfoundintheshipwormassociatedsulfide-oxidizingspeciesT.teredinicola.Oneofthese,GCF_17,isshowninFig.7,whereitisfoundinthesymbiontmetagenomesofKuphusandD.mannii.ItisclearthatthecellulolyticsymbiontsaremuchricherinBGCs,andinadditiontheBGCsvarymuchmoreextensively.Stochasticpathwayoccurrenceinshipwormgills.Manypathwaysinbothgenomesandmetagenomeswerefoundonlyonceoroccurredrelativelyrarely,sothattrendscouldnotbediscerned.InFig.7,only18GCFsfoundinmorethanonespeciesofshipwormaredisplayed.Theremaining114GCFsoccurrarelyoronlyonce.Thisindicatesthatmostbiosyntheticpathwaysoccurstochastically.ThistrendisreinforcedinFig.6,wheremostGCFsinthediagramoccuronlyonce(single,unlinkedspots).Whileseveralbiosyntheticpathwaysareconservedandthuslikelyhaveanimportantconservedroleinthesymbiosis,mostarenotconserved.Furthersamplingofshipwormspecimens,species,andcultivatedisolateswillyieldmanyfurther,unanticipatedBGCs.VariabilityinconservedshipwormGCFsincreasespotentialcompounddiversity.EvenamongconservedGCFs,thereisvariabilityinsomeofthepathways.ThiscanbeobservedintheBGCnetwork,wheretherelationshipsbetweenclusterscanbegraphicallyobserved.(Fig.6).Forsomeofthesepathways,wecouldobservedifferencesingenecontentconsistentwithdifferentchemicalsthatwouldbeproduced.ExamplesincludetheuniversalGCF_3andsiderophorepathwayGCF_8describedabove.Discussion.Marineinvertebratesoftenusesmallmoleculesinchemicaldefense(38).Thesuiteofdefensivechemicalsinanindividualanimalisusuallyfairlysimple.Theproductionofpotentlybioactivecompoundsinlargeabundance(>0.1%ofanimaldryweight)hasenabledthediscoveryoftensofthousandsofcompounds,someofwhichareclinicallyuseddrugs(39).Becausemanyofthecompoundsresemblebacterialmetabolites,chemistsspeculatedthatthetrueoriginofcompoundsmightbebacterial.Laterworkrelyingongeneticsandmetagenomicsidentifieduncultivatedbacteriaaskeyproducersofdefensivemetabolites(40,41).Thesymbiosesappeartobeobligateandspecies-specific.Todate,noneofthesedefensivesymbiontshasbeenstablycultivated,possiblybecauseofthelongrelianceonhostmetabolism.

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Ontheotherendofthespectrum,humansalsocontainmanybacteriathatproducepotentiallybioactivesecondarymetabolites(15,42).Mostofthesehavecultivatedrepresentatives.Thebacteriaarenotobligateandarehighlyvariablewithinourspecies.Becausetheresultingcompoundsarelikelypresentinlowabundancewithinaverycomplexmicrobiota,itisdifficulttostudythebiologyofsecondarymetabolisminhumans.Here,wedescribeasymbioticsystemthatisintermediatebetweenthesetwoextremes.Thestorybeganwiththebiologyofshipworms,inwhichcellulolyticbacteriawerelongknowntospecificallyinhabitgillsandhypothesizedtobethecauseofanevolutionarypaththatleadstowood-specializationinmostofthefamily,alongwithdrasticmorphophysiologicalmodifications(1,5,43).Thesesymbiontscouldbecultivated,althoughonlyrecentlyhavewebeenabletosamplethefullspectrumofmajorsymbiontspresentingills.TheunexpectedfindingthatT.turneraeT7901wasexceptionallyrichinBGCs–proportionatelydenserinBGCcontentthanStreptomycesspp.(13,15)–ledustoinvestigateshipwormsasasourceofnewbioactivecompounds.Likeaconsiderableportionofthehumanmicrobiota,shipwormsymbiontsareamenabletocultivation.Here,weshowthat,likedefensivesymbiosesinseveralmarineinvertebrates,thegillmicrobiotaisrelativelysimple,leadingtoarelativelydefinedsuiteofpotentialmetabolites.TheBGCsincultivatedisolatesarefaithfullyfoundasmajorpathwayswithintheshipwormgillmetagenome.Theabilitytoexperimentallymanipulatethegillcommunitywillprovideagoodsystemtounderstandtheroleofsecondarymetabolisminanimalsymbioses.OnlyfourGCFsarewidelyconservedincellulolyticshipworms.Twoofthesepathwayshaveobviouspotentialrolesinsymbiosis.Siderophoressuchasturnerbactinareimportantinsequesteringiron,buttheyarealsowidelyusedinbacterialcompetition(34,35).Hopanoidsareknowntobeimportantinsymbiosis(44).Twoofthepathways(GFC_2andGCF_3)areexcitingintermsofnewchemistryandbiology.Theseencodeverycomplexbiosyntheticpathwaystotrans-ATpolyketides(45).Suchcompoundsareoftenpotentlyactiveagainsthumandiseasetargets.Insymbiosesbetweenfungiandbacteria,trans-ATPKSproductsarehighlytoxicandlikelydefendtheholobiontfrompredation(46).Thus,isolationofthesecompoundsisapriorityofourproject.Manypathwaysarespecifictocertaincladesofshipwormsymbionts,andthereforetothehostanimalsthatharborthem.Thesealsoencodeverycomplexmetabolites,mostofwhicharepolyketides,nonribosomalpeptides,orhybridsofthetwo.TherearemanyothersuchcomplexpathwaysbeyondthePKS/NRPS,includingtabtoxin-likepathwaysinspecificshipworms.Mostofthesehaveyettobecharacterized,withtheexceptionofthetartrolonD/EpathwaythatisfoundinmanyT.turnerae-containingshipworms.Tartrolonsarepicomolarantiparasiticcompounds(37),implyingthattheymaybeimportantinshapingthemicrobialenvironmentinhosts.ThespecificityofBGCstohosttypessuggestthatthesepathwaysareimportanttothebiologyofsymbiosis.Finally,thereisalargedegreeofstochasticoccurrenceofbiosyntheticpathways.Chemicaldefensivetheorysuggeststhatdifferentiationordiversityofsecondarymetabolitesincreases

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fitness(47).Thismaybeespeciallyimportantinhighlybiodiverseenvironmentssuchasthosefoundinmangrovehabitats.ThisexperimentalsystemwillenableustoexaminetheeffectsofthesevariableBGCs,aswellasthecommon/universalBGCs,onthebiologyoftheshipwormhosts.Insummary,hereweshowthatthebiosyntheticrichnessofcultivatedshipwormsymbiontsisalsofoundinthegillsofthehostanimals.Theseresultsrevealpotentiallyimportantchemicalinteractionsthatwouldaffectavarietyofmarineecosystemsandanovelandunderexploredsourceofbioactivemetabolitesfordrugdiscovery.MethodsCollectionandprocessingofbiologicalmaterial.Shipwormsamples(TablesS1andS2)werecollectedfromfoundwood.Briefly,infestedwoodwascollectedandtransportedimmediatelytothelaboratoryorstoredintheshadeuntilextraction(<1day).Specimenswerecarefullyextractedtoavoiddamageusingwoodworkingtools.Extractedspecimenswereprocessedimmediatelyorstoredinindividualcontainersoffilteredseawaterat4°Cuntilprocessing.Specimenswerecheckedforviabilitybysiphonretractioninresponsetostimulationandobservationofheartbeat,andlivespecimensselected.Specimenswereassignedauniquecode,photographedandidentified.Specimensweredissectedusingadissectingstereoscope.Taxonomicvouchers(valves,pallets,andsiphonaltissueforsequencinghostphylogeneticmarkers),wereretainedandstoredin70%ethanol.Thegillwasdissected,rinsedwithsterileseawater,anddividedforbacterialisolationandmetagenomicsequencing.Oncethegillwasdissecteditwasprocessedimmediatelyorflash-frozeninliquidnitrogen.BacterialDNAextractionandanalysis.Teredinibacterturneraestrains(withTprefix)wereisolatedusingthemethoddescribedinDistelelal.2002(12),whileBankiasetaceasymbionts(withBsprefix)wereobtainedusingthetechniqueindicatedinO’Connoretal.2014(9).Sulfur-oxidizingsymbiontswereisolatedusingtheprotocolspecifiedinAltamiaetal.2019(21).Forthisstudy,additionalT.turneraeandnovelcellulolyticsymbiontsfromPhilippinespecimens(withprefixPMS)wereisolated(TablesS1andS2).Briefly,dissectedgillorcecawerehomogenizedinsterile75%naturalseawaterbufferedwith20mMHEPES,pH8.0usingaDouncehomogenizer.Tissuehomogenateswereeitherstreakedonshipwormbasalmediumcellulose(5)plates(1.0%BactoAgar)orstabbedintosoftagar(0.2%BactoAgar)tubesandincubatedat25°Cuntilcellulolyticclearingsdeveloped.Cellulolyticbacterialcoloniesweresubjectedtoseveralroundsofrestreakingtoensureclonalselection.Contentsofsoftagartubeswithclearingswerestreakedonfreshcelluloseplatestoobtainsinglecolonies.Purecolonieswerethengrownin6mLSBMcelluloseliquidmediumin16×150mmtesttubesuntilthedesiredturbiditywasobserved.Forlong-termpreservationoftheisolates,aturbidmediumwasaddedto40%glycerolat1:1ratioandfrozenat-80°C.Bacterialcellsintheremainingliquidmediumwerepelletedbycentrifugationat8,000gandthensubjectedtogenomicDNAisolation.Thesmall-subunitribosomal(SSU)16SrRNAgeneoftheisolateswasthenPCRamplifiedusing27Fand1492RfromthepreparedgenomicDNAandsequenced.Phylogeneticanalysesof16SrRNAsequenceswasperformedusingprogramsimplementedinGeneious,

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version10.2.3.Briefly,sequenceswerealignedusingMAFFT(version7.388)byusingtheE-INS-ialgorithm.Thealignedsequencesweretrimmedmanually,resultinginafinalaligneddatasetof1,125nucleotidepositions.PhylogeneticanalysiswasperformedusingFastTree(version2.1.11)usingtheGTRsubstitutionmodelwithoptimizedGamma20likelihoodandratecategoriespersitesetto20.GenomicDNAusedforwholegenomesequencingofnovelisolatesandselectT.turneraestrainswerepreparedusingCTAB/phenol/chloroformDNAextractionmethoddetailedinhttps://www.pacb.com/wp-content/uploads/2015/09/DNA-extraction-chlamy-CTAB-JGI.pdf.ThepurityoftheextractedgenomicDNAwasthenassessedspectrophotometricallyusingNanodropandthequantitywasestimatedusingagarosegelelectrophoresis.SamplesthatpassedthequalitycontrolstepsweresubmittedtoJointGenomeInstitute–DepartmentofEnergy(JGI-DOE)forwholegenomesequencing.ThesequencingplatformandassemblymethodusedtogeneratethefinalisolategenomesequencesusedinthisstudyaredetailedonTableS1.MetagenomicDNAextraction.GilltissuesamplesfromPhilippineshipwormspecimens(TableS2)wereflash-frozeninliquidnitrogenandstoredat-80°Cpriortoprocessing.BulkgillgenomicDNAwaspurifiedbyQiagenBloodandTissueGenomicDNAKitusingthemanufacturer’ssuggestedprotocol.GilltissuesamplesfromBrazilshipwormspecimenswerepulverizedbyflash-frozeninliquidnitrogenandsubmittedtometagenomicDNApurificationbyadaptingaprotocolpreviouslyoptimizedfortotalDNAextractionfromcnidariatissues(48,49).Briefly,shipwormsgillswerecarefullydissected(takingcarenottogetintersectionswithotherorgans),submittedtoaseriesoffivewasheswith3:1sterileseawater/distilledwaterforremovalofexternalcontaminants,andmacerateduntilpowderedinliquidnitrogen.Powderedtissues(~150mg)werethentransferredto2mLmicrotubescontaining1mLoflysisbuffer[2%(m/v)cetyltrimethylammoniumbromide(SigmaAldrich),1.4MNaCl,20mMEDTA,100mMTris-HCl(pH8.0),withfreshlyadded5μgproteinaseK(v/v;Invitrogen),and1%2-mercaptoethanol(SigmaAldrich)]andsubmittedtofivefreeze-thawingcycles(-80°Cto65°C).Proteinswereextractedbywashingtwicewithphenol:chloroform:isoamylalcohol(25:24:1)andoncewithchloroform.MetagenomicDNAwasprecipitatedwithisopropanoland5Mammoniumacetate,washedwith70%ethanol,andelutedinTEbuffer(10mMTris-HCl,1mMEDTA).MetagenomiclibrarieswerepreparedusingtheNexteraXTDNASamplePreparationKit(Illumina)andsequencedwith600-cycle(300bppaired-endruns)MiSeqReagentKitsv3chemistry(Illumina)attheMiSeqDesktopSequencer.Metagenomesequencingandassembly.BankiasetaceametagenomeswereobtainedfromtheJGIdatabase(foraccessionnumbers,seeTableS1).Kuphuspolythalamiusgillmetagenomes(KP2132GandKP2133G)wereobtainedfromapreviousstudy(7).MetagenomesfromKuphussp.specimenKP3700GandDicyathifermanniiandBactronophorusthoracitesspecimensweresequencedusinganIlluminaHiSeq2000sequencerwith~350bpinsertsand125bppaired-endrunsattheHuntsmanCancerInstitute’sHighThroughputGenomicsCenterattheUniversityof

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Utah.IlluminafastqreadsweretrimmedusingSickle(50)withtheparameters(pesanger-q30–l125).ThetrimmedFASTQfileswereconvertedtoFASTAfilesandmergedusingthePerlscript‘fq2fq’inIBDA_udpackage(51).MergedFASTAfileswereassembledusingIDBA_udwithstandardparametersintheCenterforHighPerformanceComputingattheUniversityofUtah.FormetagenomesamplesfromBrazil,allNeroterdoreyneigillmetagenomicsamplespreviouslyanalyzedwerere-sequencedheretocoveragedepth(25).Teredosp.andBankiasp.gillmetagenomesweresequencedusingIlluminaMiseq.TherawreadswereassembledusingeitherthemetaspadespipelineofSPAdes(52,53)orIDBA-UD(51).RawreadsweremergedusingBBMerge(54).Non-mergedreadswerefilteredandtrimmedusingFaQCs(55).Bothmergedandprocessednon-mergedreadswereusedinassemblyusingthemetaspadespipeline.Identificationofbacterialsequencesinmetagenomicdata.Assembly-assistedbinningwasusedtosortandanalyzetrimmedreadsandassembledcontigsintoclustersputativelyrepresentingsinglegenomesusingMetaAnnotator(56).EachbinnedclusterwasretrievedusingSamtool(57,58).Toidentifybacterialclusters,genesforeachbinwereidentifiedwithProdigal(59).Proteinsequencesforbinswithcodingdensity>50%weresearchedagainstNCBInrdatabasewithDIAMOND(60).Binswith60%ofthegeneshittingbacterialsubjectinthenrdatabasewereconsideredtooriginatefrombacteria.Eachbacterialbinwascomparedtothe23shipwormisolategenomesusinggANIandAFvalues(61).Withacut-offofAF>0.6andgANI>0.95,thebacterialbinsfromeachmetagenomeweremappedtocultivatedbacterialgenomes.Binsthatmappedtoasinglebacterialgenomewerecombinedintoamega-bin.Readsmappingtoeachmega-binwereretrievedusingMetaAnnotator.FormetagenomesamplesfromBrazil,structuralandfunctionalannotationswerecarriedoutusingDFAST(62),includingonlycontigswithlength≥500bp.AllmetagenomeswerebinnedusingAutometa(63).First,eachcontig’staxonomicidentitywaspredictedusingmake_taxonomy_table.py,includingonlycontigs≥1000bp.Predictedbacterialandarchaealcontigswerebinned(withrecruitmentviasupervisedmachinelearning)usingrun_autometa.py.BuildingBGCsimilaritynetworks.BGCswerepredictedfromthebacterialcontigsofeachmetagenomeandfromcultivatedbacterialgenomesusingantiSMASH4.0(27).Fromthepredictions,onlyBGCsforPKSs,NRPSs,siderophores,terpenes,homoserinelactones,andthiopeptides(aswellascombinationsofthesebiosyntheticenzymefamilies)wereincludedinsucceedinganalyses.Anall-versus-allcomparisonoftheseBGCswasperformedusingMultiGeneBlast(28)followingtheprotocolpreviouslyreported(64).BidirectionalMultiGeneBlastBGC-to-BGChitswereconsideredtobereliable.Inmetagenomedata,sometruncatedBGCsonlyshowedsingle-directionalcorrelationtoafulllengthBGC.Thosesingle-directionalhitswererefinedasfollows:proteintranslationsofallcodingsequencesfromtheBGCswerecomparedinanall-versus-allfashionusingblastpsearch.Onlyproteinhitsthathadatleast60%identityandatleast80%coveragetobothqueryandsubjectwereconsideredasvalidhits.Asingle-directionalMultiGeneBlastBGC-to-BGChitwasretainediftherewereatleastn-2numberofproteins(nisthenumberofproteinsinthetruncatedBGC)passingthe

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blastprefining.TheremainingMultiGeneBlasthitswereusedtoconstructanetworkinCytoscape(65).Finally,eachBGCcluster(GCF)thathasrelativelownumberofbidirectionalcorrelationsweremanuallycheckedbyexaminingtheMultiGeneBlastalignment.OccurrenceofGCFsinmetagenomes.BasedontheGCFsidentifiedinpreviousstep,thecorebiosyntheticproteinsfromeachGCFwereextractedandqueried(NCBItblastn)againsteachmetagenomeassembly.Athresholdofquerycoverageof>50%andidentity>90%wasappliedtoremovethenonspecifichits,andtheremininghits,incombinationwiththeMultiGeneBlasthits,wereusedtomakethematrixofGCFsoccurrenceinmetagenomes.Acknowledgments.AllcollectionsfollowedNagoyaProtocolrequirements;BraziliansamplingwereperformedunderSISBIOlicensenumber48388,andgeneticresourcesaccessedundertheauthorizationoftheBrazilianNationalSystemfortheManagementofGeneticHeritageandAssociatedTraditionalKnowledge(SisGenpermitnumberA2F0DA0).WethanktheGenomicsandBioinformaticsCenterofDrugResearchandDevelopmentCenterofFederalUniversityofCearafortechnicalsupport.TheworkwascompletedundersupervisionoftheDepartmentofAgriculture-BureauofFisheriesandAquaticResources,Philippines(DA-BFAR)incompliancewithallrequiredlegalinstrumentsandregulatoryissuancescoveringtheconductoftheresearch.AllPhilippinespecimenswerecollectedunderGratuitousPermitnumbersFBP-0036-10,GP-0054-11,GP-0064-12,GP-0107-15,andGP-0140-17.WethankthegovernmentsandmunicipalitiesofthePhilippinesandBrazilforaccessandhelp.ThisworkwasalsosupportedbytheNationalCounselofTechnologicalandScientificDevelopment(CNPq)(http://cnpq.br)andbytheCoordinationfortheImprovementofHigherEducationPersonnel(CAPES)(http://www.capes.gov.br)underthegrantnumbers473030/2013-6and400764/2014-8toAETSResearchreportedinthispublicationwassupportedbytheFogartyInternationalCenteroftheNationalInstitutesofHealthunderAwardNumberU19TW008163.ThecontentissolelytheresponsibilityoftheauthorsanddoesnotnecessarilyrepresenttheofficialviewsoftheNationalInstitutesofHealth.TheworkwassupportedinpartbyUSNOAAOERaward#NA190AR0110303References1. DistelDL,etal.(2011)MolecularphylogenyofPholadoideaLamarck,1809supportsa

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Figure1.

Figure1.Top,diagramofgenericshipwormanatomy.InsetsarefromBetcheretal.,PLoSOne,2012Figure2,panelsBandD,andareequalmagnification(8).Red:signalfromafluorescentuniversalbacterialprobeindicatinglargenumbersofbacterialsymbiontsinthebacteriocytesofthegill,andpaucityofbacteriainthececum.Greenisbackgroundfluorescence.Bottom,collectionlocationsofspecimensincludedinthisstudy.

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Figure2.Cultivatedbacterialisolatesrepresentthemajorshipwormgillsymbionts.A)Isolatedbacteriaanalyzedinthisstudyareshowninabstractedschematicofa16SrRNAphylogenetictree.ThecompletetreewithaccuratebranchlengthsandbootstrapnumbersisshowninFigureS1.T.turnerae,withinGroup1comprised11sequencedstrains,forothergroupsindividualstrainsareshown.EachcolorindicatesdifferentbacteriaappearinginthemetagenomesinB.B)Speciescompositionofshipwormgillsymbiontcommunitybasedonshotgunmetagenomesequenceanalysis.They-axisindicatesthepercentofreadsoriginatingfromeachbacterialspecies,whilethex-axisindicatesindividualshipwormspecimensusedinthestudy.Colorsindicatetheoriginofbacterialreads;grayisminor,sporadic,unidentifiedstrains.Formoredetails,seefigureS1

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Figure3.MostBGCsfoundinthemetagenomesandinthebacterialisolategenomesareshared.401BGCsfrommetagenomesequenceswerecomparedtothebacterialisolategenomes,ofwhich305couldbefoundinisolates.Conversely,148of168BGCsfromsequencedbacterialisolatescouldbefoundinthemetagenomes.Thesharednumberslikelydifferbecausethecontigsassembledfromthemetagenomesequenceswereshorteronaverage,sothatseveralmetagenomefragmentsmaymaptoasingleBGCinanisolate.

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Figure4.GCFsfoundinA)bacterialgenomesandB)gillmetagenomes.A)AlistofGCFsfoundincultivatedbacterialgenomesisprovidedinthex-axis,whilethenumberoftimesthattheGCFoccursindifferentsequencedstrainsisshowninthey-axis.ColorsindicatebacteriafromFigure2A.Becausethereare11isolatesofaT.turneraeinGroup1,thenumberofGCFsinthisgroup(darkbluebars)arecomparativelyoverrepresentedinthediagram.B)GCFs(x-axis)foundineachmetagenome(y-axis)areshown.TheinsetexpandsaregioncontainingthemostcommonGCFsfoundinourspecimens.Colorsindicateshipwormhostspecies.SeeTableS3foracompletelistofGCFsusedinthisfigure.

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Figure5.ApossibletabtoxinpathwayisfoundintheD.mannimetagenome.Tabtoxinisaphytotoxinβ-lactaminitiallydiscoveredinPseudomonasspp.(top).Strain2719Kcontainedatabtoxin-likeclusterthatwaspseudogenized(shownasaninsertionintabB;middle).Anon-pseudogenizedtabtoxin-likeclusterwasfoundintheD.mannimetagenomegill(bottom)supportingtheobservationthatmultiplevariantsofeachsymbiontgenomearerepresentedineachmetagenome.

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Figure6.GCFdistributionacrossshipwormspecies.Shownisasimilaritynetworkdiagram,inwhichcirclesindicateindividualBGCsfromsequencedisolates(gray)andgillmetagenomes(colorsindicatespeciesoforigin;seelegend).LinesindicatetheMultiGeneBlastscoresbetweenidentifiedBGCs,withthinnerlinesindicatingalowerdegreeofsimilarity.Forexample,theclusterlabeled“GCF_8”encodesthesiderophoreturnerbactin,thestructureofwhichisshownatright.Themaincluster,circledbyalightblueoval,includesBGCsthatareverysimilartotheoriginallydescribedturnerbactingenecluster.MoredistantlyrelatedBGCs,withfewerlinesconnectingthemtothemajoritynodesinGCF_8,mightrepresentothersiderophores.GCF_11likelyallrepresenttartrolonD/E,aboronatedpolyketideshownatright.FordetailedalignmentsofBGCs,seeFig.S3.

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Figure7.PatternofoccurrenceofmostfrequentlyobservedGCFsinisolatesandmetagenomes.ThevaluesineachboxindicatetheBGCoccurrenceperspecimenforeachGCF.Forexample,GCF_5occursintwooutofthreeTeredosp.specimens.Whenthenumberisgreaterthan1,asfoundwithGCF_3,thisresultsfromhavingmorethanoneofthegeneclustertypespresentinthemetagenome(seeFig.8).Whenthenumberequals1,theGCFisfoundinallspecimenssampled.ThesedatawerecompiledfromanalysisofGFCsinindividualbacterialstrainsandsamples(seeFig.S4).

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Figure8.ThreetypesofGCF_3geneclustersaredistributedinallcellulolyticshipwormsinthisstudy.tBLASTxwasusedtocomparetheclusters,demonstratingthepresenceofthreecloselyrelatedGCF_3genefamiliesfoundinallcellulolyticshipwormgills.

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SupportingInformation.SupportingTables:SeeattacheddocxfilesforTablesS1-S3.

FigureS1.Phylogenyofshipwormgillsymbiontsandrelatedfree-livingbacteriabasedonapproximatemaximum-likelihoodtreeof16SrRNAsequences.Thetreewasreconstructedusing1,125nucleotidepositionsemployingGTRsubstitutionmodelinFastTreeversion2.1.11withoptimizedGamma20likelihoodandratecategoriespersitesetto20.Supportvaluesareindicatedforeachnode.Thescalebarrepresentsnucleotidesubstitutionratepersite.Cultivatableshipwormsymbiontsandrelatedbacteriaareinboldface.TheexcerptedversionofthistreeisshowninFigure2A.

T. turnerae PMS-991H.S.0a.06 from Lyrodus pedicellatusT. turnerae T8513 from Teredo navalis (KF959891)T. turnerae from Lyrodus sp. PMS-1133Y.S.0a.04T. turnerae PMS-1675L.S.0a.01 from Kuphus polythalamiusT. turnerae T0609 from Lyrodus pedicellatus (EU604079)T. turnerae T7902 from Lyrodus pedicellatus (NR_027564)T. turnerae T8402 from Teredora malleolus (KF959886)T. turnerae T8412 from Lyrodus bipartitus (KF959887)T. turnerae T7901 from Bankia gouldi (EU604078)T. turnerae T8415 from Bankia gouldi (KF959888)T. turnerae T8602 from Dicyathifer mannii (EU604077) PMS-1120W.S.0a.04 from Teredo fulleri

PMS-2753L.S.0a.02 from Infanta Bactronophorus thoracites PMS-2052S.S.stab0a.01 from Butuan Bactronophorus thoracites

Bsc2 from Bankia setacea (KJ836296) OTU 07 from Bankia setacea (KJ836286)

OTU 11 from Bankia setacea (KJ836290) OTU 06 from Bankia setacea (KJ836285)

OTU 10 from Bankia setacea (KJ836289) Bs12 from Bankia setacea (KJ836295)

Bs08 from Bankia setacea (KJ836294) Bs31 from Bankia setacea

Bs02 from Bankia setacea (KJ836293) OTU 09 from Bankia setacea (KJ836288)

OTU 13 from Bankia setacea (KJ836292) OTU 15 from Bankia setacea (KJ836284)

OTU 12 from Bankia setacea (KJ836291) OTU 08 from Bankia setacea (KJ836287)

Endosymbiont RT17 of Lyrodus pedicellatus (DQ272304) Endosymbiont RT18 of Lyrodus pedicellatus (DQ272313)

Symbiont LP3 of Lyrodus pedicellatus (AY150578) Endosymbiont RT14 of Lyrodus pedicellatus (DQ272315) Endosymbiont RT24 of Lyrodus pedicellatus DQ272312

Symbiont LP1 of Lyrodus pedicellatus (AY150183) Endosymbiont RT20 of Lyrodus pedicellatus (DQ272307)

PMS-1162T.S.0a.05 from Lyrodus sp.Agarilytica rhodophyticola 017 (KR610527)

PMS-1081L.S.0a.03 from Bankia sp. Symbiont LP2 of Lyrodus pedicellatus (AY150184)

Saccharophagus degradans 2-40 (AF055269)Cellvibrio japonicus NCIMB 10462 (AF452103)

Cellvibrio mixtus ACM 2601 (AF448515)Sedimenticola thiotaurini SIP-G1 (JN882289)

Sedimenticola selenatireducens AK4OH1 (AF432145)Thiosocius sp. PMS-2719K.STB50.0a.01 from Dicyathifer mannii

Thiosocius teredinicola PMS-2141T.STBD.0c.01a from Kuphus polythalamius (KY643661) Endosymbiont Alviniconcha sp. Lau Basin (AB235229)

Endosymbiont of scaly-foot snail (AP012978) Sulfur-oxidizing bacterium ODIII6 (AF170422)

Candidatus Thiobios zoothamnicoli (EU439003) Ectosymbiont Zoothamnium niveum (AB544415)

Acidothiobacillus ferrooxidans ATCC 23270 (NC_011761)

0.98

0.99

0.90

0.99

0.880.92

0.90

0.67

0.90

0.94

0.83

11

0.930.97

0.97

0.73

0.98

0.980.91

0.900.89

0.620.95

1

0.250.57

0.81

1

0.97

0.99

1

1

0.900.99

0.990.89

0.93

0.99

0.99

0.050

Order Cellvibrionales

Order Chromatiales

Outgroup

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FigureS2.Strainvariationinshipwormgillsymbiontbacterialspecies.ThisfigurewasmadeaspreviouslyreportedforKuphussymbionts(7),usingDNAgyraseBin10bpframesandexaminingSNPvariation.DifferentcolorsindicatereadswithdifferentSNPsalongthegyrasesequence.ThespecificexampleshownisfromthegillofB.thoracitesspecimen2771.Theresultsindicatethat,eventhoughCellvibrionales2753ListhemajorspeciespresentinB.thoracites2771,thereareatminimum2majorand4minorstrainvariantsrelatedto2753L.They-axisrepresentsnumberofreadsobserved,whilethex-axisindicateseach10bpregion.

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FigureS3.RepresentativealignmentsshowingactualdataunderlyingtheclustersshowninFigures3,4,6,and7.A)representativealignmentofGCF_3fromgenomesandmetagenomes.Threesubtypeswereindicatedbyredblueandgreencolors;forexample,theNR03metagenomecontainstwocopiesofbluesubtype.DM2858GandDM2722Gcontainblueandredsubtypes.B)alignmentofGCF_2.C)alignmentofGCF_5.D)alignmentofGCF_8.

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FigureS4.OccurrenceofGCFsinindividualsamples,expandingwhatisshowninFig.7.A)GCFsfoundinbacterialstrains.B)GCFsfromindividualshipwormspecimens.A

Ga0198945

BS12

BS02

BS08

2052S

1162T

2719K

2141T

BSC2

BS31

T8602

991H

T7901

T0609

T8412

T8402

T8415

T7902

1133Y

T8513

1675L

1120W

2753L

GCF_8GCF_3GCF_11GCF_9GCF_1GCF_4GCF_5GCF_2GCF_22GCF_33GCF_119GCF_17GCF_25GCF_77GCF_31GCF_14GCF_6GCF_10GCF_30GCF_16GCF_12GCF_13GCF_122GCF_117GCF_76GCF_79GCF_74GCF_106GCF_105GCF_66GCF_58GCF_60GCF_120GCF_113GCF_51GCF_49GCF_50GCF_35GCF_80GCF_114GCF_75GCF_34GCF_52GCF_27GCF_115GCF_24GCF_73GCF_7GCF_116GCF_20GCF_23

group groupgroup1group2group3group4group5group6

0

0.2

0.4

0.6

0.8

1

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B

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35

TableS3.ListofGCFsfoundinthisstudy.GCF_1 cf_fatty_acid-t1pks-

nrps GCF_62 terpene

GCF_2 bacteriocin-transatpks-t1pks-nrps

GCF_63 terpene

GCF_3 cf_fatty_acid-transatpks-t1pks-nrps

GCF_64 terpene

GCF_4 t1pks-cf_saccharide-nrps

GCF_65 terpene

GCF_5 terpene-arylpolyene GCF_66 t1pks GCF_6 transatpks-

cf_saccharide-nrps GCF_67 t1pks

GCF_7 nrps GCF_68 t1pks GCF_8 cf_fatty_acid-

nrps_(tunerbactin) GCF_69 t1pks

GCF_9 t1pks GCF_70 t1pks-PUFA GCF_10 hserlactone-

transatpks-nrps GCF_71 t1pks-nrps

GCF_11 transatpks_(tartrolon) GCF_72 t1pks-nrps GCF_12 transatpks-nrps GCF_73 t1pks-nrps GCF_13 t1pks-nrps GCF_74 t1pks-nrps GCF_14 siderophore GCF_75 t1pks-nrps GCF_15 transatpks-otherks GCF_76 t1pks-cf_saccharide-

nrps GCF_16 t1pks-nrps GCF_77 t1pks-cf_saccharide-

nrps GCF_17 nrps GCF_78 t1pks-cf_fatty_acid GCF_18 transatpks GCF_79 siderophore GCF_19 t1pks GCF_80 nrps-transatpks-otherks GCF_20 nrps GCF_81 nrps GCF_21 transatpks GCF_82 nrps GCF_22 t1pks GCF_83 nrps GCF_23 siderophore GCF_84 nrps GCF_24 nrps GCF_85 nrps GCF_25 nrps GCF_86 nrps GCF_26 transatpks GCF_87 nrps GCF_27 transatpks-otherks-

nrps GCF_88 nrps

GCF_28 nrps GCF_89 nrps GCF_29 nrps GCF_90 nrps GCF_30 nrps GCF_91 nrps GCF_31 nrps GCF_92 nrps

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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36

GCF_32 transatpks GCF_93 nrps GCF_33 transatpks-t1pks-nrps GCF_94 nrps GCF_34 thiopeptide-

hserlactone GCF_95 nrps

GCF_35 t1pks-cf_saccharide-nrps

GCF_96 nrps

GCF_36 t1pks-nrps GCF_97 nrps GCF_37 t1pks-nrps GCF_98 nrps GCF_38 t1pks-cf_saccharide-

nrps GCF_99 nrps

GCF_39 nrps GCF_100 nrps GCF_40 nrps GCF_101 nrps GCF_41 nrps GCF_102 nrps GCF_42 nrps GCF_103 nrps GCF_43 nrps GCF_104 nrps GCF_44 nrps GCF_105 nrps GCF_45 nrps GCF_106 nrps GCF_46 nrps GCF_107 nrps GCF_47 nrps GCF_108 nrps GCF_48 nrps GCF_109 nrps GCF_49 nrps GCF_110 nrps GCF_50 nrps GCF_111 nrps GCF_51 hserlactone-t1pks-

nrps GCF_112 nrps

GCF_52 cf_saccharide-nrps GCF_113 nrps GCF_53 transatpks GCF_114 nrps GCF_54 transatpks GCF_115 nrps GCF_55 transatpks GCF_116 nrps GCF_56 transatpks GCF_117 hserlactone-transatpks-

cf_fatty_acid GCF_57 transatpks GCF_118 hserlactone-nrps GCF_58 transatpks-t1pks-nrps GCF_119 cf_saccharide-nrps GCF_59 transatpks-otherks GCF_120 cf_fatty_acid-t1pks GCF_60 transatpks-

cf_saccharide GCF_121 bacteriocin-lantipeptide

GCF_61 transatpks-cf_fatty_acid

GCF_122 arylpolyene-nrps_(butunamide)

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TableS1.Shipwormgillmetagenomesusedinthisstudy.

#Gillmetagenome

PMS-ICBGsamplecodes

Sourceshipwormspecies

Location Coordinates SequencingcenterSequencingplatform

AssemblerReads,posttrim

SizeinbpNo.ofcontigs

N50 %GCIMGGenomeID

1 DM2722G PMS-2722P DicyathifermanniispecimenPMS-2717Y

Infanta,Quezon,Philippines

N14.68367°,E121.63690°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000 IDBA_ud 187291588 1235295176 924064 2095 34.9

2 BT2771G PMS-2771XBactronophorusthoracitesspecimenPMS-2769U

Infanta,Quezon,Philippines

N14.68367°,E121.63690°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000 IDBA_ud 177392546 1056707310 813604 2023 35.4

3 BT2849G PMS-2849YBactronophorusthoracitesspecimenPMS-2839H

Infanta,Quezon,Philippines

N14.68367°,E121.63690°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000 IDBA_ud 193099534 1059162705 814617 2024 35.4

4 DM2858G PMS-2858W DicyathifermanniispecimenPMS-2823T

Infanta,Quezon,Philippines

N14.68367°,E121.63690°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000 IDBA_ud 186697500 1236681788 928980 2083 34.9

5 DM3770G PMS-3770U DicyathifermanniispecimenPMS-3768S

Infanta,Quezon,Philippines

N14.68367°,E121.63690°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000 IDBA_ud 297553066 1328488478 1067922 1946 34.9

6 BT3790G PMS-3790S

BactronophorusthoracitesspecimenPMS-3779S

Infanta,Quezon,Philippines

N14.68367°,E121.63690°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000

IDBA_ud 309554332 1127330840 927279 1873 35.4

10 KP3700G PMS-3700MKuphussp.specimenPMS-3696Y(wood-boring)

Mabini,Batangas,Philippines

N13.75843°,E120.92586°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000 IDBA_ud 82015762 734092095 358482 4300 37.6

11 KP2132G PMS-2246KandPMS-2249P

KuphuspolythalamiusspecimenPMS-2132W(mud-dwelling)

Kalamansig,SultanKudarat,Philippines

N6.53631°,E124.048365°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000 IDBA_ud 318294870 772720664 424816 4530 37.6

12 KP2133G

PMS-2157H,PMS-2116M,andPMS-2110W

KuphuspolythalamiusspecimenPMS-2133X(mud-dwelling)

Kalamansig,SultanKudarat,Philippines

N6.53631°,E124.048365°

HuntsmanCancerInstitute,UniversityofUtah

IlluminaHiSeq2000 IDBA_ud 329174268 795400237 500141 3879 37.4

13 BSG1 - Bankiasetacea PugetSound,Washington,USA

N47.85072°,W122.33843°

JointGenomeInstitute-DepartmentofEnergy

IlluminaHiSeq2000

SOAPdenovo,Newbler,andMinimus2

- 563042012 761912 985 35.0 3300000111

14 BSG3 - Bankiasetacea PugetSound,Washington,USA

N47.957498°,W122.529373°

JointGenomeInstitute-DepartmentofEnergy

IlluminaHiSeq2000

SOAPdenovo,Newbler,andMinimus2

- 620222960 648493 1550 34.9 3300000024

15 BSG2 - BankiasetaceaPugetSound,Washington,USA

N47.957498°,W122.529373°

JointGenomeInstitute-DepartmentofEnergy

IlluminaHiSeq2000

SOAPdenovo,Newbler,andMinimus2

- 540217764 793976 860 34.8 3300000110

17 BSG4 - BankiasetaceaPugetSound,Washington,USA

N47.85072°,W122.33843°

JointGenomeInstitute-DepartmentofEnergy

IlluminaHiSeq2000

SOAPdenovo,Newbler,andMinimus2

- 574332630 692986 1194 34.6 3300000107

19 BS_sunk - Bankiasetacea PugetSound,Washington,USA

N47.85072°,W122.33843

JointGenomeInstitute-DepartmentofEnergy

Illumina,454GSFLXTitanium

NewblerandVelvet - 26539887 38227 1943 45.2 2070309010

20 NR01 - Neoteredoreynei

CoroagrandeMangrove-Sepetibabay,RiodeJaneiroState,BR

22.9081670°S43.8756390°W CEGENBIO IlluminaMiSeq SPAdes 9224156 313630826 413893 779 37.3

21 NR02 - Neoteredoreynei

CoroagrandeMangrove-Sepetibabay,RiodeJaneiroState,BR

22.9081670°S43.8756390°W CEGENBIO IlluminaMiSeq SPAdes 18338062 416566737 468503 986 37.2

22 NR03 - Neoteredoreynei CoroagrandeMangrove-Sepetiba

22.9081670°S43.8756390°W

CEGENBIO IlluminaMiSeq SPAdes 13078802 309408486 414159 769 38.2

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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bay,RiodeJaneiroState,BR

23 TBF02 - Teredosp.

EnvironmentalPreservationAreaofPacotiriver,CearáState,Brazil

S3.843111,W38.422695(3°50'35.2"S38°25'21.7"W)

CEGENBIO IlluminaMiSeq SPAdes 356571174108472

236018 1037 40.1

24 TBF03 - Bankiasp.

EnvironmentalPreservationAreaofPacotiriver,CearáState,Brazil

S3.843111,W38.422695(3°50'35.2"S38°25'21.7"W)

CEGENBIO IlluminaMiSeq SPAdes 2205607 33524312 75538 1123 41.2

25 TBF05 - Bankiasp.

EnvironmentalPreservationAreaofPacotiriver,CearáState,Brazil

S3.843111,W38.422695(3°50'35.2"S38°25'21.7"W)

CEGENBIO IlluminaMiSeq SPAdes 3632367 107179837 230494 995 37.4

26 TBF07 - Teredosp.

EnvironmentalPreservationAreaofPacotiriver,CearáState,Brazil

S3.843111,W38.422695(3°50'35.2"S38°25'21.7"W)

CEGENBIO IlluminaMiSeq SPAdes 3731031 78684542 258368 965 38.6

27 TBF09 - Teredosp.

EnvironmentalPreservationAreaofPacotiriver,CearáState,Brazil

S3.843111,W38.422695(3°50'35.2"S38°25'21.7"W)

CEGENBIO IlluminaMiSeq SPAdes 4029653 108441874 340072 948 38.1

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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TableS2:Shipwormsymbiontgenomes.# Codeinthe

manuscriptIsolatename Metabolic

typeHostshipworm Location Coordinates Sequencing

centerSequencingplatform

Sequenceassembler

Estimatedgenomesize

No.ofcontigs/scaffolds

N50 %GC IMGGenomeID

1 T7901 T.turneraestrainT7901

Cellulolytic Bankiagouldi Beaufort,NorthCarolinaUSA

N34.71737°,W76.67198°

J.CraigVenterInstitute

454,Sanger CeleraAssemblerandcustomsoftware

5,193,164 1(closedcircular) Notapplicable

50.89 2541046951

2 T8415 T.turneraestrainT8415

Cellulolytic Bankiagouldi FortPierce,Florida,USA

N27.48063°,W80.30967°

JGI-DOE Illumina ALLPATHS 5,158,349 50 ScaffoldN/L50:5/398.1KbpContigN/L50:6/395.4kbp

50.78 2510917000

3 T8602 T.turneraestrainT8602

Cellulolytic Dicyathifermannii Townsville,Queensland,Australia

S19.27631°,E147.05784°

JGI-DOE Illumina ALLPATHS 5,097,488 59 ScaffoldN/L50:6/291.7kbpContigN/L50:2/291.7kbp

51.03 2513237135

4 T7902 T.turneraestrainT7902

Cellulolytic Lyroduspedicellatus

LongBeach,California,USA

N33.76138°,W118.17281°

JGI-DOE Illumina ALLPATHS 5,387,817 72 ScaffoldN/L50:11/176.4kbpContigN/L50:11/176.4kbp

50.81 2513237099

5 T8402 T.turneraestrainT8402

Cellulolytic Teredoramalleolus FloatingwoodintheAtlanticOcean

N38.30667°,W69.59333°

JGI-DOE Illumina Velvet(1.1.04)andALLPATHS-LG

5,166,130 27 ScaffoldN/L50:6/348.4kbpContigN/L50:7/315.4kbp

50.86 2519899652

6 T8412 T.turneraestrainT8412

Cellulolytic Lyrodusbipartitus JimIsland,FortPiece,Florida,USA

N27.476944°,W80.311944°

JGI-DOE Illumina Velvet(1.1.04)andALLPATHS-LG

5,147,360 58 ScaffoldN/L50:10/205.3kbpContigN/L50:10/205.3kbp

51.07 2519899664

7 T0609 T.turneraestrainT0609

Cellulolytic Lyroduspedicellatus

LongBeach,California,USA

N33.76138°,W118.17281°

JGI-DOE Illumina Velvet(1.1.04)andALLPATHS-LG

5,069,061 49 ScaffoldN/L50:7/246.6kbpContigN/L50:7/246.6kbp

51.15 2519899663

8 991H T.turneraestrainPMS-991H.S.0a.06

Cellulolytic LyroduspedicellatusspecimenPMS-988W

Panglao,Bohol,Philippines

N9.54558°,E123.76030°

JGI-DOE Illumina ALLPATHS-LG 5,279,031 13 ScaffoldN/L50:2/1.8MbpContigN/L50:3/888.4kbp

51.07 2524614873

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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9 T8513 T.turneraestrainT8513

Cellulolytic Teredonavalis SãoPaulo,Brazil

S23.81992°,W45.40517°

JGI-DOE Illumina Velvet(1.1.04)andALLPATHS-LG

5,268,281 84 ScaffoldN/L50:9/189.8kbpContig:8/189.8kbp

50.92 2523533596

10 1133Y T.turneraestrainPMS-1133Y.S.0a.04

Cellulolytic Lyrodussp.specimenPMS-1128S

Panglao,Bohol,Philippines

N9.59670°,E123.74990°

JGI-DOE Illumina ALLPATHS-LG 5,134,977 6 ScaffoldN/L50:1/3.2MbpContigN/L50:4/607.0kbp

50.85 2540341229

11 1675L T.turneraestrainPMS-1675L.S.0a.01

Cellulolytic KuphuspolythalamiusspecimenPMS-1672Y

Kalamansig,SultanKudarat,Philippines

N6.53631°,E124.04836°

JGI-DOE PacBio HGAP2.1.1 5,283,781 1(closedcircular) Notapplicable

51.05 2571042908

12 2753L PMS-27553L.S.0a.02 Cellulolytic BactronophorusthoracitesspecimenPMS-2749X

Infanta,Quezon,Philippines

N14.68367°,E121.63690°

JGI-DOE PacBio HGAP2.1.1 6,056,039 2 ScaffoldN/L50:1/4.4Mbp

47.96 2579779156

13 1120W PMS-1120W.S.0a.04 Cellulolytic TeredofullerispecimenPMS-1114L

Panglao,Bohol,Philippines

N9.59670°,E123.74990°

JGI-DOE PacBio HGAP2.0.1 5,699,307 1(closedcircular) Notapplicable

50.39 2558309032

14 2052S PMS-2052S.S.stab0a.01

Cellulolytic BactronophorusthoracitesspecimenPMS-1959H

Butuan,AgusandelNorte,Philippines

N8.98650°,E125.45768°

JGI-DOE Illumina ALLPATHS-LG 5,635,926 3 ScaffoldN/L50:1/5.6MbpContig:3/981.6kbp

54.68 2541046951

15 Bs12 Bs12 Cellulolytic Bankiasetacea PugetSound,Washington,USA

N47.95749°,W122.52937°

JGI-DOE PacBio HGAP2.0.0 4,921,245 3 Contig:1/4.7Mbp

45.72 2545555829

16 Bs08 Bs08 Cellulolytic Bankiasetacea PugetSound,Washington,USA

N47.95749°,W122.52937°

JGI-DOE Illumina Velvetv.DEC-2010

4,814,259 90 ScaffoldN/L50:7/255.3MbpContig:14/112.2kbp

47.18 2767802764

17 Bsc2 Bsc2 Cellulolytic Bankiasetacea PugetSound,Washington,USA

N47.95749°,W122.52937°

NewEnglandBiolabs

PacBio HGAP2.0.1 5,414,953 10 4.2Mbp 47.31 2531839719

18 Bs31 Bs31 Cellulolytic Bankiasetacea PugetSound,Washington,USA

N47.95749°,W122.52937°

JGI-DOE PacBio Velvet1.1.04andALLPATHS-LG

5,017,353 46 ScaffoldN/L50:5/341.1kbpContig:8/260.1kbp

47.60 2528768159

19 Bs02 Bs02 Cellulolytic Bankiasetacea PugetSound,Washington,USA

N47.95749°,W122.52937°

JGI-DOE Illumina Velvetv.DEC-2010

3,886,134 141 Contig:8/176.2kbp

47.76 2503982003

20 1162T PMS-1162T.S.0a.05 Cellulolytic Lyrodussp.specimenPMS-1157K

Talibon,Bohol,Philippines

N10.30748°,E124.40168°

JGI-DOE IlluminaandPacBio

ALLPATHS-LG 4,404,964 1(closedcircular) Notapplicable

47.72 2524614822

21 1081L PMS-1081L.S.0a.03 Agarolytic Bankiasp.specimenPMS-1083P

Panglao,Bohol,Philippines

N9.59670°,E123.74990°

JGI-DOE PacBio HGAP2.1.1 4,255,513 13 ScaffoldN/L50:568.3kbp

53.67 2574179784

22 2141T ThiosociusteredinicolaPMS-2141T.STBD.0c.01a

Sulfur-oxidizing

Kuphuspolythalamius

Kalamansig,SultanKudarat,Philippines

N6.53631°,E124.048365°

JGI-DOE PacBio HGAP2.0.1 4,790,451 1(closedcircular) Notapplicable

60.08 2751185674

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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specimenPMS-2133X

23 2719K Thiosociussp.PMS-2719K.STB50.0a.01

Sulfur-oxidizing

DicyathifermanniispecimenPMS-2715W

Infanta,Quezon,Philippines

N14.68367°,E121.63690°

JGI-DOE PacBio HGAP2.0.1 5,077,565 1(closedcircular) Notapplicable

58.55 2574179721

24 Ga0198945 Agarilyticarhodophyticolastrain017

Agarolytic AssociatedwiththeseaweedGracilariablodgettii

LingshuiCounty,Hainan,China

N18.40828°,E110.0623°

JGI-DOE IlluminaandPacBio

SOAPdenovo2.04;CeleraAssembler8.0

6,878,829 1(closedcircular) Notapplicable

40.97 2751185671

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted October 31, 2019. . https://doi.org/10.1101/826933doi: bioRxiv preprint

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XXX XXXX DEGRADE XXX DEGRADE s/borde TRANSPARENCIA XXX TRANSP. s/borde Posibles combinaciones de autoforma con texto PALETA TNX

XXX XXXX DEGRADE XXX DEGRADE s/borde TRANSPARENCIA XXX TRANSP. s/borde Posibles combinaciones de autoforma con texto PALETA TNX

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