Seeing!the!Light:!the!Origin!and!Evolution!of!Plant!Photoreceptors! by! Fay<Wei!Li! Department!of!Biology! Duke!University! Date:! Approved:!

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1 SeeingtheLight:theOriginandEvolutionofPlantPhotoreceptors by Fay<WeiLi DepartmentofBiology DukeUniversity Date: Approved: KathleenM.Pryer,Supervisor MengChen SönkeJohnsen CorbinJones SarahMathews Dissertationsubmittedinpartialfulfillmentof therequirementsforthedegreeofdoctorofphilosophyinthedepartmentof BiologyintheGraduateSchool ofdukeuniversity 2015

2 ABSTRACT SeeingtheLight:theOriginandEvolutionofPlantPhotoreceptors by Fay<WeiLi DepartmentofBiology DukeUniversity Date: Approved: KathleenM.Pryer,Supervisor MengChen SönkeJohnsen CorbinJones SarahMathews Anabstractofadissertationsubmittedinpartialfulfillmentof therequirementsforthedegreeofdoctorofphilosophyinthedepartmentof BiologyintheGraduateSchool ofdukeuniversity 2015

3 Copyrightby Fay<WeiLi 2015

4 Abstract Plantsuseanarrayofphotoreceptorstomeasurethequality,quantity,anddirectionof lightinordertorespondtoever<changinglightenvironments.photoreceptorsnotonlydetermine howandwhenindividualplantscompletetheirlifecycles,buttheyalsohaveaprofoundand long<termmacroevolutionaryinfluenceonspeciesdiversification.despitetheirsignificances, verylittleisknownaboutphotoreceptorsacrossplantsaswhole,andwelackacomprehensive viewofphotoreceptorevolution. Inmydissertation,Iinvestigatetheoriginandevolutionofthreeofthemostprominent photoreceptorgenefamiliesinplants:phytochromes,phototropinsandneochromes.using newlyavailabletranscriptomicandgenomicdata,icompletedthefirstin<depthsurveyofthese photoreceptorfamiliesacrosslandplants,greenalgae,redalgae,glaucophytes,cryptophytes, haptophytes,andstramenopiles. Phytochromesarered/far<redphotoreceptorsthatplayessentialrolesinseed germination,seedlingphotomorphogenesis,shade<avoidance,dormancy,circadianrhythm, phototropism,andflowering.here,ishowthatthecanonicalplantphytochromesoriginatedina commonancestorofstreptophytes(charophytegreenalgaepluslandplants),andiidentifythe mostlikelysequencewherebytheplantphytochromestructureevolvedfromitsancestral phytochrome.phytochromesincharophytealgaearestructurallydiverse,includingcanonical andnon<canonicalforms,whereasinlandplants,phytochromestructureishighlyconserved. Liverworts,hornworts,andSelaginellaapparentlypossessasinglephytochromegenecopy, whereasindependentgeneduplicationsoccurredwithinmosses,lycopods,ferns,andseed plants,leadingtodiversephytochromefamiliesintheseclades.mydetailedphylogeny iv

5 encompassesallofgreenplantsandenablesmetonotonlyuncovernewphytochromelineages, butalsotomakelinkstoourcurrentunderstandingofphytochromefunctioninarabidopsisand Physcomitrella5(themajormodelorganismoutsideoffloweringplants).Basedonthisrobust evolutionaryframework,iproposenewhypothesesanddiscussfuturedirectionstostudy phytochromemechanisms. Phototropinsareblue<lightphotoreceptorsthatregulatekeyadaptivephysiological responses,includingshoot<positivephototropism,root<negativephototropism,chloroplast accumulation/avoidance,stomatalopening,circadianrhythm,leafexpansion,andseedling elongationishowthatphototropinsoriginatedinthecommonancestorofviridiplantae(all greenalgae[charophytes,chlorophytes,prasinophytes]pluslandplants).phototropins repeatedlyunderwentindependentduplicationsinallmajorplantlineages(mosses,lycopods, fernsandseedplants),exceptforliverwortsandhornworts,wherephototropinisasingle<copy gene.followingeachmajorduplicationevent,phototropinssubsequentlydifferentiatedin parallel,resultingintwospecialized(yetpartiallyoverlapping)functionalformsthatprimarily mediateeitherlow<orhigh<lightresponses.mygenephylogenyfurthersuggeststhat phototropinshaveco<evolvedwithphytochromes,asisevidentfromtheirmolecularinteractions andstrikinglysimilargeneduplicationpatterns.ihypothesizethattheco<evolutionof phototropinswithphytochromes,togetherwiththeirsubsequentconvergentfunctional divergencesinphototropicresponses,contributedtothesuccessofplantsinadaptingtodiverse andheterogeneoushabitats. Neochromesarechimericphotoreceptorsthat,byfusingphytochromeandphototropin modulesintoasingleprotein,areabletousebothred/far<redandbluelighttomodulate v

6 phototropicresponses.neochromeswerefirstdiscoveredinferns,andtheevolutionof neochromeswasimplicatedasakeyinnovationthatfacilitatedferndiversificationunderthe low<lightangiospermcanopies.despiteitssignificancefromanevolutionarystandpoint,the originofneochromeshasremainedamystery.hereipresentthefirstevidenceforneochromein hornworts(abryophytelineage)anddemonstratethatfernsacquiredneochromefromhornworts viahorizontalgenetransfer(hgt).fernneochromesarenestedwithinhornwortneochromesin mylarge<scalephylogeneticreconstructionsofphototropinandphytochromegenefamilies. DivergencedateestimatesfurthersupporttheHGThypothesis,withfernandhornwort neochromesdiverging179mya,longafterthesplitbetweenthetwoplantlineages(atleast400 MYA).ByanalyzingthedraftgenomeoftheAnthocerospunctatushornwort,Ialsodiscovereda novelphototropingenethatlikelyrepresentstheancestrallineageoftheneochromephototropin module.thus,aneochromeoriginatinginhornwortswashorizontallytransferredtoferns,where itmayhaveplayedasignificantroleinthediversificationofmodernferns. Insummary,mystudiesidentifiedthemolecularoriginsofphytochromes,phototropins andneochromes,andreconstructedtheirrespectiveevolutionaryhistories.thisnewframework forphotoreceptorevolutionwillstimulatenewresearchlinkingecology,evolution,and photochemistrytounderstandhowplantsadapttovariablelightenvironments. vi

7 Tomyparentswhoshowedmethewonderofferns vii

8 Contents Abstract...iv ListofTables...xi ListofFigures...xii Acknowledgements...xiv Introduction Theoriginandevolutionofphytochromes Introduction Results Namesforphytochromegenelineages Stramenopilesandhaptophytes Redalgae Glaucophytes Cryptophytes Viridiplantae Neochromes Bryophytes Lycophytes Ferns Seedplants Discussions MaterialsandMethods Transcriptome<andgenome<miningforphytochrome...25 viii

9 1.4.2Sequencealignment Phylogeneticreconstruction Confirminggenecopynumberinhornwortsbytargetenrichment Theoriginandevolutionofphototropins Introduction Results Theoriginofphototropins Phototropinphylogeny Discussions MaterialsandMethods Miningphototropinsfromtranscriptomesandgenomes Sequencealignmentandphylogeneticreconstruction Targetenrichmentforconfirmingphototropincopynumberinhornworts Theoriginandevolutionofneochromes Introduction ResultsandDiscussions Algalneochrome Novelneochromeinhornworts NeochromeHGTfromhornwortstoferns Recurrentfern<to<fernHGT Evolutionaryandphysiologicalimplicationsofneochromeinhornworts Evolutionarysignificanceofplant<to<plantHGT MaterialsandMethods...57 ix

10 3.3.1Miningtranscriptomesandwholegenomesequencesforhomologsofneochrome, phototropinandphytochrome AssemblingandmininganAnthoceros5punctatusdraftgenomeforhomologsof neochrome,phototropinandphytochrome Cloningofneochrome,phototropinandphytochrome Genomewalkinginhornwortphototropinandneochrome Sequencealignmentforneochrome,phototropinandphytochrome Phylogeneticanalysesofphototropinandneochrome Phylogeneticanalysesofphytochrome Topologytest Phylogeneticanalysisofimidazoleglycerol<phosphatedehydratasegene(IGPD) Divergencetimeestimationofthephototropingenefamily InferringepisodicselectionandGCcontentvariationinneochromeevolution...65 AppendixA:SupplementaryFiguresforChapterOne...67 AppendixB:SupplementaryTablesforChapterOne...73 AppendixC:SupplementaryTablesforChapterTwo...78 AppendixD:SupplementaryFiguresforChapterThree...82 AppendixE:SupplementaryTablesforChapterThree...88 x

11 List of Tables Table1:ReclassificationofPhyscomitrella5patensphototropinsbasedongeneorthology...33 Table2:Listoftranscriptomesandgenomesscreenedforphytochromes...73 Table3:SourcesandGenBankaccessionnumbersofthephytochromesusedinphylogenetic analyses...75 Table4:Listoftranscriptomesandgenomesscreenedforphototropins...78 Table5:SourcesandGenBankaccessionnumbersofthephototropinsusedinphylogenetic analyses...80 Table6:Listoftranscriptomesandgenomesequencesscreenedforneochrome,phototropinand phytochromegenes...88 Table7: Thecalibrationsusedindatingthedivergenceofphototropingenefamily...89 Table8:TheprimersandPCRprotocolsusedinthisstudy...90 Table9:TheprimersequencesusedinPCR...91 xi

12 List of Figures Figure1:Plants see lightthroughphotoreceptors.domainstructuresofphytochromes, phototropinsandneochromesareshownontheright...2 Figure2: Theorganismallineagesscreenedforphytochromehomologs...6 Figure3:Phylogenyofphytochrome...8 Figure4:ThediversityandevolutionofphytochromeC<terminaloutputmodule...10 Figure5: Phylogeneticrelationshipofneochromesandphytochromes...13 Figure6: Phytochromephylogenyforbryophytes...16 Figure7: Phytochromephylogenyforfernsandlycophytes...19 Figure8:Organismallineagesscreenedforphototropinhomologs...30 Figure9:Phylogenyofseed<plantandfernphototropins...36 Figure10:Phylogenyoflycophyteandbryophytephototropins...38 Figure11:Phylogenyofalgalphototropins...39 Figure12:Theoriginoffernneochrome...47 Figure13:Phylogeneticrelationshipsoffernneochrome(NEO),hornwortneochromeand phototropin(phot)...51 Figure14: Phylogeneticincongruencebetweenfernneochromegenetreeandfernspeciestree..52 Figure15: Phylogeny,selectionprofileandGCcontentoffernneochromes...54 Figure16: Hornwortchloroplastscontractunderstronglight...56 Figure17:Thephylogenyofphytochromesreconstructedfrom423proteinsequences...67 Figure18:Phylogeneticrelationshipsoflandplantandalgalphototropin(PHOT)andthe correspondingdomainsfromhornwort,fern,andalgalneochrome(neo)...83 Figure19:Phylogeneticrelationshipsoflandplantandalgalphytochrome(PHY)andthe correspondingdomainsfromhornwortandfernneochrome(neo)...85 Figure20:Phylogenyoflandplantimidazoleglycerol<phosphatedehydratase(IGPD)...86 xii

13 Figure21:Chronogramoflandplantandalgalphototropin(PHOT)andthecorresponding domainsfromhornwort,fern,andalgalneochrome(neo)...87 xiii

14 Acknowledgements WhenIlookedbackthepastfiveyears,Irealizedhowmygraduatestudywouldbe impossiblewithoutthegeneroushelpfromsomanypeople.iwouldliketobeginbythanking CarlRothfels,whogotmeinvolvedintheOneThousandPlant(1KP)project,fromwhereIgot mostofmytranscriptomedatafor$freeby,literally,writingafewlinesofbatchdownloading scripts.iamindebtedtoallthe1kpcontributors,andofcoursetothevisionaryleaderganekai ShuWong.IalsowanttothankSteveKelly,EftychiosFrangedakisandJaneLangdaleforsharing theirhornwortgenomicdata,andtojoshderforsharinghispteridiumtranscriptome,bothof whichhavebeeninstrumentaltomystudy. JuanCarlosVillarealhasbeenmybest hornwortbuddy,whohassharedeverybitof hisincrediblehornwortknowledgewithme.daveswoffordtaughtmehowtomakephylogeny treesandthetheoriesbehindit,andsavedmyassmanytimeswhenwetaughtthephylogenetics course.tommitchellioldsintroducedmetotheworldofpython,alanguageicannotlive without.jonshawgavemeacrashcourseonbryophytephylogeny,sothaticanpretendiknow somethingwhendiscussinggeneduplicationsinmosses.paulmanosofferedmetheopportunity toorganizedukesystematicsdiscussiongroup(i.e.sdg),duringwhichigottoknowsome incredibleevolutionarybiologists.laynehuietisawalkingwikipediaofmolecularbiologyand givesthebesttipseverindoinglabexperiments.karlbatesisthewizardofscience communications,andmademefeellikea celebrityofferns foradayortwo. Iappreciatethepeople,professionalsocieties,andfundingagenciesthatbelievedinmy researchpotentialandgavemethefundingtopursue.theseinclude:americansocietyofplant Taxonomists,DukeBiology,NationalScienceFoundation(DEB andGRFP),SigmaXi, xiv

15 SocietyofSystematicBiologists,TorreyBotanicalSociety,andanangeldonorwhosenameIshall notreveal. Iamverygratefultomycommitteemembers.IenjoythediscussionswithMengChen aboutphytochromefunctionandsignaltransduction,andwithcorbinjonesaboutchimericgene evolutionandtheemerginggenomictools.sönkejohnsenpromptedmetothinkaboutplant visionfromanotherangleandofferedmemanyrealisticcareeradvices.mydissertationwork wouldbeawfullytawdrywithoutsarahmathew sguidanceandhercriticalthinking.ihave alwaysfeltenlightenedafterdiscussingphotoreceptorswithsarahoverskype. ThePryerlabisawesome.ErinSigel,AmandaGrusz,CarlRothfels,KathrynPicard,Tzu< TongKaoandLayneHuiethavebeenthegreatestcheerleadersandalwayssosupportive.They havealsobeentirelesslyteachingmenewenglishwords,andfeltembarrassed(orjoyful?)when Iusedthemindevastatinglyinappropriateways. KathleenPryerandMikeWindhamarethebestadvisorsever.Achapterofitsown wouldnotsufficemygratefulness.mikegavemean America101 courseduringatwo<month fieldtripacrosstheus;itwasnotjustonfernsandmustards,butalsoaboutthecultures,history, andpolitics perhapseverythingineededtoknowasa fresh<off<the<boat Taiwanese.Kathleen alwayshasthewildest,unconventionalideas,andienjoydeeplypursuingthecrazinesswithher. Forthesefiveyears,theygavemetheabsolutefreedomtodowhateverIfindinteresting,whichI amterriblygratefulfor Well,that snottrue,butallthestufftheyinflictedonmeturnedoutto beamazinglyawesome FinallyIwanttothankYu<Hsuan,mywifeandmybestfriend,for everything xv

16 Introduction Light5exerts5a5powerful5influence5on5most5vegetable5tissues,5and5there5can5be5no5doubt5that5it5 generally5tends5to5check5their5growth CharlesDarwin,1880 Lightistheultimatesourceofenergyformuchoflifeonearth,andinevitablygoverns thegrowthandphysiologyofphotosyntheticorganisms.plants see lightthrough photoreceptors(figure1).fivephotoreceptorgenefamiliesaregenerallypresentinplants: phytochromes,phototropins,cryptochromes,zeitlupes,anduvr8(möglichetal.,2010;heijde andulm,2012).amongthem,phytochromesandphototropinsareperhapsthemostprominent, giventheirprevalenceincontrollingalmosteveryaspectoftheplantlifecycle:fromthe dormancyandgerminationofseeds/spores,morphogenesisandgrowth,toflowering(franklin andquail,2010;christie,2007).ampleevidencehasshownthatthesetwophotoreceptorsexert anadaptivesignificanceonindividualfitness(galenetal.,2004),localadaptation(ikedaetal., 2009;IkedaandSetoguchi,2010),and/orlong<termmacroevolutionarysuccess(Mathewsetal., 2003).Furthermore,theirchimericderivativeneochrome/(partphytochromeandpart phototropin),wasalsoimplicatedasakeyinnovationthatfacilitatedfernradiationunderlow< lightenvironments(schneideretal.,2004;schuettpelzandpryer,2009;kawaietal.,2003). Tounderstandhowplantsadaptedto,andthrivedin,thediverseenvironmentsthey inhabit,therolesofphotoreceptorscannotbeignored.however,photoreceptorgenesequence datahavebeenscarceandmostlylimitedtoseedplants,thusimpedingdetailedreconstructions ofphotoreceptorevolutionaryhistories.themajorgoalofmydissertationresearchwasto leveragerecentgenomicandtranscriptomicdatatoinvestigatetheoriginandevolutionofthree photoreceptorfamilies:phytochromes(chapter1),phototropins(chapter2),andneochromes (Chapter3). 1

17 Chapter3onneochromeswaspublishedfirst(Lietal.,2014),alongwithpreliminary datafromchapters1and2.morecomprehensiveanalysesonphytochromes(chapter1)and phototropins(chapter2)arebeingpublishedinseparate,dedicatedpapers. UVR8 Cryptochrome Phototropin Zeitlupe LOV LOV PKC Neochrome PAS GAF PHY LOV LOV PKC Phytochrome PAS GAF PHY PAS PAS KD Figure/1:/Plants/ see /light/through/photoreceptors./domainstructuresofphytochromes, phototropinsandneochromesareshownontheright.neochromeisachimericphotoreceptorfusing phytochromeandphototropinmodules,andisabletorespondtobothblueandred/far<redlight.domain names:gaf(cgmpphosphodiesterase/adenylatecyclase/fhla);kd(histidine<kinase<related<domain);pas (Per/Arnt/Sim);PHY(Phytochrome);PKC(ProteinKinaseC). 2

18 1. The origin and evolution of phytochromes Li,F.<W.,M.Melkonian,C.J.Rothfels,J.C.Villarreal,D.W.Stevenson,S.W.Graham, G.K.S.Wong,K.M.Pryer,andS.Mathews.Novel/phytochrome/lineages/and/complex/ evolutionary/histories/revealed/across/extant/plant/diversity.naturecommunications, in5review 1.1 Introduction Phytochromesarered/far<redlightsensors,particularlyprominentfortheircontrolof seedgermination,seedlingphotomorphogenesis,shade<avoidance,dormancy,circadianrhythm, phototropism,andflowering(möglichetal.,2010;rockwelletal.,2006;franklinandquail, 2010).Becauseoftheirbiologicalsignificance,phytochromeshavebeenamajorfocusinplant research.phytochromephotochemistry,function,anditsassociatedsignaltransduction mechanismshavebeeninvestigatedextensively,mostlyusingthemodelfloweringplant Arabidopsis5thaliana5(Möglichetal.,2010;Rockwelletal.,2006;FranklinandQuail,2010;Chenand Chory,2011). CanonicalplantphytochromescompriseanN<terminalphotosensorycoremodule (PCM)andaC<terminalregulatorymodule(FranklinandQuail,2010;Rockwelletal.,2006).The PCMcontainsthreeconserveddomainsinthelinearsequencePAS,GAF,andPHY.Itisessential forlightreceptionandphotoconversionbetweenreversibleconformationsthatabsorbmaximally inthered(650<670nm)orfar<red(705<740nm)regionsofthespectrum,referredtoasprandpfr, respectively.thec<terminalmoduleconsistsofapas<pasrepeatfollowedbyahistidine<kinase< relateddomain(hkrd).thehkrdresemblesahistidinekinasedomainbutlackstheconserved histidinephosphorylationsite,exhibitingserine/threoninekinaseactivityinstead(yehand Lagarias,1998;Fankhauser,2000). 3

19 Plantphytochromesoccurasasmallnuclear<encodedgenefamily,andinseedplants theyfallintothreedistinctclades:phya,phyb/e,andphyc5(mathews,2010).thephylogenetic relationshipsamongthesecladesarewellresolved,allowingfortheformulationoffunctional hypothesesforseed<plantphytochromesbasedontheirorthologywitharabidopsisphytochromes (Mathews,2010).Phytochromediversityinnon<seedplants,however,isverypoorlyunderstood, withthelimitedavailabledatabeingderivedfromthephyscomitrella5(moss)andselaginella (lycophyte)genomeprojects(banksetal.,2011;rensingetal.,2008),andafewcloningstudies (Schneider<Poetschetal.,1994;Nozueetal.,1997;Okamotoetal.,1993;Pasentsisetal.,1998; Suzukietal.,2001).Thelackofacomprehensivephytochromeevolutionaryframeworkforall landplantsisanobstacletounderstandingtheevolutionofphytochromefunctionaldiversity, andmakesitdifficult,forexample,tointerpretcorrectlyresultsfromcomparisonsoffunctionin Arabidopsis5thalianaandPhyscomitrella5patens. Anespeciallyremarkableplantphytochromederivativeisneochrome,achimeric photoreceptorcombiningaphytochromepcmandabluelight<sensingphototropin(nozueet al.,1998).neochromeshavebeendetectedonlyinzygnemetaleanalgae,fernsandhornworts (Suetsuguetal.,2005;Lietal.,2014).Whileithasbeenshownthatthephototropincomponentof neochromeshastwoindependentorigins(oneinzygnemetaleanalgaeandtheotherin hornworts;seechapter3andlietal.,2014),theancestryofthephytochromeportionremains unclear. Inadditiontoplants,phytochromesarepresentinprokaryotes,fungi,andseveral protistanandalgallineages(ulijaszandvierstra,2011;rockwelletal.,2014).these phytochromessharewithcanonicalplantphytochromesthepcmdomainarchitectureatthen< terminal,buttheydifferintheirc<terminalregulatorymodules.prokaryoticandfungal phytochromes,forexample,lackthepas<pasrepeat,andhaveafunctionalhistidine<kinase 4

20 domainwiththeconservedhistidineresidue.recently,rockwelletal.(2014)andduanmuetal. (2014)examinedthephytochromesinseveralalgallineages(brownalgae,cryptophytes, glaucophytes,andprasinophytes),anddiscoveredthatsomeofthemnotonlyexhibitgreat spectraldiversity,butalsohavenoveldomaincombinationswithinthec<terminalmodule. Despitetheseimportantfindings,phytochromesremainunreportedfromthemajorityofalgal lineages.duanmuetal.(2014)proposedthatthecanonicalplantphytochromemayhave originatedamongcharophytealgae,buttheywereunabletoconfirmthis. Inthisstudy,Iinvestigatednewlyavailablegenomicandtranscriptomicresourcesto discoverphytochromehomologsoutsideofseedplants.iexaminedatotalof300genomesand transcriptomesfromferns,lycophytes,bryophytes,charophytes,chlorophytes,and prasinophytes(allinviridiplantae),andfromotherplastid<bearingalgallineages,the glaucophytes,cryptophytes,rhodophytes,haptophytes,andstramenopiles(figure2,table2).i usedthesedatatoreconstructthefirstdetailedphytochromephylogenyfortheeukaryotic branchesofthetreeoflife,andtomapallthemajorgeneduplicationeventsanddomain architecturetransitionsontothisevolutionarytree. 1.2 Results Idiscoveredatotalof350phytochromehomologsin148transcriptomeassembliesand 12whole<genomesequences(Table2,Table3)spanningextantplantandalgaldiversity.Inthe remaining140assembliesandgenomesequences,idetectednophytochromehomologs.i inferredaphytochromephylogenyfromanaminoacidmatrixthatincludedthesequencesi discovered,togetherwithpreviouslypublishedsequencesfromgenbank.toimproveour understandingofphytochromeandneochromeevolution,especiallywithinfernsand bryophytes,ialsoassembledthreenucleotidematrices.thefernandbryophytematrices 5

21 included113and97phytochromesequences,respectively.theneochromematrixincluded16 neochromesand95phytochromesfromselectedbryophytesandcharophytes. Seed plants Ferns Archaeplastida* Viridiplantae Streptophytes Lycophytes Mosses Liverworts Hornworts Desmidiales Zygnematales Coleochaetales Charales Klebsormidiales Mesostigmatales Trebouxiphyceae Ulvophyceae Chlorophyceae Pedinophyceae Prasinophytes Rhodophytes Glaucophytes Cryptophytes Stramenopiles Chlorophytes Charophytes Land plants Haptophytes Figure/2: The/organismal/lineages/screened/for/phytochrome/homologs./Thephylogenetic relationshipswerederivedfromwickettetal.(2014)andmarin(2012).lineagesthatlackphytochromeare ingrey.*traditionalarchaeplastidadoesnotincludecryptophytes. Thetopologiesofthephytochromegenetreescorrespondwellwithpublished organismalrelationships(wickettetal.,2014;kuoetal.,2011;coxetal.,2010;gontcharovand Melkonian,2010;Cavalier<Smithetal.,2014;Burkietal.,2012;GrantandKatz,2014;Marin,2012; VillarrealandRenner,2012;Forrestetal.,2006),allowingmetopinpointthephylogenetic positionsofgeneduplicationeventsanddelineatenovelphytochromeclades.belowireport resultsonphytochromediversity,phylogeneticstructure,anddomainarchitectureinthe stramenopiles,cryptophytesandarchaeplastida(or Plantae :redalgae+glaucophytes+ Viridiplantae;Adletal.,2005). 6

22 1.2.1 Names for phytochrome gene lineages ThehighdiversityofphytochromesIdiscoveredincharophytes,mossesandferns resultingfrommultiple,independentgeneduplications(figure2) demandedasensiblesystem fornamingthegenelineages.withineachmajororganismalgroupofarchaeplastida(except seedplants,whereasystemfornamingphyhasalreadybeenwellestablished),iusednumerical labelsforthephytochromecladesthatresultedfrommajorgeneduplicationevents(e.g.,fern PHY1E4andcharophytePHY1E2).Subcladesresultingfrommorelocalduplicationswerethen namedalphabeticallywithinclades(e.g.,polypodialesphy4aebanddesmidialesphy2aec).it shouldbestressedthatthisalphanumericsystemdoesnotimplyorthologyacrossorganismal groups;forexamplefernphy1sharesalowerdegreeofrelatednesstocharophytephy15thanto fernphy2.charophytephyx1andphyx2weresonamedherebecausetheyarenotcanonical plantphytochromeslikecharophytephy1e2,andtheirevolutionaryoriginislessclear.forthe cryptophytephytochromeswithc<terminalserine/threoninekinase,ifollowedduanmuetal. (2014)andcalledthemphytochromeeukaryotickinasehybrids(PEK) Stramenopiles and haptophytes Stramenopilesarealargeeukaryoticcladethatincludesbrownalgae(suchaskelps), goldenalgae,anddiatoms,thelatterbeinganimportantcomponentofplankton.withinthis group,phytochromesareknownsofaronlyfrombrownalgae,someoftheirviruses,and diatoms.theirsequencesformacladethatissistertofungalphytochromes(figure3,appendix Figure17).Interestingly,thephytochromefromthebrownalgalvirusEsV<1(Delaroqueetal., 2001)doesnotgroupwithbrownalgae5phytochromes,butinsteadismorecloselyrelatedto thoseofdiatoms.thisrelationshipwasnotsupportedinabootstrappinganalysis(appendix Figure17);itwas,however,alsoobtainedbyDuanmuetal.(2014)(butwithoutsupport). 7

23 Summary of phytochrome gene phylogeny Domain architecture Archaeplastida* Inferred gene duplication Origin of canonical plant phytochrome Viridiplantae Streptophytes A Land plants 2/4 H 1 2_4/5 D 1_3 4 I 2 4A J 4B 1 G 2 5A F 5 5B E 2_4 C C B B Angiosperm Gymnosperm Angiosperm Gymnosperm Angiosperm Angiosperm Gymnosperm Polypodiales Polypodiales Cyatheales Salviniales Schizaeales Polypodiales Cyatheales Salviniales Schizaeales Gleicheniales Osmundales Marattiales Ophioglossales Psilotales Ophioglossales Equisetales Polypodiales Cyatheales Salviniales Gleicheniales Osmundales Equisetales Marattiales Lycopodiales Lycopodiales Isoetales Selaginellales Bryopsida Bryopsida Polytrichopsida Bryopsida Polytrichopsida Andreaeopsida Sphagnopsida Takakiopsida Bryopsida Andreaeopsida Jurgemanniopsida Marchantiopsida Notothyladales Dendrocerotales Anthocerotales Hornworts+Ferns Hornworts Zygnematales Desmidales C Desmidales B Desmidales A Zygnematales Coleochaetales Klebsormidiales Desmidiales Klebsormidiales Mesostigmatales Zygnematales Coleochaetales Zygnematales Coleochaetales Prasinophyte Cryptophyte Cryptophyte Glaucophyte Cyanobacteria Diatom Brown algae Fungi Cyanobacteria Bacteria Bacteria PHYA PHYN PHYC PHYO PHYB PHYE PHYP Fern PHY4 Fern PHY2 Fern PHY2/4 Fern PHY1 Lycophyte PHY Moss PHY5 Moss PHY2_4 Moss PHY2_4/5 Moss PHY1_3 Liverwort PHY Hornwort PHY Neochrome Charophyte PHY2 Charophyte PHY1 Charophyte PHY1/2 Charophyte PHYX2 Charophyte PHYX1 Prasinophyte PHY Cryptophyte PHY Cryptophyte PEK Glaucophyte PHY Cyano PAS-less Stramenopile PHY Fungi PHY Bacteria PHY PAS GAF PHY PAS GAF PHY PAS GAF PHY PAS GAF PHY PAS PAS LOV LOV PKC PAS PAS PAS PAS GAF PHY PAS PAS H PKC RING KD KD KD PAS GAF PHY PAS PAS H KD REC PAS GAF PHY PAS H KD REC GAF PHY GAF H KD Phototropin portion PAS GAF PHY H KD REC Figure/3:/Phylogeny/of/phytochrome./Terminalcladesarecollapsedintohighertaxonomicalunits (usuallyordersorclasses)fordisplaypurposes;thedetailedtreeisshowninsupplementaryfig.1.orange circlesindicateinferredgeneduplications.italicizedletterswithineachcirclecorrespondstotheduplication eventmentionedinthetext,andthenumbers/lettersadjacenttoeachorangecirclearethenamesofthegene duplicates.canonicalplantphytochromesoriginatedinthecommonancestorofstreptophyta(greenstar), andsomecharophytesretainnon<canonicalphytochromes(phyx1,phyx2).domainarchitecturesare shownontheright.domainsthatarenotalwayspresentareindicatedbydashedoutlines.domainnames: 8

24 GAF,cGMPphosphodiesterase/adenylatecyclase/FhlA;H/KD,histidinephosphorylationsite(H)inthe histidinekinasedomain(kd);pas,per/arnt/sim;phy,phytochrome;pkc,proteinkinasec;rec, ResponseRegulator;andRING,ReallyInterestingNewGene.*TraditionalArchaeplastidadoesnotinclude cryptophytes(adletal.,2005). Additionalphytochromedatafromstramenopileswillbenecessarytoclarifytheoriginofthese viralphytochromes.ialsoexaminedhaptophytes,apredominantlymarinelineageof phytoplankton(theirrelationshipswithstramenopilesandotherprotistsareunclear(grantand Katz,2014;Burkietal.,2012).Nophytochromecouldbefoundinthehaptophytetranscriptomes Red algae Redalgaearemostlymulticellular,marinespeciesthatincludesmanycorallinereef< buildingalgae.nophytochromeswerefoundinthe28redalgaltranscriptomesiexamined,nor inthepublishedgenomesofporphyridium5purpureum,chondrus5crispus,cyanidioschyzon5merolae, Galdieria5sulphuraria,andPyropia5yezoensis5(AppendixTable2).Thisresult,basedondatafromall Rhodophytaclasses(Yoonetal.,2006),providescompellingevidencefortheabsenceof phytochromesfromredalgae(figure2) Glaucophytes Glaucophytesareasmallcladeoffreshwater,unicellularalgaewithunusualplastids referredtoascyanelles,which,unlikeplastidsinrhodophytesandgreenplants,retaina peptidoglycanlayer(keeling,2004).phytochromesarepresentinglaucophytes,andwhenthe treeisrootedonthebranchtoprokaryote/fungus/stramenopilephytochromes,glaucophyte phytochromesareresolvedassistertocryptophyte+viridiplantaephytochromes(figure3, AppendixFigure17).Glaucophytephytochromes,incontrastwithcanonicalplantphytochromes, haveasinglepasdomaininthec<terminalmodule,andtheconservedhistidineresidueis presentinthekinasedomain,suggestingitretainshistidinekinaseactivity(duanmuetal.,2014). 9

25 Organismal phylogeny Land plants C-terminal output module PAS Canonical plant PHY PAS KD Desmidiales PAS PAS KD Archaeplastida* Viridiplantae Streptophytes Zygnematales Coleochaetales Charales Klebsormidales Mesostigmatales Prasinophytes PAS PAS H KD REC PAS PAS KD PAS PAS H KD REC PAS PAS KD PAS PAS KD PAS PAS KD PAS PAS KD PAS PAS H KD REC Glaucophytes PAS H KD REC Cryptophytes Stramenopiles H KD REC PAS PAS PAS PKC H RING KD REC Fungi H KD REC Cyanobacteria H KD REC Bacteria H KD REC Figure/4:/The/diversity/and/evolution/of/phytochrome/CCterminal/output/module./Thetree depictstherelationshipofallthephytochrome<containinglineages.foreachlineage,thedomain architectureofthec<terminalregulatorymoduleisshownontheright(connectedbydashedlines).then< terminalphotosensorymoduleisomitted.thesubstitutionofthehistidinephosphorylationsite(h)inthe histidinekinasedomain(kd)occurredsubsequenttothedivergenceofprasinophytes.thecanonicalplant phytochromeisrestrictedtostreptophytes(ingreybox),althoughzygnematalesandcoleochaetalesalso havenon<canonicalplantphytochromes.domainnames:pas,per/arnt/sim;pkc,proteinkinasec;rec, ResponseRegulator;andRING,ReallyInterestingNewGene.*TraditionalArchaeplastidadoesnotinclude cryptophytes32. FulllengthphytochromefromCharalesisnotavailableandthedomaincompositionwas inferred Cryptophytes Thephylogeneticpositionofcryptophytesremainscontroversial.Theywereonce thoughttoberelatedtostramenopilesandhaptophytes(belongingtothekingdom Chromalveolata),butsomerecentphylogenomicstudiesplacethemeitherasnestedwithin,or sisterto,archaeplastida(cavalier<smithetal.,2014;burkietal.,2012;grantandkatz,2014).in myanalyses,cryptophyte+viridiplantaephytochromesformacladethatissistertoglaucophyte phytochromes(figure3,appendixfigure17).also,phytochromesfromviridiplantaeandfrom somecryptophytessharethecharacteristicpas<pasrepeatinthec<terminus(figure4).these cryptophytephytochromesdifferfromthecanonicalphytochromesintheirretentionofthe 10

26 conservedhistidinephosphorylationsiteinthekinasedomain(figure3,figure4).some cryptophytephytochromesdonothavethepas<pasrepeatinthec<terminus,butinstead possessasinglepasfollowedbyaserine/threoninekinasedomain( PKC infigure3,figure4). DespitethisvariationintheC<terminus,theN<terminalphotosensorymodulesofallcryptophyte phytochromesaremonophyletic(figure3,appendixfigure17) Viridiplantae Viridiplantaecomprisetwolineages,ChlorophytaandStreptophyta.Chlorophyta includechlorophytes(trebouxiophyceae+ulvophyceae+chlorophyceae+pedinophyceae)and prasinophytes(figure2).chlorophytesappeartolackphytochromesentirely;ididnotfind homologsinanyofthechlorophytetranscriptomesexamined,including14trebouxiophyceae,21 Ulvophyceae,59Chlorophyceae,and2Pedinophyceae.Thisresultisconsistentwithavailable whole<genomesequencedata;thegenomesofchlamydomonas5reinhardtii,volvox5carteriand Chlorella5variabilis(Chlorophyceae)lackphytochromes.Prasinophytes,ontheotherhand,dohave phytochromes.mostofthesehaveapas<pasrepeat,ahistidinekinasedomain,andaresponse< regulatordomainatthec<terminus(duanmuetal.,2014).prasinophytephytochromesare monophyleticandarethesistergrouptostreptophytephytochromes(figure3,appendixfigure 17). Streptophyta(orstreptophytes)areanassemblageofthecharophytes(aparaphyletic gradeofalgae)andthelandplants(wickettetal.,2014)(figure2).ifoundphytochrome homologsinalllandplantclades,aswellasinallcharophytelineages:mesostigmatales (includingchlorokybales),klebsormidiales,coleochaetales,charales,zygnematales,and Desmidiales(Figure1,Figure2).TheCharalesphytochromeswerenotincludedinmyfinal phylogeneticanalysesbecausethetranscriptomecontigs(andalsothedatacurrentlyavailableon GenBank)aretooshorttobeinformativeabouttheirrelationships.Allstreptophyteshave 11

27 canonicalplantphytochromes,includingmesostigmatales,theearliest<divergingcharophyte lineage(figure2,figure3,figure4).thisresultsuggeststhattheoriginofthecanonicalplant phytochrometookplaceintheancestorofextantstreptophytes. WithincharophytealgaeIidentifiedseveralgeneduplicationevents.Iinferone duplicationtohaveoccurredaftermesostigmatalesdiverged( A infigure3),resultingintwo clades:oneissmallandcharophyte<specific(charophytephy1),whereastheotherislargeand includescharophytephy2,andthelandplantphytochromes.membersofthecharophytephy1 cladearenotcommoninthealgaltranscriptomes,andwerefoundonlyindesmidialesandin Entransiaoftheearly<divergingKlebsormidiales(AppendixFigure17).Ontheotherhand,the charophytephy2homologisfoundconsistentlyacrossalgaltranscriptomes.itexperienced additionalduplications( B and C infigure3)thatresultedinthreephytochromesubclades withindesmidiales(desmidialesphy2aec).relationshipsrecoveredwithineachofthese phytochromesubcladescorrespondwelltospeciesphylogeniesfordesmidiales(gontcharovand Melkonian,2010). IfoundthatZygnematalesandColeochaetales(charophytes)alsohavetwonon< canonicalphytochromeclades(charophytephyx1andphyx2,figure3).somephyx1hasa responseregulatordomainatthec<terminus,similartoprasinophyte,cryptophyte,and glaucophytephytochromes(figure2,figure3).intriguingly,phyx1lacksalltheknown conservedcysteineresidues(cysa<d;rockwelletal.,2014)inthepas<gafregionofthen< terminusthatbindbilinchromophores,indicatingthateitherthisproteinmaynotbindabilin,or thatanon<conservedbindingsiteisused. 12

28 Lycophyte PHY Liverwort PHY Legend for support values: MLBS-nucGTR / PP-nucGTR / alrt-codon / MLBS-aaJTT / PP-aaJTT Moss PHY Hornwort HornPHY /1.0/97// 99/1.0/95/82/ 96/1.0/86/77/ * * * 76/1.0/94/-/.99 -/.99/-/-/- Dipteris conjugata Blechnum spicant Adiantum capillus veneris Hemidictyum marginatum Megaceros flagellaris Nothoceros aenigmaticus Phaeoceros carolinianus Paraphymatoceros hallii Anthoceros punctatus * 89/1.0/92/85/ /1.0/99// Fern NEO Hornwort NEO 99/1.0/99/97/ * Cylindrocystis sp Cylindrocystis brebissonii * Cylindrocystis brebissonii Cylindrocystis sp Zygnemopsis sp Mougeotia scalaris Mougeotia scalaris Zygnematales NEO Desmidiales CharoPHY2A-C Zygnematales PHY2 Coleochaetales PHY2 Klebsormidiales PHY2 0.3 substitution/site Figure/5: Phylogenetic/relationship/of/neochromes/and/phytochromes./Thesupportvaluesare shownfortheneochromebranchesonly,inthefollowingorder:maximumlikelihoodbootstrapsupport (MLBS)fromGTRnucleotidemodel/Bayesianposteriorprobabilities(PP)fromGTRnucleotidemodel/ alrtsupportfromcodonmodel/maximumlikelihoodbootstrapvaluesfromjttaminoacidmodel/ BayesianposteriorprobabilitiesfromJTTaminoacidmodel. * indicatesallthesupportvalues=or1.0. < denotesmlbs<70,alrt<70,orpp<0.95.branchesarethickenedwhenmlbs>70,alrt>70,andpp > Neochromes Mydatasuggestthatthephytochromemoduleofneochromehadasingleorigin(Figure 3,AppendixFigure17).Publisheddataindicatethatthephototropinmoduleofneochromes,in contrast,hadindependentoriginsinalgaeandhornworts(lietal.,2014;chapter3),implying twoseparatefusioneventsinvolvingphytochromesthatsharedacommonancestor.tofurther explorethisfinding,ianalyzedtheneochromenucleotidedataset(seeabove)usingseveral nucleotide,codonandaminoacidmodels,andperformedatopologytest.iconsistently recoveredthemonophylyofthephytochromemoduleofneochromes,andusuallywithhigh support,fromanalysesusingallmodels(figure5).althoughanthoceros(ahornwort)neochrome wasresolvedassistertoazygnematales(algal)neochrome,thisrelationshipwasnotsupported 13

29 (exceptinthemrbayesanalysisofthenucleotidedataset).ithenusedtheswofford<olsen< Waddell<Hillis(SOWH)testtocomparethetopologywithallneochromes(thephytochrome module)formingasingleclade,againstanalternativeinwhichneochromesofzygnematales wereforcedtonotgroupwithhornworts+ferns.thealternativehypothesiswasrejected(p< ),andthemonophylyofthephytochromemoduleofneochromeswasfavored Bryophytes Phytochromesfrommosses,liverworts,andhornwortseachformamonophyleticgroup (Figure6).Idetectedsinglephytochromehomologsinhornwortandliverworttranscriptomes. Thegenephylogeniesmatchthespeciesrelationships(VillarrealandRenner,2012;Forrestetal., 2006),consistentwiththepresenceofsingleorthologousgenesinthesetaxa.Indeed,asingle phytochromehasbeenidentifiedviacloningmethodsintheliverwort,marchantia5paleacea5var.5 diptera5(suzukietal.,2001).ialsosearchedthelow<coveragedraftgenomeofthehornwort Anthoceros5punctatus(20X;Lietal.,2014,Chapter3)andfoundonlyonephytochrome.Tofurther evaluategenecopynumber,ihybridizedtheanthoceros5punctatus5genomicdnawith phytochromernaprobes,andusedilluminamiseqtosequencethecaptureddnafragments. Thesamephytochromecontig(andonlythatcontig)wasrecovered,suggestingthatthis hornwortdoesnotharboradditional,divergentphytochromecopies. Incontrast,phytochromesinmossesarediverse,withatleastfourdistinctclades resultingfromthreegeneduplications(figure6).thephylogenyrevealsthosemoss phytochromesthatareorthologoustothepreviouslynamedphyscomitrella5patensphytochromes, PpPHY1E5.ThePhyscomitrellaphytochromesandtheirorthologsformthefollowingclades:moss PHY1_3(includingPpPHY1andPpPHY3),mossPHY2_4(includingPpPHY2andPpPHY4),and mossphy5(includingppphy5aec).anancientduplication( D infigure6)gaverisetomoss PHY1_3andmossPHY2_4+PHY5clades.Thetimingofthisduplicationisdependentonthe 14

30 phylogeneticpositionofthetakakiaphytochrome,whichwasresolvedhereassistertothemoss PHY2_4+PHY5cladebutwithoutsupport(Figure6).BecauseTakakia(Takakiopsida)represents theearliest<diverginglineageinthemossspeciesphylogeny(changandgraham,2011),thefirst phytochromeduplicationprobablypredatestheoriginofallextantmosses.inthemossphy2_4+ PHY5clade,anotherduplication( E infigure6)occurredfollowingthesplitofandreaea (Andreaeopsida)butbeforeAtrichum(Polytrichopsida)diverged,separatingmossPHY2_4and PHY5.ThemossPHY5cladehadanadditionalduplication( F infigure6),probablyafter Physcomitrelladiverged,thatresultedinmossPHY5DandPHY5Esubclades. MyresultsshowthatthephytochromecopiespreviouslyclonedfromCeratodon5 purpureus,whichwerenamedcpphy1e45(mittmannetal.,2009),havethefollowingrelationships withthemossphytochromes:cpphy15andcpphy2areeachothersclosestrelatives,andare membersofthemossphy1_3lineage;cpphy3andcpphy4aremembersofthemossphy5 lineage5(fig.4).theseresultssuggestthatthefourknownc.purpureus5phytochromes CpPHY1, CpPHY2, CpPHY3 and CpPHY4 (Figure6)shouldberenamedtoCpPHY1_3A, CpPHY1_3B,5CpPHY5DandCpPHY5E,respectively,andthatthenovelC.5purpureus5phytochrome discoveredhereshouldbedesignatedascpphy2_ Lycophytes Lycophytephytochromesareresolvedasmonophyleticandaresistertothefernplus seedplantphytochromes(figure7).selaginellaandisoetes(isoetopsida)eachhaveasingle phytochrome,withtheexceptionofs.5mollendorffii,wheretwonearlyidenticalphytochromesare apparentinthewhole<genomesequencedata.theirhighdegreeofsimilaritysuggeststhatthey mightbeproductsofaspecies<specificgeneduplication.incontrast,lycopodialeshavetwo distinctphytochromecladesthatinamelycopodialesphy1andlycopodialesphy2.becauseall thelycopodialeslineages(wikstrom,2001)arerepresentedineachphytochromeclade,iinfer 15

31 thattheduplicationoflycopodialesphy1/2( G infigure1)predatesthecommonancestorofall extantlycopodiales. Inferred gene duplication Projected attachment of vascular plant PHY. See Supplementary Fig. 1 99/1.0 Rhynchostegium serrulatum Anomodon rostratus Hypnum subimponens 99/1.0 Anomodon attenuatus PHY5 /.99 Neckera douglasii Rhytidiadelphus loreus E Schwetschkeopsis fabronia 85/.99 Leucodon sciuroides PHY2_4 94/1.0 Pseudotaxiphyllum elegans Aulacomnium heterostichum Bryum argenteum Bryopsida PHY2_4 Hedwigia ciliata Philonotis fontana 99/1.0 Ceratodon purpureus Leucobryum albidum Racomitrium varium Physcomitrella patens PHY4 99/1.0 96/1.0 Physcomitrella patens PHY2 81/1.0 Scouleria aquatica Atrichum angustatum Polytrichopsida PHY2_4 Andreaea rupestris Andreaeopsida PHY2_4/5 Sphagnum palustre Sphagnopsida PHY2_4/5 Takakia lepidozioides Takakiopsida PHY2_4/5 92/1.0 Rhynchostegium serrulatum 76/.92 Anomodon rostratus 94/1.0 Hypnum subimponens 98/1.0 Neckera douglasii 81/.99 Rhytidiadelphus loreus Pseudotaxiphyllum elegans Schwetschkeopsis fabronia 96/1.0 Leucodon sciuroides 74/1.0 Aulacomnium heterostichum Bryum argenteum Bryopsida PHY1_3 Hedwigia ciliata Philonotis fontana 99/1.0 Ceratodon purpureus PHY1 99/1.0 Ceratodon purpureus PHY2 Leucobryum albidum Racomitrium varium Physcomitrella patens PHY1 99/1.0 Physcomitrella patens PHY3 Andreaea rupestris Andreaeopsida PHY1_3 PHY2_4/5 D PHY1_3 92/1.0 85/1.0 /.63 78/.65 Scapania nemorosa Odontoschisma prostratum Bazzania trilobata Porella pinnata Schistochila sp Metzgeria crassipilis Pellia neesiana Riccia berychiana Conocephalum conicum Marchantia paleacea Lunularia cruciata Sphaerocarpos texanus 99/1.0 Rhynchostegium serrulatum Anomodon rostratus Rhytidiadelphus loreus 92/1.0 89/1.0 Neckera douglasii Anomodon attenuatus Pseudotaxiphyllum elegans Leucodon sciuroides Schwetschkeopsis fabronia Aulacomnium heterostichum 2 Aulacomnium heterostichum 1 Bryopsida PHY5E Hedwigia ciliata Philonotis fontana 80/1.0 Ceratodon purpureus PHY4 Leucobryum albidum Syntrichia princeps 78/.99 Racomitrium varium Scouleria aquatica 99/1.0 Anomodon rostratus Rhynchostegium serrulatum PHY5E 79/1.0 Anomodon attenuatus 90/.95 Rhytidiadelphus loreus F Pseudotaxiphyllum elegans PHY5D Leucodon sciuroides Aulacomnium heterostichum Bryum argenteum Bryopsida PHY5D Hedwigia ciliata Philonotis fontana 86/1.0 Ceratodon purpureus PHY3 75/.98 Syntrichia princeps Leucobryum albidum Racomitrium varium Scouleria aquatica Physcomitrella patens PHY5C Physcomitrella patens PHY5B Bryopsida PHY5A-C Physcomitrella patens PHY5A Atrichum angustatum 5 Polytrichopsida PHY5 Jurgemanniopsida Marchantiopsida Moss Liverwort 94/.99 Nothoceros aenigmaticus Megaceros tosanus Phaeomegaceros coriaceus Paraphymatoceros hallii Phaeoceros carolinianus 82/1.0 Anthoceros punctatus Leiosporoceros dussii 0.08 substitutions/site Dendrocerotales Notothyladales Anthocerotales Leiosporocerotales Hornwort Figure/6: Phytochrome/phylogeny/for/bryophytes./Phytochromespreviouslyidentifiedarein bold.supportvaluesassociatedwithbranchesaremaximumlikelihoodbootstrapvalues(bs)/bayesian posteriorprobabilities(pp);theseareonlydisplayed(alongwiththickenedbranches)ifbs>70andpp> 16

32 0.95.Thickenedbrancheswithoutnumbersare/1.0.Thepositionoforangecirclesestimatestheoriginof inferredgeneduplications.italicletterswithineachcirclecorrespondtotheduplicationeventmentionedin thetext,andthenumbers/lettersadjacenttoeachcircleindicatethenamesofthegeneduplicates Ferns Fernphytochromesformacladethatissistertotheseed<plantphytochromes(Figure3, Figure7,AppendixFigure17).WithinfernsIuncoveredfourphytochromecladesthatIdesignate fernphy1,phy2,phy4a,andphy4b.thenamephy3wasusedpreviouslytodenotethe chimericphotoreceptorthatisnowrecognizedasneochrome(suetsuguetal.,2005;lietal., 2014).ThedeepevolutionarysplitbetweenthefernPHY1andPHY2/4cladespredatesthemost recentancestorofextantferns( H infigure7).fernphy2andphy4probablyseparatedafter Gleichenialesdiverged( I infigure7),andtheearliest<divergingfernlineages(i.e., Gleicheniales,Osmundales,Psilotales,Ophioglossales,Marattiales,andEquisetales)havethe pre<duplicatedphy2/45copy.itshouldbenotedthatmybroad<scaleaminoaciddatasetresolved aslightlydifferenttopology,placinggleichenialesphy2/4closertophy4(appendixfigure17). However,theaminoaciddatasetincludedfewersequencesfromferns,whichcouldreduce phylogeneticaccuracy(hillis,1998).itislikelythatthatthephylogeny(figure7)inferredfrom rigorousanalysesofnucleotidedatamoreaccuratelyreflectsgenerelationships. IfoundthatOphioglossalesandOsmundaleseachhavetwoPHY2/4copies,whichlikely arosefromindependentgeneduplications(figure7).theduplicationofophioglossalesphy2/4a andphy2/4boccurredeitherattheancestorofophioglossalesorofophioglossales+psilotales, butthehistoryofphy2/4inosmundalesisunclear.theosmundalesphy2/4aandphy2/4b werenotresolvedasmonophyletic,andthephylogeneticpositionofosmundalesphy2/4bis incongruentwithpublishedfernspeciesrelationships(kuoetal.,2011). AfterthesplitoffernPHY25andPHY4,PHY4duplicatedagain,givingrisetofernPHY4A andphy4b( J infigure7),andbotharefoundinpolypodiales.icannotpreciselydeterminethe 17

33 timingofthisduplicationeventbecausetherelationshipsamongpolypodialesphy4aeb, CyathealesPHY4andSalvinialesPHY4areresolvedwithoutsupport.Interestingly,PHY4Awas previouslyknownonlyfromadiantum5capilluseveneris5(asacphy4).itsfirstintronincorporated aninsertedty3/gypsyretrotransposonandthedownstreamexonsequencewasunknown (Nozueetal.,1997).Ifoundfull<lengthPHY4AtranscriptsinawiderangeofPolypodiales, suggestingthatphy4alikelyisfunctionalinmostotherspecies,ifnotina.5capilluseveneris. PHY4Bisanovelphytochromecladethathasnotbeendocumentedbefore;itisnotcommonin theferntranscriptomesiexamined Seed plants Seed<plantphytochromesclusterintothreeclades(AppendixFigure17)correspondingto PHYA,PHYB/E,andPHYC,inaccordancewithpreviousstudies(Mathews,2010).Organismal relationshipswithinthegenesubcladeslargelyareconsistentwiththoseinferredinphylogenetic studiesofangiosperms(bremeretal.,2009).notably,however,supportforthemonophylyof gymnospermswaslow.ifoundtwodivergenttranscriptsofphyeinranunculales,represented byaquilegia(ranunculaceae;fromwholegenomedata)andcapnoides(papaveraceae;from transcriptomedata)(figure17inappendixa),suggestingthatageneduplicationeventoccurred deepinranunculales;however,moreextensivesamplinginranunculalesisneededtoresolve thetimingofthisduplication. 18

34 81/1.0 Inferred gene duplication Projected attachment of seed plant PHY. See Supplementary Fig. 1 97/1.0 H 98/1.0 PHY2/4 PHY1 G PHY1 PHY2 98/ /1.0 84/1.0 Woodsia ilvensis Woodsia scopulina 96/1.0 Blechnum spicant Athyrium filix-femina Diplazium wichurae 97/1.0 Deparia lobato-crenata 82/1.0 Gymnocarpium dryopteris Cystopteris fragilis Homalosorus pycnocarpos Asplenium platyneuron Polypodium hesperium 92/1.0 Polystichum acrostichoides 89/1.0 Leucostegia immersa Pteridium aquilinum 98/1.0 Gaga arizonica Notholaena montieliae 74/1.0 Myriopteris rufa 90/1.0 Argyrochosma nivea Vittaria appalachiana Adiantum capillus-veneris Cryptogramma acrostichoides Pteris ensigormis 88/1.0 Pityrogramma trifoliata 78/1.0 Ceratopteris thalictroides Lindsaea microphylla Lonchitis hirsuta Culcita macrocarpa Plagiogyria japonica Thyrsopteris elegans Pilularia globulifera Anemia tomentosa 1 Anemia tomentosa 2 Polypodiales PHY2 Cyatheales PHY2 Salviniales PHY2 Schizaeales PHY2 Vittaria appalachiana 80/.99 98/1.0 Adiantum capillus-veneris Adiantum tenerum 97/1.0 Argyrochosma nivea Myriopteris rufa Gaga arizonica Ceratopteris thalictroides PHY2 Pteris ensigormis Cryptogramma acrostichoides Polypodium hesperium Polypodiales PHY4A 96/1.0 I Polystichum acrostichoides 89/1.0 Leucostegia immersa 99/1.0 Asplenium platyneuron PHY4 Blechnum spicant Cystopteris fragilis Lonchitis hirsuta Lindsaea microphylla 82/1.0 Azolla caroliniana Salviniales PHY4 Pilularia globulifera PHY4A /.99 Athyrium filix-femina Diplazium wichurae J 76/1.0 Woodsia ilvensis 92/1.0 Gymnocarpium dryopteris PHY4B Cystopteris fragilis Polypodiales PHY4B Blechnum spicant Polystichum acrostichoides Pteridium aquilinum Thyrsopteris elegans Cyathea spinulosa Plagiogyria japonica Cyatheales PHY4 77/1.0 Lygodium japonicum Anemia tomentosa Schizaeales PHY4 Dipteris conjugata Osmunda javanica Gleicheniales PHY2/4 Osmunda sp Osmundastrum cinnamomeum Osmundales PHY2/4A Osmunda javanica Osmundastrum cinnamomeum Osmundales PHY2/4B Psilotum nudum Psilotales PHY2/4 Sceptridium dissectum Botrypus virginianus Ophioglossales PHY2/4B Botrypus virginianus Sceptridium dissectum Ophioglossales PHY2/4A Marattia attenuata Danaea nodosa Marattiales PHY2/4 Equisetum diffusum Equisetum diffusum Equisetales PHY2/4 Blechnum spicant 96/1.0 Deparia lobato-crenata Woodsia scopulina Polypodium glycyrrhiza Cystopteris protrusa Asplenium platyneuron Adiantum capillus-veneris Polypodiales PHY1 Vittaria appalachiana Ceratopteris thalictroides Pteridium aquilinum Lonchitis hirsuta Thyrsopteris elegans Plagiogyria japonica Cyatheales PHY1 Cyathea spinulosa 86/1.0 70/1.0 Azolla filiculoides 96/1.0 Pilularia globulifera Salviniales PHY1 Dipteris conjugata Gleicheniales PHY1 Osmunda sp Osmundales PHY1 Danaea nodosa Marattia howeana Marattiales PHY1 Equisetum diffusum Equisetales PHY1 Selaginella lepidophylla 94/1.0 Selaginella apoda Selaginella moellendorffii Selaginella willdenowii Selaginellales Selaginella kraussiana Selaginella wallacei Isoetes tegetiformans Isoetales 98/1.0 Dendrolycopodium obscurum Pseudolycopodiella caroliniana Lycopodiales PHY1 Huperzia lucidula 92/1.0 Dendrolycopodium obscurum Pseudolycopodiella caroliniana Lycopodiales PHY2 Huperzia selago 0.1 substitutions/site Figure/7: Phytochrome/phylogeny/for/ferns/and/lycophytes./Phytochromesthatwerepreviously reportedareshowninbold.supportvaluesassociatedwithbranchesaremaximumlikelihoodbootstrap values(bs)/bayesianposteriorprobabilities(pp);theseareonlydisplayed(alongwiththickenedbranches) ifbs>70andpp>0.95.thickenedbrancheswithoutnumbersare/1.0.thepositionoforangecircles estimatestheoriginofinferredgeneduplications.italicletterswithineachcirclecorrespondtothe duplicationeventmentionedinthetext,andthenumbers/lettersadjacenttoeachcircleindicatethenames ofthegeneduplicates. 19

35 1.3 Discussions Myphylogeneticresultsrefuteprevioushypothesessuggestingthatplantsacquired phytochromefromcyanobacteriaviaendosymbioticgenetransfer(karnioletal.,2005;herdman etal.,2000),becausestreptophyteandcyanobacterialphytochromesarenotclosestrelativesin myphytochrometrees(figure3,appendixfigure17),aresultalsorecentlyobtainedbyduanmu etal.5(2014).instead,plantphytochromesevolvedfromaprecursorsharedwithother Archaeplastida.Iclearlyplacedtheoriginofcanonicalplantphytochromesinacommonancestor ofextantstreptophytes(figure3,figure4).mydata,moreover,showthattheoriginofthis structurerequiredmultiplesteps.thegainoftheinternalpas<pasrepeattookplacefirst,inthe ancestorofviridiplantae,orofviridplantae+cryptophytes(figure4).asnotedabove,the positionofcryptophytesisuncertain,anditsinclusioninarchaeplastidaisnotstrongly supportedinpublishedstudies(burkietal.,2012;grantandkatz,2014;cavalier<smithetal., 2014).Thetopologyofmyphytochrometreesisconsistentwithasister<grouprelationship betweenviridiplantaeandcryptophytes,butthetopologyalsocouldresultfromendosymbiotic orhorizontalgenetransfer(egtorhgt).thelossofthehistidinephosphorylationsiteinthe histidinekinasedomain hencetheattainmentofthecanonicalform occurredlater,inthe ancestorofstreptophytes,andseemstohavebeenaccompaniedbyapermanentdissociation withtheresponseregulatoratthec<terminalend(figure4).somestreptophyteshaveadditional, non<canonicalphytochromes.charophytephyx1andphyx2,bothfoundinzygnematalesand Coleochaetales,havetheconservedhistidineresidue,andsomePHYX1alsohavearesponse regulatordomain(figure3,figure4).thefactthatcharophytephy1,phyx1,andphyx2arenot foundinallstreptophytesimpliesthatduplicationsearlyinthehistoryofstreptophyteswere followedbymultiplelossesofcharophytephy1andthenon<canonicalcharophytephy. 20

36 MyfindingshighlightthedifferentevolutionaryhistoriesofthephytochromeN<andC< terminalmodules.then<terminalphotosensorymoduleisdeeplyconservedacrosseukaryotes andprokaryotes,andthelineardomainsequenceofpas<gaf<phyisfoundinthemajorityof knownphytochromes(figure3).incontrast,theevolutionofthec<terminalregulatorymodule hasbeenmuchmoredynamic(figure4).forexample,thec<terminalpasmaybeabsent,may occursingly,ormayoccurasatandemrepeat(figure4).serine/threoninekinaseortyrosine kinasedomainshavealsobeenindependentlyrecruitedintotheregulatorymoduleinthe cryptophyteandceratodon5purpureus(moss)phytochromes(thümmleretal.,1992)(figure3).the successfullinkageofthephytochromephotosensorymodulewithavarietyofc<terminal moduleshaspromotedphytochromefunctionaldiversity.certainlythemostcompelling exampleisthatoftheneochromes.itcombinesphytochromeandphototropinmodulesintoa singleproteintoprocessblueandred/far<redlightsignalsinthecontrolofphototropism (Kanegaeetal.,2006).Neochromewasfirstdiscoveredinferns(Nozueetal.,1998)and postulatedtobeadriverofthemodernfernradiationunderlow<light,angiosperm<dominated forestcanopies(kawaietal.,2003;schneideretal.,2004;schuettpelzandpryer,2009).suetsugu etal.(2005)laterdiscoveredasimilarphytochrome<phototropinchimerainmougeotia5scalaris (zygnemataleanalga),andproposedthatneochromehadindependentlyevolvedtwice.arecent studyidentifiedyetanotherneochromefromhornworts,anddemonstratedthatfernsacquired theirneochromesfromhornwortsviahorizontalgenetransfer(lietal.,2014;chapter3).by placingthephototropinportionofneochromeintoabroadphylogenyofphototropins,lietal. (2014)alsoshowedthatphototropinmodulesofneochromeshadtwoseparateorigins,oncein hornwortsandonceinzygnemataleanalgae.incontrast,thephytochromeportionofneochrome hashadadifferentevolutionaryhistory,withzygnematales,hornworts,andfernsforminga singlemonophyleticgroup(figure5).thisresultisrobust,andissupportedbymostofthe 21

37 analysesandbyatopologytest.myresultsthussuggestthatneochromesoriginatedviatwo separatefusionevents,involvingtwodistinctsourcesofphototropinbutthesamephytochrome progenitor.thisisafascinatingextensionofthecapacityandpropensityofthephytochrome photosensorymoduletobelinkedwithfunctionallydistinctdownstreamdomains./ Themajorcladesoflandplantsdiffermarkedlywithrespecttophytochromegene diversity.itappearsthatphytochromesaresingle<copyinmostliverworts,hornwortsand Isoetopsida(IsoetaceaeandSelaginellaceae),whereastheyhaveindependentlydiversifiedin Lycopodiales,mosses,fernsandseedplants(Figure3).Inferns,apatternofearlygene duplicationfollowedbygenelossescouldexplainthephylogeneticpositionsoftwoosmundales PHY2/4,whichareincongruentwithknownspeciesrelationshipsinferns(Figure7).Interestingly, Iobservedarelationshipbetweenphytochromecopynumberandspeciesrichness.Forinstance, thepolypodferns(polypodiales),whichaccountfor90%ofextantferndiversity(schuettpelzand Pryer,2009),havefourphytochromecopies,whereasotherspecies<poorfernlineageshaveonly twoorthree(figure7).likewise,mossspeciesbelongingtothehyper<diversebryopsida containing95%ofextantmossdiversity haveexperiencedthehighestnumberofphytochrome duplicationscomparedtootherbryophytelineages(figure6).itispossiblethattheevolutionof phytochromestructuralandfunctionaldiversityenhancedtheabilityofpolypodfernsand Bryopsidamossestoadapttodiverselightenvironments.Indeed,seedplants,ferns,andmosses eachhaveatleastonephytochromeduplicatethatconvergentlyevolvedorretainedtheroleof mediatinghigh<irradianceresponses(cookeetal.,1993;possartandhiltbrunner,2013;mathews, 2006;MathewsandTremonte,2012),atraitlikelytobeimportantforsurvivingunderdeep canopyshade(yanovskyetal.,1995)(seebelow).this phytochrome<drivenspecies diversification hypothesis,however,needsrigoroustestingbyphylogeneticcomparative 22

38 methodsandfunctionalstudiesinnon<seedplantsthatidentifythegeneticbasesofphytochrome functions. Theindependentphytochromediversificationeventsinseedplants,ferns,mossesand Lycopodialeshavesignificantimplicationsforphytochromefunctionalstudies.Moss phytochromes,forexample,aremorecloselyrelatedtoeachotherthantoanyoftheseed<plant phytochromes(andthesameistrue,ofcourse,forphytochromesfromferns,andthosefrom Lycopodiales).Seed<plantphytochromeshaveundergonesignificantdifferentiationintotwo majortypes.oneisrepresentedbyphyaofarabidopsis5thaliana,whichistheprimarymediatorof red<lightresponsesindeepshadeandbeneaththesoilsurface.itdegradesrapidlyinlight, mediatesvery<low<fluenceandhigh<irradianceresponses,anddependsonproteinpartners FHY1(FAR<REDELONGATEDHYPOCOYTL1)andFHL(FHY1LIKE)fornuclear translocation.theotherisrepresentedbyphyb<eofa.5thaliana,whicharetheprimarymediators ofred<lightresponsesinopenhabitats.theyarelight<stable,mediatelow<fluenceresponses;and inthecaseofphyb,dependonaninternalnuclearlocalizationsignalfornucleartranslocation (FranklinandQuail,2010;ChenandChory,2011).Asimilarpartitioningoffunctionhasbeen documentedinsomefernandmossphytochromeduplicates(sineshchekovetal.,2013;possart andhiltbrunner,2013;possartetal.,2014),demonstratingacaseofconvergentdifferentiation followingindependentgeneduplications.infuturestudies,itwouldbeofparticularinterestto infertheancestralpropertiesoflandplantphytochrome:wasitlight<labileorstable?whatkinds ofphysiologicalresponsesdiditmediate?howwasnucleartranslocationexecuted?studiesof liverwort,hornwortandselaginellaphytochromes,whichexistasasingle<copygene,couldserve as baselinemodels forunderstandingthegeneticbasisofphytochromefunctional diversification. 23

39 Recentfunctionalstudiesonasmallbutvariedsetofalgalphytochromesrevealeda surprisingdegreeofspectraldiversity,whichmightreflectadaptationstoarangeofmarineand aquaticenvironments(rockwelletal.,2014;duanmuetal.,2014).forexample,photoreversible phytochromesinprasinophytealgaeincludeorange/far<redreceptorsaswellasred/far<red receptors,andinalgaeoutsideofviridiplantae,therealsoareblue/far<redandred/bluereceptors (Duanmuetal.,2014).Thissharplycontrastswiththeverylimitedspectraldiversityincanonical plantphytochromes,allofwhicharered/far<redreceptors.itappearsthen,thatthetransitionof plantsfrommarineoraquatictoterrestrialenvironmentsinvolvedacenteringofphytochrome evolutiononalimitedmodel.thenovelalgalphytochromecladesiuncoveredherearea potentialtreasuretrovefordiscoveringthestepsduringthistransition,andforcharacterization ofnewbiochemicalvariants,someofwhichmayhaveimplicationsforunderstandingtheroleof phytochromeevolutioninrecolonizationofmarineandaquaticenvironmentsbyterrestrial plants. Insummary,mystudyhasrevealedthatthediversityofViridiplantaephytochromesis fargreaterthanwasrealized,andpointstoexcitingopportunitiestolinkthisstructuraldiversity withfunctionandecology. 24

40 1.4 Materials and Methods Transcriptome- and genome-mining for phytochrome ThetranscriptomesandgenomessampledinthisstudyarelistedinAppendixTable2.I usedthepythonpipelinebluedevilfollowinglietal.(2014)tominetranscriptomes.tosearch thewhole<genomedata,iusedblastpimplementedinphytozome(goodsteinetal.,2012)or individualgenomeprojectportals(appendixtable2).theproteindomaincompositionforeach ofthephytochromesequencewasdeterminedbyqueryingthencbiconserveddomain Database(Marchler<Baueretal.,2011) Sequence alignment Inadditiontothephytochromehomologsminedfromtranscriptomesandgenomes,I alsogatheredselectedgenbankaccessionsandasequenceclonedfrommarattia5howeana (voucher:s.w.graham&s.mathews15,depositedinnsw;primers:110f 5 GTNACNGCNTAYYTNCARCGNATG3,788r 5 GTMACATCTTGRSCMACAAARCAYAC3 ). Iassembledfoursequencedatasets,onewastranslatedintoanaminoacidalignmentand theotherswereanalyzedasnucleotidematrices.theaminoaciddatasetincludedthemajorityof thesequencesihave(423sequencesintotal;appendixfigure17).thesequenceswereinitially alignedusingmuscle(edgar,2004),andimanuallycuratedthealignmentbasedonknown domainboundariesandproteinstructures.theunalignableregionswereexcludedandthefinal alignmentincluded1,106aminoacidsites.thenucleotidedatasetswereassembledtoprovide higherphylogeneticresolutionwithinfern+lycophytephytochromes(113sequences;figure7), bryophytephytochromes(97sequences;figure6),andneochromes(111sequences;figure5). 25

41 Sequenceswerealignedasaminoacidsandthenback<translatedtonucleotides,andthe alignmentwasrefinedbymanualediting.thefern+lycophyte,andbryophytephytochrome alignmentscontained3,366and3,429nucleotidesites,respectively.theneochromealignment includedonlythen<terminalphotosenorymodule(pas<gaf<phydomains;1,920nucleotide sites).allalignmentsareavailablefromdryad( Phylogenetic reconstruction Forthebroad<scaleaminoacidalignment,JTT+I+Gwasselectedasthebest<fitting empiricalmodelbyprotestunderakaikeinformationcriterion(darribaetal.,2011).iused Garliv2.0(Zwickl,2006)tofindthemaximumlikelihoodtree,withtenindependentrunsand genthreshfortopotermsetto,000.thestartingtreeforgarlicamefromaraxml(stamatakis, 2006)run.Toobtainbootstrapbranchsupports,RAxMLwasranrunwith1,000replicatesusing JTT+G. Forthenucleotidealignments,IusedPartitionFinder(Lanfearetal.,2012)toinferthe bestcodonpositionpartitionschemesandsubstitutionmodels,underakaikeinformation Criterion.Maximumlikelihoodtreesearchingandbootstrapping(1,000replicates)weredonein RAxML.BayesianinferencewascarriedoutinMrBayes(Ronquistetal.,2012),withtwo independentmarkovchainmontecarlo(mcmc)runsandfourchainseach.iunlinkedthe substitutionparametersandsettheratepriortovaryamongpartitions.themcmcoutputwas inspectedusingtracer(rambautanddrummond,2013)toensureconvergenceandmixing (effectivesamplesizesall>200);25%ofthetotalgenerationswerediscardedasburn<inbefore analyzingtheposteriordistribution. 26

42 Additionalanalyseswereappliedtotheneochromedataset.First,IusedCodonPhyML (Giletal.,2013)toinferthetreetopologyandtoassesssupport(SH<likeaLRTbranchsupport), usingacodonsubstitutionmodel.fourcategoriesofnon<synonymous/synonymoussubstitution rateratiosweredrawnfromadiscretegammadistribution,andcodonfrequencieswere estimatedfromthenucleotidefrequenciesateachcodonposition(f3 4).Second,Itranslatedthe nucleotidesintoaminoacids,andcarriedoutmaximumlikelihoodtreesearchingand bootstrapping(inraxml),aswellasbayesianinference(inmrbayes)underthejtt+i+g model.finally,iusedtheswofford Olsen Waddell Hillis(SOWH)test,implementedin SOWHAT(Churchetal.2015),toinvestigatewhethertheinferredtreetopology(phytochrome portionofneochromeformingaclade)issignificantlybetterthanthealternativetopology (neochromenotmonophyletic).insowhat,iusedthedefaultstoppingcriterionandapplieda topologicalconstraintforcinglandplantandzygnemataleanneochrometobenon<monophyletic Confirming gene copy number in hornworts by target enrichment Iusedatargetenrichmentstrategytotestwhetherhornwortshaveasinglephytochrome locus.inthisapproach,specificrnaprobesarehybridizedtogenomicdnatoenrichthe representationofparticulargenefragments.targetenrichmenthasseveraladvantagesoverthe traditionalsouthernblottingapproach.inparticular,itusesthousandsofdifferenthybridization probes(ratherthanjustafew),andtheendproductsarenotdnabands,butactualsequence data. Idesignedatotalof7,502120<merRNAprobestotargetphytochrome,phototropinand neochromegenes,withaspecialfocusonthoseofhornwortsandferns(probesequences availablefromdryadhttp://dx.doi.org/ /dryad.[nnnnn]).theprobesoverlapevery60bp 27

43 (a2xtilingstrategy),andweresynthesizedandbiotin<labeledbymycroarray.genomicdnaof thehornwortanthoceros5punctatuswasextractedusingamodifiedctabprotocol,andsheared bycovariswithfragmentspeakat300bp.librarypreparationforilluminasequencingwasdone usingakapabiosystemkit,incombinationwithnebnextmultiplexoligos.toenrichfor potentiallydivergenthomologs,iusedthetouchdownprocedureoflietal.(2013),inwhichthe genomicdnalibraryandtheprobeswerehybridizedat65 Cfor11hoursfollowedby60 C(11 hours),55 C(11hours)and50 C(11hours).ThehybridizedDNAfragmentswerecapturedby streptavidinbeadsandwashedfollowingtheprotocolofmycroarray.thefinalproductwas pooledwithnineotherlibrariesinequimolarandsequencedonilluminamiseq(250bppaired< end).toprocessthereads,iusedcutadapt(martin,2011)toremovetheadaptorsequences,and usedsickle(joshiandfass,2011)totrimlow<qualitybases.theresultingreadswerethen assembledbysoapdenovo2(luoetal.,2012),andthephytochromecontigwasidentifiedby tblastn(camachoetal.,2009).therawreadsweredepositedinncbisra(srp055877). 28

44 2. The origin and evolution of phototropins Li,F.<W.,C.J.Rothfels,M.Melkonian,J.C.Villarreal,D.W.Stevenson,S.W.Graham, G.K.S.Wong,S.Mathews,andK.M.Pryer.On/the/origin/and/evolution/of/phototropins. inreview. 2.1 Introduction Despitetheirsedentarynature,plantsarenotstaticandarecapableofasurprisingrange ofmotion(darwinanddarwin,1880).plantshaveevolvedsophisticatedphototropic responses involvingmovementofshootsand/orchloroplasts tooptimizetheirexposureto light.charlesdarwinpioneeredmodernphototropismresearchbydemonstratingthattheshoot tipisthepointoflightperceptionfromwhere fluence istransducedtoinitiatetropic movements(darwinanddarwin,1880).subsequentstudiessoonledtothediscoveryoftheplant hormoneauxin(darwin s fluence ),andlatertheidentificationoftheblue<light photoreceptors phototropins(liscumetal.,2014). Phototropinsregulatekeyadaptivephysiologicalresponsesthatareunderlightcontrol, includingshoot<positivephototropism,root<negativephototropism,chloroplast accumulation/avoidance,stomatalopening,circadianrhythm,leafexpansion,andseedling elongation(christie,2007).ourcurrentunderstandingofthefunctionandbiochemistryof phototropinsoriginatesfrombasicresearchonarabidopsis5thaliana(afloweringplant),andtoa muchlesserextentonadiantum5capilluseveneris(afern)andphyscomitrella5patens(amoss).despite thephylogeneticspanencompassedbythesemodelorganisms,theorthologyofphototropin geneshasbeenambiguous,confoundingnotonlyfunctionalhomologyassignments,butalsoour 29

45 understandingoftheirroleinallowingplantstoadapttoheterogeneouslightenvironments throughtime. a Origin of PHOT PHOT duplication PHY duplication Viridiplantae Seed plants Ferns Lycophytes Mosses Liverworts Hornworts Desmidiales Zygnematales Coleochaetales Charales Klebsormidiales Mesostigmatales Chlorophytes Prasinophytes Rhodophytes Glaucophytes Cryptophytes Stramenopiles Land plants Charophytes Seed plant D b Ferns + lycophytes c Leptosporangiate ferns Mosses B E C F G Polypodiales incl. A. capillus-veneris Cyatheales Salviniales Schizaeales Gleicheniales Hymenophyllales Osmundales Marattiales Ophioglossales Psilotales Equisetales Selaginella Isoetes Lycopodiales Bryidae Dicranidae Funariidae incl. P. patens Timmiidae Diphysciidae Buxbaumiidae Tetraphidopsida Polytrichopsida Oedipodiopsida* Andreaeopsida Sphagnopsida Takakiopsida Ferns Lycophytes Bryopsida Figure/8:/Organismal/lineages/screened/for/phototropin/homologs./(A)Viridiplantaeandalgae.Lineages thatlackphototropinaredepictedingrey.topologyderivedfromwickettetal.(2014)andburkietal. (2012).Phototropinandphytochromeduplicationsareonlyshownonlandplantbranches(withingrey box).(b)fernsandlycophytes;topologyderivedfromwickettetal.(2014)andkuoetal.(2011)(c)mosses; topologyderivedfromcoxetal.(2010).capitallettersabovebluesquaresdenotephototropinduplication eventsmentionedinthetextandinfig.2. indicatesthattheexactphylogeneticpositionofthegene duplicationeventisambiguous. 2.2 Results The origin of phototropins Topinpointtheoriginofphototropinsandreconstructtheirevolutionaryhistory,I examined215transcriptomesandgenomesspanningallextantplantandalgallineages (AppendixTable4,Table5).Ishowherethatphototropinsarepresentinallmajorlandplant 30

46 lineages(seedplants,ferns,lycophytes,mosses,liverworts,andhornworts),aswellasingreen algae(charophytes,chlorophytes,andprasinophytes;figure8).incontrast,ididnotrecover phototropinsfromglaucophytes,redalgae,cryptophytes,haptophytesandstramenopiles, indicatingthattheoriginofphototropinmostlikelytookplaceinanancestorofviridiplantae (greenalgae+landplants;figure8).becausethechlorophytealgachlamydomonas5reinhardtiiis knowntousephototropinstomodulateitssexualprocesses(huangandbeck,2003),itispossible thatthefunctionofearlyphototropinsmaynothaveinvolvedphototropicresponses. Unfortunately,solittleisknownaboutphototropinfunctioningreenalgallineagesthatwe cannotdeterminewhenphototropinswererecruitedtodirecttrophicresponses Phototropin phylogeny Seed<plantphototropinsformamonophyleticgroupthatissistertofernphototropins (Figure9).HereIinferasinglegeneduplicationeventinseedplants,oneleadingtoArabidopsis PHOT1andtheothertoArabidopsisPHOT25(Christie,2007).BecausethePHOT1andPHOT2 cladeseachincludeangiospermsandgymnosperms,theduplicationeventthatgaverisetothese twohomologspredatesthedivergenceofallextantseedplants( A infigure9).ialsofindstrong evidenceforthemonophylyoffernphototropins( B infigure9).leptosporangiatefernshave twophototropinhomologsthatwedesignatephot1andphot2,inreferencetoadiantum5 capillusevenerisphot1andphot25(kagawaetal.,2004),respectively.theearliest<divergingfern lineages,equisetales,psilotalesandophioglossales,eachhaveonephototropingene, representingthepre<duplicatedversionoffernphot1andphot2.theexactphylogenetic positionastowherefernphot1andphot2divergedisambiguousduetoalackofbranch support,althoughitprobablywaspriortoleptosporangiatefernsdivergingfrommarattiales 31

47 ( B infigure9). Aswithseedplantsandleptosporangiateferns,Iinferasingleduplicationeventinthe lycophyteselaginella,correspondingtophot1andphot2basedonthegenomeannotationofs.5 moellendorffii5(banksetal.,2011).thephylogeneticpositionofthisduplicationisalsounclear( C infigure10),butitmustpredatethecommonancestorofextantselaginellabecausethephot1 cladecontainsallknownmajorselaginellalineages(korallandkenrick,2002).forisoetalesand Lycopodiales,wefoundonlyonephototropinhomolog,butwhetherornotitisindeedasingle< copygeneintheselineageswillrequirefurtherconfirmation. Allliverworttranscriptomesweexaminedcontainedonlyonephototropin(Figure10),a resultconsistentwiththerecentdemonstrationthatphototropininmarchantia5polymorphaisa single<copygene(komatsuetal.,2014).hornwortphototropinsalsoappeartobesingle<copy, basedonourscreeningofhornworttranscriptomesandalow<coveragegenomedraftof Anthoceros5(Lietal.,2014;Chapter3).Tofurtherconfirmthegenecopynumberinhornworts,we usedatargetenrichmentstrategytosequenceallphototropin<likegenomicfragmentsin Anthoceros5punctatus,andfoundnoadditionaldivergentcopies. Mossphototropins,ontheotherhand,haveasignificantlymorecomplexevolutionary history.ideterminedthatthephototropinannotationsfromthemossphyscomitrella5patens genome(ppphota1e4,ppphotb1e3)donotreflectgeneorthology.because PHOTAs and PHOTBs areintermingled,ireclassifiedthemossphototropinsbasedontheirphylogenetic relationshipsshownhere(table1,figure10).priortothedivergenceofallextantmosses,agene duplicationevent( D infigure10)tookplace,givingrisetomossphot1andphot2.in PHOT1,asecondduplicationoccurredinthecommonancestorofBryopsidaandPolytrichopsida 32

48 ( E infigure10)thatsplitphot1intophot1aandphot1b.inphot2,twoadditional duplicationstookplace( F and G infigure10)subsequenttothedivergenceofbuxbaumiidae (Bryopsida),resultinginPHOT2AEC.PHOT2AandPHOT2BarebothpresentinDicranidaeand Bryidae,whereasPHOT2CisonlyknowninPhyscomitrella5patens(Funariidae).Physcomitrella5 patensmayalsohavelostthephot2a5homolog.ingreenalgae,mostofthetranscriptomesand genomesrevealedasinglephototropingene(figure11).thesingularexceptioniszygnematales, wheretwophototropinhomologsarepresent(photaandphotb). Table/1:Reclassification/of/Physcomitrella.patens/phototropins/based/on/gene/ orthology./ Proposed(new(name Previous(annotation Genbank(No. PpPHOT1A(1 PpPHOTA1 XM_ PpPHOT1A(2 PpPHOTA2 XM_ PpPHOT1A(3 PpPHOTB3 XM_ PpPHOT1B PpPHOTA3 XM_ PpPHOT2B PpPHOTB2 XM_ PpPHOT2C(1 PpPHOTB1 XM_ PpPHOT2C(2 PpPHOTA4 XM_ Neochrome(NEO,Figure10,Figure11)isauniquephototropinvariantthatpossesses supplementaryred/far<red<sensingdomainsfromphytochromes(nozueetal.,1998).recent studieshaverevealedtwoindependentoriginsofneochromes,oneinzygnemataleanalgaeand theotherinhornworts(suetsuguetal.,2005;lietal.,2014;chapter3),andthattheneochromes foundinfernswerederivedfromhornwortsviahorizontalgenetransfer(lietal.,2014;chapter 3).Neochromeperceivesbothblueandred/far<redlighttomediatephototropismandchloroplast movement(kanegaeetal.,2006;kawaietal.,2003)inferns,anditappearstohaveplayeda significantroleintheirdiversification(schneideretal.,2004;schuettpelzandpryer,2009). Neochromefunctioninzygnemataleanalgae,however,isstillunclear.Becausezygnematalean 33

49 algaehaveplate<likechloroplaststhatrotateinresponsetobothblueandred/far<redlight irradiation(hauptandscheuerlein,1990),itwashypothesizedthatalgalneochrome,originally discoveredinmougeotia5scalaris,isthegenecandidateresponsible(suetsuguetal.,2005). However,neochromeinM.5scalarisrespondsonlytored/far<redlightandnottobluelight (Suetsuguetal.,2005).ToexplorewhetherM.5scalarismightbeanoutlieramongzygnematalean algaeinperhapshavinga defective neochrome,ifurtherinvestigatedallthealgalneochromes thatimined.idiscoveredthatnoneofthemhastheconservedcysteineresidueinthelov2 domain,thatisessentialforflavinmononucleotidechromophoreadductformationandbluelight signaltransduction(christie,2007).therefore,itislikelythatallzygnemataleanalgaeuse neochromeonlyforsensingred/far<redlight,anduseotherblue<lightphotoreceptors (phototropinsorcryptochromes)tomaneuverchloroplastrotations. 34

50 Arabidopsis thaliana Solanum lycopersicum Fragaria vesca Fig. 2a 75/+ Medicago truncatula 78/+ Citrus clementina Vitis vinifera Aquilegia coerulea Austrobaileya scandens Illicium floridanum Amborella trichocarpa Angiosperm PHOT1 Magnolia grandiflora Fig. 2b Goodyera pubescens 99/+ Zea mays Smilax bona-nox 86/+ Cunninghamia lanceolata Thuja plicata Fig. 2c 83/+ 99/+ Cephalotaxus harringtonia Gymnosperm Podocarpus rubens Gnetum montanum PHOT1 Welwitschia mirabilis Stangeria eriopus Seed 77/+ Arabidopsis thaliana Citrus clementina Fragaria vesca plants PHOT1 Medicago truncatula A 88/+ Solanum lycopersicum Vitis vinifera PHOT2 Aquilegia coerulea Angiosperm Goodyera pubescens 94/+ Smilax bona-nox PHOT2 Zea mays Magnolia grandiflora Austrobaileya scandens Illicium floridanum Amborella trichocarpa 94/+ Thuja plicata Cunninghamia lanceolata Cephalotaxus harringtonia Gymnosperm Podocarpus rubens Welwitschia mirabilis PHOT2 Gnetum montanum Stangeria eriopus Polystichum acrostichoides 91/+ Polypodium hesperium 90/+ Leucostegia immersa Cystopteris reevesiana 86/+ Pteridium aquilinum Polypodiales 93/+ Adiantum capillus-veneris PHOT1 Gaga arizonica Ceratopteris thalictroides 95/+ Lonchitis hirsuta Salviniales Leptosporangiate ferns Pilularia globulifera Dipteris conjugata Gleicheniales PHOT1 PHOT1 Osmunda sp. 98/+ Osmundales PHOT1 Danaea nodosa Danaea nodosa Marattiales PHOT1 77/+ Danaea nodosa Onoclea sensibilis Blechnum spicant Athyrium filix-femina Woodsia scopulina 70/+ Homalosorus pycnocarpos Gymnocarpium dryopteris Cystopteris reevesiana Polypodium hesperium Davallia fejeensis To Fig. 2b Leucostegia immersa Polystichum acrostichoides Asplenium platyneuron PHOT1 Dennstaedtia davallioides Polypodiales Pteridium aquilinum B 82/.99 Adiantum capillus-veneris PHOT2 93/+ PHOT2 Adiantum aleuticum Vittaria lineata Gaga arizonica 74/.99 Pityrogramma trifoliata Pteris vittata 99/+ Pityrogramma trifoliata Cryptogramma acrostichoides Ceratopteris thalictroides 88/+ Lindsaea linearis Lonchitis hirsuta 98/+ Thyrsopteris elegans Plagiogyria japonica Salviniales PHOT2 Pilularia globulifera Leptosporangiate Azolla caroliniana Cyatheales PHOT2 Anemia tomentosa ferns Lygodium japonicum Schizaeales PHOT2 97/+ Dipteris conjugata Gleicheniales PHOT2 Ferns Osmunda sp. Osmundales PHOT2 Botrypus virginianus 99/+ Sceptridium dissectum Ophioglossum Psilotum nudum Tmesipteris parva Ophioglossales PHOT1/2 Psilotales PHOT1/2 Equisetum hyemale Equisetales PHOT1/2 0.1 substitution/site 35

51 Figure/9:/Phylogeny/of/seedCplant/and/fern/phototropins./Orangecirclesindicateinferred photropinduplicationevents.theitalicizedcapitalletterwithineachcirclecorrespondstotheduplication eventmentionedinthetext,andthenumbers/lettersadjacenttoeachorangecirclearethenamesofthegene duplicates.thevaluesassociatedwithbranchesaremaximumlikelihoodbootstrapvalues/bayesian posteriorprobabilities. indicatesthattheexactphylogeneticpositionofthegeneduplicationeventis ambiguous. 36

52 To Fig. 11 Fig. 9 Fig. 10 Fig. 11 Hornworts To Fig. 9 Selaginella moellendorffii 1-2 Selaginella moellendorffii 1-1 Selaginella willdenowii Selaginella 96/.98 Selaginella kraussiana Selaginella acanthonota PHOT1 Selaginella selaginoides Selaginella moellendorffii /+ Selaginella moellendorffii 2-2 Selaginella willdenowii Selaginella C Selaginella kraussiana PHOT2 Selaginella acanthonota Isoetes tegetiformans Isoetes PHOT 84/+ Dendrolycopodium obscurum 96/+ Lycopodium deuterodensum Diphasiastrum digitatum Lycopodiales Pseudolycopodiella caroliniana Huperzia lucidula PHOT Phylloglossum drummondii 81/+ Porella pinnata Radula lindenbergia Scapania nemorosa 85/+ Bazzania trilobata Schistochila sp Metzgeria crassipilis Liverwort Pellia neesiana PHOT 96/+ Marchantia polymorpha 72/+ 99/+ Conocephalum conicum Lunularia cruciata Sphaerocarpos texanus Liverworts 93/+ Rhynchostegium serrulatum Neckera douglasii 95/+ Loeskeobryum brevirostre Bryidae Leucodon brachypus Orthotrichum lyellii Bryum argenteum 91/+ Bryopsida Scouleria aquatica Ceratodon purpureus PHOT1A 98/+ Dicranidae Aulacomnium heterostichum 95/+ Physcomitrella patens PHOTA2 Physcomitrella patens PHOTB3 Funariidae 74/+ 82/+ Physcomitrella patens PHOTA1 Buxbaumiidae Buxbaumia aphylla Polytrichopsida Atrichum angustatum PHOT1A Neckera douglasii PHOT1A Rhynchostegium serrulatum 99/+ E 71/.98 Leucodon brachypus Loeskeobryum brevirostre PHOT1B Bryopsida Bryidae Aulacomnium heterostichum 99/+ Orthotrichum lyellii PHOT1B Bryum argenteum Funariidae Physcomitrella patens PHOTA3 Polytrichopsida 93/+ Atrichum angustatum PHOT1B Sphagnum lescurii Sphagnopsida PHOT1 Neckera douglasii 89/+ Loeskeobryum brevirostre Rhynchostegium serrulatum 81/+ Leucodon brachypus Aulacomnium heterostichum Bryopsida Bryidae Orthotrichum lyellii 99/+ PHOT2A Bryum argenteum PHOT1 98/+ Ceratodon purpureus 73/.98 Scouleria aquatica D Dicranidae PHOT2A 94/+ Leucodon brachypus PHOT2 71/+ G Bryidae Neckera douglasii PHOT2B Rhynchostegium serrulatum 87/+ Orthotrichum lyellii Bryopsida Dicranidae Scouleria aquatica F PHOT2B Fissidens adianthoides Funariidae 2C Physcomitrella patens PHOTB2 Funariidae Physcomitrella patens PHOTB1 Bryopsida Physcomitrella patens PHOTA4 PHOT2C Buxbaumiidae Buxbaumia aphylla Bryopsida PHOT2 Atrichum angustatum Polytrichopsida PHOT2 97/+ Andreaea rupestris Andreaeopsida PHOT2 Sphagnum lescurii Sphagnopsida PHOT2 Takakia lepidozioides Takakiopsida PHOT2 Dennstaedtia punctilobula 73/+ Plagiogyria distinctissima 97/+ Phegopteris hexag Diplazium wichurae 95/+ Fern NEO Allantodia dilatata Dipteris conjugata Adiantum capillus veneris 95/+ Hemidictyum marginatum 89/+ Adiantum tenerum Nothoceros aenigmaticus Megaceros flagellaris Phaeomegaceros coriaceus 87/.99 Phymatoceros phymatodes Hornwort NEO 99/+ Paraphymatoceros hallii 95/+ Phaeoceros carolinianus Anthoceros punctatus Anthoceros bhardwajii Anthoceros puncatatus 97/+ Megaceros tosanus 93/+ Megaceros aenigmaticus 99/+ Phaeomegaceros coriaceus Hornwort PHOT 99/+ Phymatoceros phymatodes Phaeoceros carolinianus 0.1 substitution/site Paraphymatoceros hallii Lycophytes Mosses Neochrome 99/+ 37

53 Figure/10:/Phylogeny/of/lycophyte/and/bryophyte/phototropins./Orangecirclesindicateinferred photropinduplicationevents.theitalicizedcapitalletterwithineachcirclecorrespondstotheduplication eventmentionedinthetext,andthenumbers/lettersadjacenttoeachorangecirclearethenamesofthegene duplicates.thevaluesassociatedwithbranchesaremaximumlikelihoodbootstrapvalues/bayesian posteriorprobabilities. indicatesthattheexactphylogeneticpositionofthegeneduplicationeventis ambiguous. 38

54 98/+ To Fig /+ Mesotaenium kramstei 75/+ Zygnemopsis sp Cylindrocystis cushleckae Fig. 9 Mesotaenium caldariorum 98/+ Mougeotia scalaris Zygnematales Cylindrocystis brebissonii 2 PHOTB PHOTB Cylindrocystis sp 2 98/+ Cylindrocystis brebissonii 1 F Cylindrocystis sp 1 Mesotaenium kramstei PHOTA 96/+ Fig. 10 Zygnemopsis sp Cylindrocystis cushleckae Zygnematales Mougeotia scalaris Mesotaenium caldariorum PHOTA Desmidium aptogonum 81/+ Cosmarium tinctum Fig /+ Staurodesmus convergens Penium exiguum Demidiales Phymatodocis nordstedtiana Planotaenium ohtanii PHOT Gonatozygon kinahanii Roya obtusa 89/+ Mesotaenium endlicherianum Mesotaenium braunii Zygnematales PHOT Mougeotia scalaris 91/+ Mougeotia scalaris NEO1 NEO2 Mesotaenium caldariorum 99/+ Cylindrocystis cushleckae Zygnemopsis sp Cylindrocystis brebissonii 2 Cylindrocystis sp 2 Cylindrocystis brebissonii 1 Cylindrocystis sp 1 79/+ Mesotaenium braunii Mesotaenium braunii Mesotaenium endlicherianum Coleochaete irregularis Coleochaete scutata Chaetosphaeridium globosum Coleochaetales PHOT Interfilum paradoxum Klebsormidium subtile Entransia fimbriata Klebsormidioales PHOT Spirotaenia minuta Chlorokybus atmophyticus Mesostigma viride Mesostigmatales PHOT 97/+ Pediastrum duplex Scenedesmus dimorphus Cylindrocapsa geminella 93/+ Volvox carteri Chlamydomonas reinhardtii Chloromonas tughillensi 74/+ Oogamochlamys gigantea Heterochlamydomonas inaequalis Brachiomonas submarina Carteria obtusa Chlorophyceae PHOT Hafniomonas reticulata 99/+ 99/+ Aphanochaete repens Hormidiella sp Fritschiella tuberosa Oedogonium foveolatum Oedogonium cardiacu 99/+ Trebouxia arboricola 99/+ Prasiola crispa Coccomyxa pringsheimii Trebouxiophyceae PHOT Botryococcus terribilis Scherffelia dubia Tetraselmis cordiformis Prasinophyte PHOT 88/+ Persursaria percursa Entocladia endozoica Bolbocoleon piliferum Ulvophyceae PHOT Helicodictyon planctonicum Pyramimonas parkeae Scourfieldia sp Nephroselmis olivace Dolichomastix tenuilepi Ostreococcus lucimarinus Prasinophyte PHOT Ostreococcus tauri Micromonas pusilla Pycnococcus provasolii 0.1 substitution/site Streptophytes 97/+ Zygnematales NEO Figure/11:/Phylogeny/of/algal/phototropins./Orangecirclesindicateinferredphotropinduplication events.theitalicizedcapitalletterwithineachcirclecorrespondstotheduplicationeventmentionedinthe text,andthenumbers/lettersadjacenttoeachorangecirclearethenamesofthegeneduplicates.thevalues associatedwithbranchesaremaximumlikelihoodbootstrapvalues/bayesianposteriorprobabilities. indicatesthattheexactphylogeneticpositionofthegeneduplicationeventisambiguous. 39

55 2.3 Discussions Myphototropinphylogenyrefutesthepreviousassertionthat PHOT2 istheancestral phototropinandthat PHOT1 evolvedlaterinseedplants(galván<ampudiaandoffringa, 2007).Theancestralphototropinisneither PHOT1 nor PHOT2,becausetheirparalogsin seedplants,lycophytes,fernsandmosseswerederivedfromseparategeneduplicationsthatare confinedtoeachorganismallineage.inotherwords,seed<plantphot15andphot2aremore closelyrelatedtooneanotherthantofernphotsormossphots.myrevisedgeneorthologyhas importantfunctionalandevolutionaryimplications.plantsoftenresponddifferentlyunderlow< andhigh<lightlevels;chloroplasts,inparticular,aggregateunderweaklightbutretreatwhenthe intensityistoohigh.consequently,asshowninourphylogeneticreconstruction,phototropin paralogshaverepeatedly,andconvergently,specializedintomediatingeitherlow<orhigh<light responses,inseedplants,ferns,lycophytesandmosses,althoughsomeredundanciesdoexist (Christie,2007).OfthetwophototropinsknowninArabidopsis5thaliana,Atphot1mediates phototropismunderlow<lightintensity,andismoresensitivethanatphot2intriggering chloroplastaccumulation(sakaietal.,2001).atphot2,incontrast,respondspredominantlyto high<lightintensity,andissolelyresponsibleforchloroplastavoidanceunderstronglight (Kagawaetal.,2001).AsimilarfunctionaldifferentiationcanalsobeseeninthefernAdiantum5 capillusevenerisacphot1andacphot2phototropins.acphot2controlschloroplastavoidance underhigh<lightintensity,whereasacphot1playsaminorroleinthisresponse(kagawaetal., 2004).Similarly,Kasaharaetal.(2004)examinedfourphototropinsinthemossPhyscomitrella5 patens,andfoundthatppphot1a<2(seetable1)isofprimaryimportanceinchloroplast avoidancebehavior,whereastheotherscontributetothisresponsetoamuchlessextent. 40

56 Tounderstandhowphototropinfunctionaldivergences(subfunctionalizations) repeatedlyevolvedinplants,thekeyistoreconstructthefunctionofancestralphototropinthat existsasasingle<copygene.intheirrecentstudyoftheliverwortmarchantia5polymorpha phototropin(singlecopy),komatsuetal(2014)foundthatitencompassesallthefunctional characteristicsofbothatphot1andatphot2.thisfindingsuggeststhattheancestrallandplant phototropinwaslikelya general<purpose photoreceptorthatrespondedtoawiderangeof lightintensities.thesubsequentandparallelspecializationsofphototropinintolow<andhigh< lightintensityfunctionalresponsesmayhaveplayedanimportantroleintheadaptationofearly landplantstoearth schanginglandscapes.sincetheformationoftheearliestforestsbyextinct fernsandhorsetails(cladoxylopsids)about385millionyearsago(steinetal.,2007)throughto today sangiosperm<dominatedterrestrialecosystems,lightenvironmentshavebecome increasinglyheterogeneousanddynamic.possessingduplicatedphototropingenesandco< optingthemfordifferentlightintensitieswouldbeespeciallybeneficial(galenetal.,2004)and advantageousovertheancestral,general<purposephototropin.indeed,mostofthelandplant lineagesthatpossessduplicatedphototropinhomologs(seedplants,ferns,lycophytes,and mosses)aremorespeciesrichthanthosethatdonot(liverwortsandhornworts). TheevolutionaryhistorypatternthatIobservehereforphototropinsshowsastriking resemblancetothatforphytochromes.asimilarsequenceofconvergentevolutionaryevents followinggeneduplication hasalsobeenreportedforphytochromesacrossallmajorplant lineages(chapter1).bothphotoreceptors(phytochromesandphototropins)duplicated repeatedlyinseedplants,ferns,lycophytesandmosses,whiletheyremainedsingle<copyin liverwortsandhornworts(figure9).althoughthispatternofconcertedgenefamilyexpansion 41

57 andstasiscouldbeduetowholegenomeduplications(wgd),thesetwophotoreceptorsdifferin theexactevolutionarypositionsofgeneduplicationevents theydidnotallhappenalongthe samephylogeneticbranches(figure9),suggestingthatwgdisnotsolelyresponsible.ipropose herethattherehasbeenatightco<evolutionaryrelationshipbetweenphototropinsand phytochromes.recentstudieshaveshownthatthesetwophotoreceptorsnotonlysharecross< talkintheirsignaltransductionpathways(lariguetetal.,2006;decarbonneletal.,2010; Demarsyetal.,2012),butalsocanphysicallyinteract(Jaedickeetal.,2012).Inaddition,the convergentevolutionandhorizontalgenetransferofneochromes(suetsuguetal.,2005;lietal., 2014Chapter3)furtherillustratethat,throughoutplantevolutionaryhistory,atightpartnership hasresultedbetweenthetwophotoreceptors.ihypothesizethattheintegrationofbothblueand red/far<redlightinformationenabledplantstorespondoptimallytochangingenvironments throughtime.duplicationofonephotoreceptormayhavepromptedduplicationintheother,and henceresultedintheratherparallelgenefamilyevolutionaryhistories. Insummary,hereIleveragedtherecentsurgeingenomicandtranscriptomicdatato identifyphototropinsfromacrossabroadrepertoireofextantbiodiversity.mystudyrevealsthat phototropinsareuniquetoviridiplantae,andthatgenefamilyexpansionandstasishasoperated uniquelywithineachofthevariouslandplantlineages apatternsimilartothatofthe phytochromephotoreceptor.existingfunctionaldataforphototropins,interpretedinlightofmy genephylogeny,suggestsahistoryofrepeatedgeneduplicationsfollowedbyparallelfunctional divergences(subfunctionalizations).ourbroadphylogeneticapproachgreatlycomplements ongoingphotobiologyresearchfocusedonselectplantmodelorganisms,andwillenablefuture 42

58 researchlinkingecology,evolution,andphotochemistrytounderstandinghowplantsadapt(and haveadapted)tovariablelightenvironments. 2.4 Materials and Methods Mining phototropins from transcriptomes and genomes ThetranscriptomesandgenomesIusedarelistedinAppendixTable4,Table5.Tomine phototropinhomologs,iusedthebluedevilpythonpipelinefollowinglietal(2014)for transcriptomes,andforgenomesiusedblastpimplementedinphytozome(goodsteinetal., 2012)orindividualgenomeportal(AppendixTable4,Table5).Aphototropinsequencefrom Anthoceros5bhardwajii(voucher:Villarreal#6)wasobtainedbyPCRandcloning(primers: photf1970andphotr4102,seeappendixtable9) Sequence alignment and phylogenetic reconstruction IusedMUSCLE(Edgar,2004)toaligntheaminoacidsequences,andthenback< translatedthesetonucleotides.theresultingalignmentwasmanuallyimprovedbasedonknown domainandmotifboundaries,andunalignableregionswereexcludedpriortophylogenetic analyses.iusedpartitionfinder(lanfearetal.,2012)toobtaintheoptimaldatapartitionscheme (bycodonposition)andtheassociatednucleotidesubstitutionmodels.garli2.0(zwickl,2006) wasemployedtofindthebestmaximumlikelihoodtreewith genthreshfortopoterm setto 500,000and8independentruns.Icarriedoutbootstrappingtoassessbranchsupport,using RAxML(Stamatakis,2006)with1,000replicates.Thesamepartitionschemeandmodelswere usedinmrbayes3.2(ronquistetal.,2012)bayesianinference.icarriedouttwoindependent MCMCruns,eachwithfourchainsandtreessampledevery1,000generations.Iunlinked substitutionparametersandsettheratepriortovaryamongsubsets.theresultingmcmc 43

59 statisticswereinspectedintracer(rambautanddrummond,2013)toensureconvergenceand propermixing;25%ofthetotalgenerationswerediscardedasburn<inbeforecompilingthe50% majorityconsensustree.ialsocarriedoutphylogeneticreconstructionbasedoncodonmodels. CodonPhyML(Giletal.,2013)wasused,withGoldman<Yangcodonsubstitutionmodel (GoldmanandYang,1994),empiricalcodonfrequency(F1X61)andthreecategoriesofnon< synonymous/synonymoussubstitutionrateratio Target enrichment for confirming phototropin copy number in hornworts ThetargetenrichmentdatawerefromChapter1,wherebyahornwort(Anthoceros5 punctatus)dnalibrarywashybridizedwith7,502120merrnaprobestoenrichphototropin, phytochromeandneochromehomologs.thecapturedfragmentsweresequencedonone<tenthof amiseq(250bppe)run.iusedsickle(joshiandfass,2011)andcutadapt(martin,2011)toclean andtrimthereads,respectively,andassembledusingsoapdenovo(luoetal.,2012).the phototropincontigswereidentifiedbytblastn. 44

60 3. The origin and evolution of neochromes Li,F.<W., 325coEauthors,S.Mathews,andK.M.Pryer.2014.Horizontal/transfer/of/an/ adaptive/chimeric/photoreceptor/from/bryophytes/to/ferns.proceedingsofthenational AcademyofSciences,USA111:6672< Introduction Plantgrowthanddevelopmentaremodulatedbyphotoreceptorsystemsthatprovide informationaboutthesurroundingenvironment.majorpeaksintheactionspectraofthese informationalphotoreceptorslieeitherintheuv<blue(e.g.,cryptochromesandphototropins)or red/far<red(phytochromes)lightregions(möglichetal.,2010).thechimericphotoreceptor, neochrome,isaremarkableexception.itfusesred<sensingphytochromeandblue<sensing phototropinmodulesintoasinglemolecule(figure12a)thatmediatesphototropicresponses (Nozueetal.,1998;Kawaietal.,2003;Kanegaeetal.,2006).Neochromeshavearestricted occurrenceintheplanttreeoflife,andtwoindependentorigins(suetsuguetal.,2005) onein thegreenalgamougeotia5scalarisandanotherinferns suggestingthatthepossessionof neochromemaybeevolutionarilyadvantageous.thisisconsistentwithevidenceofgreatly enhancedphototropicresponsesinfernswithneochrome(kawaietal.,2003;kanegaeetal., 2006),aswellasitsphylogeneticdistributionwithinthefernlineage.Theearly<divergingfern ordersosmundalesandschizaealesdonotpossessneochrome(kawaietal.,2003).ithasbeen reportedonlyincyatheales(yangetal.,2010)andpolypodiales(kawaietal.,2003;yangetal., 2010),lineagesthatmostlydiversifiedfollowingtheCretaceous/Tertiaryestablishmentoflow< light,angiosperm<dominatedforestcanopies(schneideretal.,2004;schuettpelzandpryer,2009). 45

61 Asaresult,ithasbeensuggestedthattheevolutionofneochromewasakeyinnovationthat conferredaphototropicadvantageonfernsgrowingunderlow<lightconditions,facilitatingtheir moderndiversificationinthe shadowofangiosperms (Schneideretal.,2004;Schuettpelzand Pryer,2009;Kawaietal.,2003).Althoughpotentiallysignificantfromanevolutionarystandpoint, theoriginoffernneochromehasremainedamystery,andnopreviousstudyhasrevealedhowit mighthaveevolved. Inthisstudy,Iinvestigatedtheoriginofneochromebysearchingforhomologous sequencesin434transcriptomesand40wholegenomesofplantsandalgae5(appendixtable6), andsurprisinglydiscoveredneochromehomologsfromhornworts(figure12b,appendixtable 6).Analysesofthehornwortdraftgenome(Anthoceros5punctatus)suggestthatneochrome originatedinhornworts,independentfromthegreenalgae.large<scalephylogeneticanalyses anddivergencetimeestimationsfurtherdemonstratethatfernsacquiredneochromefrom hornwortsviahorizontalgenetransfer(hgt). 3.2 Results and Discussions Algal neochrome Theonlypublishedalgalneochromeisfromasinglespecies,5Mougeotia5scalaris5(Suetsugu etal.,2005).iidentifiedhomologsofneochromeinthetranscriptomesofall10sampledmembers ofthe Zygnemataceae superclade[sensugoncharovandmelkonian(2010)],including Mougeotia,5Mesotaenium,Cylindrocystis,andZygnemopsisbutinnootheralgaltranscriptomes surveyed(figure12,appendixtable6,figure18). 46

62 A 5 3 phytochrome neochrome phototropin B Angio. PHOT1 Gymno. PHOT1 C HGT Fern NEO HGT 179 MYA Angio. PHOT2 Gymno. PHOT2 Fern PHOT1 Fern PHOT2 Lycophyte PHOT Moss PHOTA Moss PHOTB Liverwort PHOT Fern NEO Hornwort NEO Hornwort PHOT D Nothoceros Megaceros Phymatoceros Hornwort Paraphymatoceros NEO Phaeoceros Anthoceros Nothoceros Megaceros Phymatoceros Paraphymatoceros Phaeoceros Anthoceros gene fusion Hornwort PHOT 0.1 substitutions/site horizontal gene transfer Algae PHOT retrotransposition Algae NEO conventional PHOT with introns Algae PHOT MYA Figure/12:/The/origin/of/fern/neochrome./(A)Neochromeisachimericphotoreceptorinwhichthe N<terminusconsistsofaphytochromesensorymodulefusedtoanalmostcompletephototropinsequenceat thec<terminus.thickandthinlinesrepresentexonsandintrons,respectively;lengthnottoscale.(b)dated phylogenyofphototropinandneochrome,showingneochromehgtfromhornwortstoferns(detailsin Figure/21).(C)Portionofthephototropinphylogeny,showingrelationshipsoffernneochrome,hornwort phototropinandneochrome,withhighlysupportedbranchesthickened(detailsinfigure/13).(d)a schematicdepictingtheoriginoffernneochrome Novel neochrome in hornworts Amonglandplants,Idocumentedtheoccurrenceofneochromein25additionalfern species(figure13,figure14).surprisingly,ialsodiscoveredneochromeinhornworts,asmall 47

63 bryophytelineagethatdivergedearlyinthehistoryoflandplants.althoughtheexactbranching orderamongthethreebryophytelineages(hornworts,mosses,liverworts)isnotresolvedwith certainty,somerecentanalyseshavesuggestedthathornwortsaresistertovascularplants (lycophytes,ferns,andseedplants;qiuetal.,2006).iempiricallyconfirmedthepresenceof neochromeinhornwortsthroughpcrandcloning,andisolatedneochromesequencesfromthe generanothoceros,megaceros,phymatoceros,phaeoceros,5paraphymatoceros5andanthoceros, representingfouroutofthefivehornwortorders(dendrocerotales,phymatocerotales, Notothyladales,andAnthocerotales).Iwasunabletoobtainadequatematerialofthemonotypic hornwortleiosporocerostotestforthepresenceofneochromeinleiosporocerotales.toconfirm thatthehornwortneochromesequencedatawereindeedderivedfromthehornwortnuclear genomeandnotfromcontaminantalgaeorferns,iperformedgenome<walkinginnothoceros aenigmaticustoobtainflankinggenomicsequences.downstreamofifoundapseudogenefor imidazoleglycerol<phosphatedehydratase(igpd)and,becauseitssequenceismostclosely relatedtootherhornwortigpdgenes(figure20),iareconfidentthatneochromeispresentinthe hornwortgenome Neochrome HGT from hornworts to ferns Thephylogeneticdistributionofneochromeinlandplants(presentonlyinhornworts andferns)couldbeexplainedby1)anancientoriginalongthebranchthatuniteshornwortsand tracheophytes,followedbylossesfromlycophytesandseedplants,2)independentoriginsin fernsandhornworts,or3)oneormoreinstancesofhorizontalgenetransfer(hgt)between hornwortsandferns.todistinguishamongthesethreepossiblescenarios,icompiled comprehensivesequencealignmentsofphototropinandphytochromefromacrossalllandplants 48

64 andalgae,whichincludedthecorrespondingdomainsfromhornwortandfernneochromes,and evaluatedtheresultantgenephylogenies.maximumlikelihoodandbayesianestimatesof phototropinandphytochromephylogeniesrevealedthatfernneochromesareembeddedwithin hornwortneochromeswithverystrongbranchsupport(figure12bandc,figure13,appendix Figure18,Figure19).Thisnestedrelationshipindicatesthatneochromewastransferred horizontallyfromhornwortstoferns,alongthestemlineageleadingtophymatoceros+nothoceros +Megaceros(Figure13,AppendixFigure18,Figure19).Thealternativepossibilities,suggesting eitheranancientverticaltransferofneochrome(i.e.,fernandhornwortneochromeswere reciprocallymonophyletic)oranindependentoriginofneochrome(i.e.,fernneochromeswere monophyleticwitheitherfernphototropinsorphytochromes)werebothrejected(p<10 30 )and wereneverobservedinthebayesianposteriortreesamples. IuseddivergencetimeestimatestofurthertesttheHGThypothesis,reasoningthat,ina caseofhgt,thesplitbetweenhornwortandfernneochromeshouldbesignificantlyyounger thanthesplitbetweenthehornwortandfernlineagesthemselves.byintegratingfossil calibrations(appendixtable7)withabayesianrelaxedmolecularclockanalysis,iestimatedthe divergencedatebetweenhornwortandfernneochrometobeapproximately179millionyears ago(mya)witha95%highestposteriordensityintervalof133and229mya(figure12, AppendixFigure21).Thisdateisfarmorerecentthanpublisheddivergenceestimatesbetween fernsandhornworts(atleast400mya;hedgesandkumar,2009),butiscongruentwiththedate estimatesforthestembranchleadingtophymatoceros5+nothoceros5+megaceros(85<244mya; VillarrealandRenner,2012).Thedisparityindivergencetimesrejectsthehypothesisinvoking multipleneochromeoriginsorlossesandreinforcesthehgtscenario. 49

65 Theoriginoflandplantneochromewithinthehornwortlineageissupportedbyits relationshiptohornwortphototropin.thesinglehornwortphototropingeneintheanthoceros5 punctatusdraftgenomecompletelylacksintrons(figure12d),andthuscloselyresemblesthec< terminalendofbothfernandhornwortneochromes.ifoundthisintron<freephototropininall hornwortsexamined,byusingpcrongenomicdna.allotherphototropinscharacterizedto date,includingthoseofferns,containmorethantwentyintrons.iexploredwhetherthismightbe apartialneochromemasqueradingasaphototropinbyusinginversepcrtoobtainthe5 upstreamgenomicregioninnothoceros5aenigmaticus.multiplestopcodonswereencountered upstreamofthenothoceros5phototropingene,andtherewasnoindicationofnearbyphytochrome domains.thesedatasuggestthathornwortsdonothaveacanonicalphototropingene.instead, hornwortphototropinsaremostcloselyrelatedtofernandhornwortneochromes(figure12, Figure13,AppendixFigure19),implyingthattheylikelyrepresenttheancestral,retrotransposed phototropinlineagethatgaverisetoneochromethroughfusionwiththephytochromemodule (Figure12D) Recurrent fern-to-fern HGT Idetectedanextraordinaryincongruencebetweenmyfernneochromegenetreeandthe publishedphylogenyofferns(figure14)(schuettpelzandpryer,2007).byexaminingtheentire Bayesianposteriortreesample,Ifoundthatnoneofthetreesresolvedneochromesfromthesame fernfamilytobemonophyletic.thisconflictingpatternisnotobservedinotherfernphylogenies basedonnucleargenes(rothfelsetal.,2013),andisnotseeninthehornwortneochrometree (Figure13),whichperfectlymirrorsthepublishedphylogenyofhornworts(VillarrealandRenner, 50

66 2012).HereIexamineanddiscussthepossiblecausesoftheincongruentgenetree/speciestreein ferns. Go to Appendix Fig. S1 Figure18 93/93/98/+/+ Phegopteris hexagonoptera KJ Athyrium filix-femina AFPO KJ Deparia acrostichoides KJ Adiantum aleuticum WCLG KJ Onoclea sensilibis KJ Thelypteris noveboracensis KJ /96/98/+/+ Macrothelypteris torresiana KJ Homalosorus pycnocarpos OCZL KJ Coniogramme intermedia var glabra FJ Plagiogyria distinctissima FJ Alsophila podophylla KJ Dennstaedtia punctilobula KJ Pronephrium lakhimpurense FJ /97/98/+/+ Dryopteris amurensis KJ Allantodia dilatata FJ /83/96/+/+ Diplazium wichurae UFJN KJ Blechnum spicant KJ /93/96/+/+ Plagiogyria japonica UWOD KJ Dipteris conjugata MEKP KJ /+/98/+/+ Lindsaea linearis NOKI KJ /99/96/+/+ Adiantum capillus-veneris AB /85/93/+/+ Hemidictyum marginatum KJ Hypolepis tenuifolia KJ Adiantum raddianum BMJR KJ /99/95/+/+ Nothoceros aenigmaticus KJ Megaceros flagellaris UCRN KJ /89/95/+/+ Phymatoceros phymatodes KJ Paraphymatoceros hallii FAJB KJ /+/98/+/+ Phaeoceros carolinianus WCZB KJ Anthoceros punctatus KJ /+/99/+/+ Nothoceros aenigmaticus KJ Megaceros flagellaris UCRN KJ /99/+/+/+ 87/89/88/+/+ Phymatoceros phymatodes KJ Paraphymatoceros hallii FAJB KJ Phaeoceros carolinianus WCZB KJ Anthoceros punctatus KJ substitutions/site Fern NEO Hornwort NEO Hornwort PHOT / Figure/13:/Phylogenetic/relationships/of/fern/neochrome/(NEO),/hornwort/neochrome/and/ phototropin/(phot)./topologyderivedfromthebestmaximumlikelihoodtree.numbersabovebranches aremaximumlikelihoodbootstrapvalues(bs)fromgarli/bsfromnhphyml/alrtsh<likesupportsunder codonmodel(alrt<sh)/bayesianposteriorprobabilities(pp)frommrbayes/ppfrombeast;theseareonly displayed(alongwiththickenedbranches)whenbs>70,sh<alrt>70andpp> denotesbs=, alrt<sh=orpp=1.00;thickenedbrancheswithoutnumbersare +/+/+/+/+.Alphanumericcodes followingspeciesnamesarethefour<letter1kptranscriptomeidentifiers,genbankaccessionsorboth; indicatesthesequencecamefromgenomesequencedata,and frompteridium5aquilinumtranscriptome. Theblue,orangeandyellowbranchesrepresenthornwortphototropin,hornwortneochromeandfern neochrome,respectively.// 51

67 Gene Tree Species Tree Hornwort NEO 94/+/.99 95/+/+ 98/95/+ 98/97/+ 99/+/+ 99/97/ substitutions/site Deparia lancea Deparia lobato-crenata Deparia acrostichoides Onoclea sensilibis Adiantum tetraphyllum Pronephrium lakhimpurense Thelypteris noveboracensis Athyrium filix-femina Phegopteris hexagonoptera Matteuccia struthiopteris Homalosorus pycnocarpos Macrothelypteris torresiana Coniogramme intermedia Tectaria zeylanica Didymochlaena truncatula Plagiogyria distinctissima Alsophila podophylla Dennstaedtia punctilobula Adiantum aleuticum Adiantum pedatum Adiantum andicola Deparia acrostichoides Deparia lancea Dryopteris expansa Dryopteris amurensis Dryopteris filix-mas Diplazium wichurae Diplazium bombonasae Allantodia dilatata Blechnum spicant Plagiogyria japonica Plagiogyria formosana Dipteris conjugata Adiantum raddianum Doodia media Adiantum hispidulum Bolbitis auriculata Hypolepis tenuifolia Hypolepis punctata Lindsaea linearis Adiantum capillus-veneris Hemidictyum marginatum Deparia lancea Deparia lobato-crenata Deparia acrostichoides Diplazium wichurae Diplazium bombonasae Allantodia dilatata Athyrium filix-femina Doodia media Blechnum spicant Matteuccia struthiopteris Onoclea sensilibis Thelypteris noveboracensis Pronephrium lakhimpurense Macrothelypteris torresiana Phegopteris hexagonoptera Homalosorus pycnocarpos Hemidictyum marginatum Dryopteris expansa Dryopteris amurensis Dryopteris filix-mas Bolbitis auriculata Tectaria zeylanica Didymochlaena truncatula Adiantum aleuticum Adiantum pedatum Adiantum andicola Adiantum capillus-veneris Adiantum tetraphyllum Adiantum hispidulum Adiantum raddianum Coniogramme intermedia Hypolepis punctata Hypolepis tenuifolia Dennstaedtia punctilobula Lindsaea linearis Plagiogyria japonica Plagiogyria formosana Plagiogyria distinctissima Alsophila podophylla Dipteris conjugata Cyatheales Gleicheniales Polypodiales Figure/14: Phylogenetic/incongruence/between/fern/neochrome/gene/tree/and/fern/species/tree./ Thegenetreetopologyisderivedfromthebestmaximumlikelihoodtreebasedonthenucleotide dataset,andthespeciestreesummarizedfromschuettpelzandpryer(1),kuoetal(2),rothfelsand Schuettpelz(3),andRothfelsetal(4).Treeinferencebasedoncodonmodels,1st+2ndand3rdcodon positionsyieldedsimilartopologies(fig.s7).closelyrelatedspecies/generaarecodedwiththesamecolor. Theneochromegenetreeisrootedwithhornwortneochromes(notshown).Numbersabovebranchesare maximumlikelihoodbootstrapvalues(bs)/alrtsupportsundercodonmodel(alrt)/bayesianposterior probabilitiesfrommrbayes(pp),andareonlydisplayed(alongwiththickenedbranches)ifbs>70,alrt> 70andPP> denotesbs=,alrt=orpp=1.00;thickenedbrancheswithoutnumbersare +/+/+.ArrowheadspointtothetwodivergentneochromecopiesfoundinDeparia5spp.Arrowspointto neochromesfromgleichenialesandcyathealesthatappearnestedamongpolypodialesneochromes.// Incompletesamplingofextantneochromehomologsisnotlikelytobetheexplanation, becauseneochromehasbeenshownbysouthernblottingtobeasingle<copygeneinadiantum5 capilluseveneris5(nozueetal.,1998).thiswascorroboratedbythecloningeffortsthatproduced mostofmysequencedata(appendixtable8).exceptfordeparia5spp.,wheretwodivergent 52

68 sequenceswerefound(figure14,arrowheads),iwasonlyabletoisolateasingleneochromefor eachfernspecies. Next,Iinvestigatedwhetheranaberrantnucleotidesubstitutionprocessmayhave misledthephylogeneticreconstruction.forexample,pervasivepositiveselectionorvariationin GCcontentcanobscuretruephylogeneticsignal(SandersonandShaffer,2002;Kapralovand Filatov,2007;Nabholzetal.,2011),therebycausingagenetreetobeincongruentwiththespecies tree.usingcodonmodelsfortreeinferencecanpotentiallyaccommodatecomplexselection profiles,byallowingdifferentnonsynonymous/synonymoussubstitutionrateratiostofallinto distinctclasses(giletal.,2013).however,ifoundthatincorporatingcodonmodelsdidnot improvetheincongruencebetweenthegenetreeandspeciestree;theresultanttreelargely matchesthatfromthenucleotidesubstitutionmodel,withcomparablebranchsupportvalues (Figure15A).Similarly,inferencesbasedonfirst+secondcodonpositions,aswellasonthird codonpositionsonly,alsoyieldedtopologiesdiscordantwiththespeciestree(figure15b,c). Ithenusedarandomeffectsbranch<sitemodeltoinferthedynamicsofpositiveselection acrosstheneochrometree(kosakovskypondetal.,2011).onlyfivefernbrancheswereidentified ashavingexperiencedsignificantepisodicpositiveselection(figure15d),andtheproportionof positivelyselectedcodonsitesalongeachofthesefivebranchesisverylow(<3%).theseresults suggestthatpositiveselectionoperatedonveryfewcodonsoveralimitednumberofbranches. Similarly,aslidingwindowanalysisofGCcontentfoundnoneofthefernsequencestobe deviantinbasecomposition(figure15e).takentogether,thenucleotidesubstitutionprocesses amongfernneochromesappeartobeunexceptional,andarenotlikelytoexplainthe incongruencebetweenthegenetreeandspeciestree. 53

69 A codon model Horwort NEO Deparia lancea Deparia lobato-crenata Deparia acrostichoides Athyrium filix-femina Phegopteris hexagonoptera Matteuccia struthiopteris Adiantum tetraphyllum Pronephrium lakhimpurense Onoclea sensilibis Deparia lancea Deparia acrostichoides Thelypteris noveboracensis Macrothelypteris torresiana Homalosorus pycnocarpos Coniogramme intermedia Tectaria zeylanica Didymochlaena truncatula Dennstaedtia punctilobula Alsophila podophylla Plagiogyria japonica Adiantum pedatum Adiantum aleuticum Adiantum andicola Dryopteris amurensis Dryopteris expansa Dryopteris filix-mas Diplazium bombonasae Diplazium wichurae Allantodia dilatata Blechnum spicant Plagiogyria formosana Plagiogyria distinctissima Dipteris conjugata Doodia media Adiantum raddianum Adiantum hispidulum Bolbitis auriculata Hypolepis punctata Hypolepis tenuifolia Lindsaea linearis Adiantum capillus-veneris Hemidictyum marginatum B 1 st + 2 nd position Horwort NEO Deparia lancea Deparia lobato-crenata Deparia acrostichoides Onoclea sensilibis Athyrium filix-femina Phegopteris hexagonoptera Matteuccia struthiopteris Deparia lancea Deparia acrostichoides Thelypteris noveboracensis Pronephrium lakhimpurense Adiantum tetraphyllum Adiantum aleuticum Adiantum pedatum Adiantum andicola Macrothelypteris torresiana Homalosorus pycnocarpos Coniogramme intermedia Alsophila podophylla Plagiogyria distinctissima Dennstaedtia punctilobula Tectaria zeylanica Didymochlaena truncatula Dryopteris amurensis Dryopteris expansa Dryopteris filix-mas Diplazium bombonasae Diplazium wichurae Allantodia dilatata Blechnum spicant Plagiogyria formosana Plagiogyria japonica Dipteris conjugata Adiantum raddianum Adiantum hispidulum Doodia media Bolbitis auriculata Hypolepis punctata Hypolepis tenuifolia Adiantum capillus-veneris Lindsaea linearis Hemidictyum marginatum C 3 rd position 0.3 substitutions/site 0.05 substitutions/site 0.3 substitutions/site Horwort NEO Deparia lancea Deparia lobato-crenata Deparia acrostichoides Thelypteris noveboracensis Pronephrium lakhimpurense Adiantum tetraphyllum Onoclea sensilibis Macrothelypteris torresiana Homalosorus pycnocarpos Athyrium filix-femina Phegopteris hexagonoptera Matteuccia struthiopteris Deparia lancea Deparia acrostichoides Coniogramme intermedia Tectaria zeylanica Didymochlaena truncatula Dennstaedtia punctilobula Alsophila podophylla Plagiogyria distinctissima Adiantum aleuticum Adiantum pedatum Adiantum andicola Dipteris conjugata Plagiogyria japonica Plagiogyria formosana Blechnum spicant Dryopteris amurensis Dryopteris expansa Dryopteris filix-mas Allantodia dilatata Diplazium bombonasae Diplazium wichurae Doodia media Adiantum hispidulum Adiantum raddianum Bolbitis auriculata Hypolepis punctata Hypolepis tenuifolia Adiantum capillus-veneris Lindsaea linearis Hemidictyum marginatum D Purifying selection Neutral or nearly neutral Positive selection Alsophila podophylla Plagiogyria japonica Dennstaedtia punctilobula Didymochlaena truncatula Tectaria zeylanica Deparia acrostichoides Deparia lancea Adiantum andicola Adiantum aleuticum Adiantum pedatum Deparia lancea Deparia lobato-crenata Deparia acrostichoides Onoclea sensilibis Thelypteris noveboracensis Pronephrium lakhimpurense Adiantum tetraphyllum Matteuccia struthiopteris Phegopteris hexagonoptera Athyrium filix-femina Coniogramme intermedia Homalosorus pycnocarpos Macrothelypteris torresiana Allantodia dilatata Diplazium wichurae Diplazium bombonasae Dryopteris filix-mas Dryopteris amurensis Dryopteris expansa Blechnum spicant Plagiogyria formosana Plagiogyria distinctissima Dipteris conjugata Hypolepis tenuifolia Hypolepis punctata Bolbitis auriculata Adiantum hispidulum Adiantum raddianuum Doodia media Hemidictyum marginatum Lindsaea linearis Adiantum capillus-veneris E GC Content Window position (bp) Phymatoceros phymatodes Megaceros flagellaris Nothoceros vincentianus Nothoceros aenigmaticus 0.02 substitutions/site Paraphymatoceros hallii Phaeoceros carolinianus Anthoceros punctatus Figure/15: Phylogeny,/selection/profile/and/GC/content/of/fern/neochromes./Maximumlikelihood reconstructionsofgenephylogenybasedon(a)codonmodel,(b)firstandsecondcodonpositions,and(c) thirdcodonposition.thickenedbranchesindicatealrtsupports(ina)orbootstrapsupports(inb,c)>70. (D)Selectionprofiledisplayedalongphylogeneticbranchesforfernandhornwortneochromes.Tree topologyderivedfromthebestmaximumlikelihoodtree(figure/13).thewidthofeachcoloralonga branchisproportionaltothenumberofcodonsitesinthecorrespondingselectionclass.thickenedbranches haveexperiencedsignificantepisodicpositiveselection(p5<0.05).(e)slidingwindowanalysisofgc contentforfernneochrome.eachlinedisplaysthegccontentforeachneochromesequence.noneofthe fernsinmystudyweredeviantinbasecompositionforneochrome.eachwindowis400bpinsizeandthe windowslidesevery50bp. 54

70 Ithereforehypothesizedthattheincongruenttreecouldbetheresultof1)multiplefern< to<fernhgtevents,2)anelevatedgeneturnoverratethatmayhavebeenselectedforafterhgt (Lindetal.,2010;Näsvalletal.,2012),or3)acombinationofboth.Ihavesomeevidence suggestingrecurringfern<to<fernhgtmighthavebeeninvolved.forexample,idiscovered neochromegenesfromtwoearly<divergingfernorders[gleicheniales(dipteris5conjugata)and Cyatheales(Alsophila5podophyllaandPlagiogyria5spp.)]thatwerelikelyderivedfromsecondary HGTevents(Figure14,arrows).Theseneochromesarenotphylogeneticallyresolvedaswouldbe predictedbasedonpublishedfernspeciesrelationships(schuettpelzandpryer,2007),butinstead arenestedamongpolypodiales(figure14).furthermore,thesplitbetweentheseandotherfern neochromes(81mya,95%highestposteriordensityinterval:59<106mya;appendixfigure21) occurredlongaftertheestimatedorganismaldivergencedatesforgleicheniales(276mya)and Cyatheales(223MYA)(SchuettpelzandPryer,2009),apatternthatmaybestbeexplainedby fern<to<fernhgt. MyhypothesisofpotentiallyrecurrentHGTeventswithinfernsisnotunprecedented.In angiosperms,rampanthgtshavebeendocumentedforthemitochondrialcox1homingintron. Thisintronisbelievedtohaveexperiencedoneinitial seedtransfer fromfungithatwas followedbyatleast80incidentsofplant<to<planthgtamong833diverseangiospermspecies (Choetal.,1998;Sanchez<Puertaetal.,2008;2011).Perhapsneochromeissimilarlyassociated withmobileelementsthatmayhavefacilitateditsmovementacrossspeciesboundaries Evolutionary and physiological implications of neochrome in hornworts Mydiscoveryofneochromeinhornwortsisanimportantsteptowardunderstandingthe evolutionofphotosensorysystemsinplants.inthemossphyscomitrella5patens,bothredandblue 55

71 lightcanelicitdirectionalchloroplastmovements,andthesearemediatedbymolecular interactionsbetweenphysicallyseparatephytochromeandphototropinproteins(jaedickeetal., 2012).Thehornwortneochromerepresentsastrikinglydifferentstrategyforintegratingthese twophotosensorysystems,combiningthemintoasingle,chimericgene.light<induced directionalchloroplastmovementhasnotyetbeenobservedinhornworts,probablybecause theirepidermalcellsusuallycontainonlyonechloroplastthatoccupiesmostofthecellularspace. However,nearly50yearsago,Burr(1968)documentedanunusualchloroplastphotoresponsein Megaceroshornworts;shediscoveredthatthelargechloroplasts contract toformcompact shapesunderstronglight.althoughiconfirmedthisphenomenoninhornworts(figure16), futurestudiesareneededtoexamineifneochromeisresponsibleforthecontractionresponse andtoexploreotherpossiblephysiologicalroles. A B Figure/16: Hornwort/chloroplasts/contract/under/strong/light./(A)Beforeirradiation,chloroplasts ofnothoceros5aenigmaticus5(arrowhead)occupymostofthecellularspace.(b)afterirradiationwithbluelight (57Ñmolm <2 s< 1 )for2hours,chloroplastsevidentlyreducedinsize.scalebar=40ñm Evolutionary significance of plant-to-plant HGT Thisstudypinpointstheoriginoflandplantneochromewithinthehornwortlineageand demonstratesthatneochromewashorizontallytransferredfromhornwortstoferns.thelife historyoffernsmayhelptoexplaintheirhypothesizedsusceptibilitytohgt.mostlandplants shareacommonsexuallifecyclethatalternatesbetweenadiploidsporophyteandahaploid 56