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Genetics: Early Online, published on October 11, 2016 as 10.1534/genetics.116.188268
Positive selection in rapidly evolving plastid-nuclear enzyme complexes
†
‡
§
Kate Rockenbach*, Justin C. Havird*, J. Grey Monroe , Deborah A. Triant , Douglas R. Taylor , Daniel B.
Sloan*.
*Department of Biology, Colorado State University, Fort Collins, CO 80523
†
Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins,
CO 80523
‡
Florida Museum of Natural History, University of Florida, Gainesville, FL 32611
§
Department of Biology, University of Virginia, Charlottesville, VA 22904
1
Copyright 2016.
Running Title: Selection on Plastid-Nuclear Complexes
Keywords: chloroplast, cytonuclear interactions, McDonald-Kreitman test, plastome
Author for Correspondence:
Daniel B. Sloan: [email protected]
Colorado State University
1878 Campus Delivery
Fort Collins, CO 80523
970.491.2256
2
ABSTRACT
Ratesofsequenceevolutioninplastidgenomesaregenerallylow,butnumerousangiospermlineages
exhibitacceleratedevolutionaryratesinsimilarsubsetsofplastidgenes.ThesegenesincludeclpP1and
accD,whichencodecomponentsofthecaseinolyticprotease(CLP)andacetyl-coAcarboxylase(ACCase)
complexes,respectively.Whethertheseextremeandrepeatedaccelerationsinratesofplastidgenome
evolutionresultfromadaptivechangeinproteins(i.e.,positiveselection)orsimplyalossoffunctional
constraint(i.e.,relaxedpurifyingselection)isasourceofongoingcontroversy.Toaddressthis,wehave
takenadvantageofthemultipleindependentaccelerationsthathaveoccurredwithinthegenusSilene
(Caryophyllaceae)byexaminingphylogeneticandpopulationgeneticvariationinthenucleargenesthat
encodesubunitsoftheCLPandACCasecomplexes.Wefoundthat,inspecieswithacceleratedplastid
genomeevolution,thenuclear-encodedsubunitsintheCLPandACCasecomplexesarealsoevolving
rapidly,especiallythoseinvolvedindirectphysicalinteractionswithplastid-encodedproteins.Amassive
excessofnonsynonymoussubstitutionsbetweenspeciesrelativetolevelsofintraspecificpolymorphism
indicatedahistoryofstrongpositiveselection(particularlyinCLPgenes).Interestingly,however,some
speciesarelikelyundergoinglossofthenative(heteromeric)plastidACCaseandputativefunctional
replacementbyaduplicatedcytosolic(homomeric)ACCase.Overall,thepatternsofmolecularevolution
intheseplastid-nuclearcomplexesareunusualforancientlyconservedenzymes.Theyinsteadresemble
casesofantagonisticco-evolutionbetweenpathogensandhostimmunegenes.Wediscussapossible
roleofplastid-nuclearconflictasanovelcauseofacceleratedevolution.
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INTRODUCTION
Plastidscarryreducedgenomesthatreflectanevolutionaryhistoryofextensivegenelossandtransfer
tothenucleussincetheirancientendosymbioticoriginroughlyonebillionyearsago(Timmisetal.2004;
Keeling2010;GrayandArchibald2012).Manyoftheproteinsencodedbygenesthathavebeen
transferredtothenucleargenomearetraffickedbackintotheplastid(Gouldetal.2008),wherethey
interactcloselywithproteinsencodedbygenesremainingintheplastidgenome.Theseinteracting
proteinsarekeynotonlytophotosynthesis,butalsototranscription,translationandcriticalnonphotosyntheticmetabolicfunctionsoftheplastid.Theinteractionsbetweenthesegeneproductscreate
theopportunityforco-evolutionbetweenplastidandnucleargenomes.Thus,studyingthenuclear
genesthatcontributetoplastidcomplexesisavaluabletoolforunderstandingtheprocessesunderlying
plastidgenomeevolutionandcytonuclearco-evolution.
Withinangiosperms,mostplastidgenomesarehighlyconservedinsequenceandstructure
(Jansenetal.2007;Wickeetal.2011),butmultipleindependentlineageshaveexperiencedaccelerated
ratesofaminoacidsubstitutioninsimilarsubsetsofnon-photosyntheticgenes(Jansenetal.2007;
ErixonandOxelman2008;Greineretal.2008b;Guisingeretal.2008,2010,2011;Straubetal.2011;
Sloanetal.2012a,2014a;Barnard-Kubowetal.2014;Wengetal.2014;Dugasetal.2015;Williamset
al.2015;Zhangetal.2016).Severalmechanismshavebeenhypothesizedtoexplaintheserepeated
accelerationsincludingpositiveselection,reducedeffectivepopulationsize(Ne),alteredDNArepair,
changesingeneexpression,andpseudogenizationfollowinggenetransfertothenucleus(seeabove
citations).Distinguishingamongthesehypotheseshasprovedchallenging,andtheultimatecauseor
causesoftheextremedifferencesinratesofmolecularevolutionamonggeneswithinplastidgenomes
remainunclear.
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Inmanycasesofextremeplastidgenomeevolution,accelerationshavedisproportionately
affectednonsynonymoussites,resultinginelevatedratiosofnonsynonymoustosynonymous
substitutionrates(dN/dS)(e.g.,ErixonandOxelman2008;Guisingeretal.2008;Barnard-Kubowetal.
2014;Sloanetal.2014a),whichindicatesthatchangesinselectionarelikelyinvolved.Inaddition,
recentstudiesshowedcorrelatedincreasesindN/dSbetweennuclear-andplastid-encodedsubunitsin
ribosomal(Sloanetal.2014b;Wengetal.2016)andRNApolymerasecomplexes(Zhangetal.2015),
providingfurtherevidenceforchangesinselectionpressures.However,thesestudiescouldnot
confidentlydistinguishbetweentwoalternativeexplanationsforincreaseddN/dS:positiveselectionand
relaxedpurifyingselection,whichcanbedifficulttodisentanglebasedonsequencedivergencedata
alone.Becausetheseselectionpressurescanhaveverydifferenteffectsonpopulationgeneticvariation,
analysesthatcombinedataonintraspecificpolymorphismandinterspecificdivergence(McDonaldand
Kreitman1991)candetectpositiveselectionevenincaseswhereitisnotreadilyidentifiablebasedonly
ondN/dS(Rausheretal.2008).However,moststudiesofacceleratedplastidgenomeevolutionand
plastid-nuclearco-evolutionhavenotincludedthenecessaryintraspecificpolymorphismdatato
performtheseanalyses.
Incontrasttorecentanalysesofplastidgeneticmachinery(i.e.ribosomalandRNApolymerase
genes;Sloanetal.2014b;Zhangetal.2015;Wengetal.2016),thepotentialformolecularco-evolution
involvingnuclear-encodedsubunitsinotherplastidcomplexesremainslargelyunexplored.Twosuch
complexesarethecaseinolyticprotease(CLP),whichisanATP-dependentproteaserequiredforproper
plastidfunction(NishimuraandvanWijk2015),andtheheteromericacetyl-coAcarboxylase(ACCase),
whichisinvolvedinfattyacidbiosynthesis(SasakiandNagano2004;SalieandThelen2016).TheCLP
complexandACCaseeachcontainasingleplastid-encodedsubunit(ClpP1andAccD,respectively)and
multiplesubunitsofnuclearorigin.Inmostangiosperms,thesequencesoftheclpP1andaccDgenesare
generallyconserved,buttheyareamongtheplastid-encodedgenesthatexhibitelevatedratesof
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sequenceevolutioninmultipleindependentlineages.TheclpP1gene,inparticular,exhibitsrecentand
dramaticallyincreasedratesofnonsynonymoussubstitutionsandindels(e.g.,ErixonandOxelman2008;
Sloanetal.2014a).
Inadditiontophylogeneticandpopulationgeneticanalyses,examiningpatternsofaminoacid
substitutionsrelativetoproteinstructurecanhelpdistinguishbetweenrelaxedandpositiveselection.In
themodelangiospermArabidopsisthaliana,theCLPcomplexismadeupoftwostackedheptameric
rings,comprisingninedifferenttypesofparalogousandstructurallyrelatedsubunitsthatarederived
fromthesinglesubunitfoundintheancestralhomotetradecamericformofthisenzyme(Peltieretal.
2004;YuandHoury2007;Olinaresetal.2011).TheP-ringisformedentirelyofthenuclear-encoded
subunitsCLPP3,4,5,6ina1:2:3:1stoichiometricratio,andtheR-ringcontainstheplastid-encoded
subunitClpP1andthenuclear-encodedsubunitsCLPR1,2,3,4ina3:1:1:1:1ratio.TheCLPPsubunitsall
containaconservedcatalyticSer-His-Asptriad,whichislackingfromtheCLPRsubunits(Peltieretal.
2004),meaningthatClpP1istheonlycatalyticsubunitwithintheR-ring.Othernuclear-encoded
subunitssuchasCLPC,CLPD,CLPF,CLPS,CLPT1,andCLPT2arephysicallyassociatedwiththecoreCLP
complexandactasadapters,chaperones,andaccessoryproteins,helpingtoregulatetheproteolytic
activityofCLP(Peltieretal.2004;NishimuraandvanWijk2015;Nishimuraetal.2015).
MostfloweringplantscontaintwodifferenttypesofACCaseenzymes:aeukaryotic-like
homomericmultidomainACCaseinthecytosolandabacterial-likeheteromericACCasewithinthe
plastids.TheheteromericACCaseconsistsofproteinsencodedbyfourdifferentgenes(ACCA,B,C,D;
SasakiandNagano2004;SalieandThelen2016).ACCCisabiotincarboxylase.InanATP-dependent
reaction,itcarboxylatesabiotinmoleculeattachedtoACCB(abiotincarboxylcarrierprotein),with
bicarbonateservingasthedonorofthecarboxylgroup(Whiteetal.2005).Thenuclear-encodedACCA
andplastid-encodedAccDcloselyinteractwitheachotherandrepresenttheαandβ
carboxyltransferasesubunits,respectively.Eachofthesesubunitsfirsthomodimerizes,andthenthey
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combineasahetero-tetramer,formingthefunctionalenzymethattransfersthecarboxylgroupfrom
biotintoacetyl-CoA(CronanandWaldrop2002).Together,theseenzymesconvertacetyl-CoAto
malonyl-CoAwithintheplastid,whichisthefirststepinthefattyacidbiosynthesispathway(Whiteetal.
2005).Insomelineages,thehomomericACCasehasundergoneaduplication,andonecopyistargeted
totheplastid,whiletheotherremainsinthecytosol(KonishiandSasaki1994;Schulteetal.1997;
Babiychuketal.2011;Parkeretal.2014).
TheangiospermtribeSileneae(Caryophyllaceae)hasemergedasmodelforstudyingorganelle
genomesunderdivergentratesofsequenceevolution(Moweretal.2007;ErixonandOxelman2008;
Sloanetal.2012a;2012b,2014a).Thisgroupcontainsmultiplelineageswithphylogenetically
independentaccelerationsinratesofplastidgenomeevolution(Sloanetal.2012a,2014a).Incontrast,
closelyrelatedSileneaelineageshavelargelymaintainedlowancestralratesofevolution.Thisrate
variationamongcloselyrelatedspeciespresentsapowerfulcontrasttoanalyzetheevolutionary
mechanismsresponsibleforacceleratedplastidgenomeevolutionandtestforcorrelatedchangesin
nuclear-encodedcounterparts.Here,weusetranscriptomesequencingdatacoupledwithstructural
informationtoidentifyvariationinnucleargenesbothwithinandamongSileneaespecieswithhighly
divergentratesofplastidgenomeevolution.Specifically,weaskedifthereisevidenceofselectionon
thesequencesofnuclear-encodedsubunitsoftheCLPandACCasecomplexesinSilenespecieswhose
plastid-encodedcounterpartshaveexperiencedrecentaccelerationsinratesevolution.
METHODS
TaxonSampling,mRNA-seq,andTranscriptomeAssembly
Sileneconica,S.noctiflora,andS.paradoxawereallpreviouslyidentifiedashavinghighlyaccelerated
ratesofnonsynonymoussubstitutionsinasubsetofplastidgenes,withthemostdramaticeffects
observedinclpP1(Sloanetal.2012a,2014a).TheaccDgenealsoexhibitedincreasednonsynonymous
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substitutionrates(albeitmuchlesspronounced)aswellastheaccumulationoflargeindelsinthese
species.Incontrast,therewaslittleornoevidenceofacceleratedsequenceevolutioninphotosynthetic
genesinthesespecies.Silenelatifolia,S.vulgaris,andAgrostemmagithagowerechosenas
representativesofcloselyrelatedlineagesthathavemaintainedlowratesofevolutionthroughouttheir
entireplastidgenomes(Sloanetal.2012a,2014a).Transcriptomesforthesesixspecies(S.conica,S.
latifolia,S.noctiflora,S.paradoxa,S.vulgaris,andA.githago)weretakenfrompreviouslydescribed
datasets(Sloanetal.2014b)thatwereeachgeneratedfromasingleindividualandassembledwith
Trinityr20120608(Grabherretal.2011).Thesedatasetswereusedforallphylogeneticanalysesand
correspondtoNCBISequenceReadArchive(SRA)accessionsSRX353031,SRX353047,SRX353048,
SRX353049,SRX353050,andSRX352988.Fortwogenes(CLPCandCLPP6),somesequenceswere
extractedfromseparateSOAPdenovo-Transv1.02(Xieetal.2014)assembliesofthesamereads
becausetheTrinityassemblieswerefragmentedorcomplex.
Seedsfrom19geographicallydispersedS.conicacollections,includingABRwhichwasusedfor
theoriginalS.conicatranscriptomereferencedabove,andonecollectionofthecloserelativeS.
macrodonta(TableS1)weregerminatedonsoilineitherJulyorAugust2014(Fafard2SVmix
supplementedwithvermiculiteandperlite)andgrownundera16-hr/8-hrlight/darkcyclewithregular
wateringandfertilizertreatmentsingreenhousefacilitiesatColoradoStateUniversity.Plantswere
grownfor7-9weeks,andtotalRNAwasextractedfrom2-3leavesofasingleindividualfromeach
collectionusinganRNeasyPlantMiniKit(Qiagen).Rosetteleaveswereusedforallindividualswiththe
exceptionoftheARZandPDAsamplesforwhichcaulineleaveswereused.TheresultingRNAwassent
totheYaleCenterforGenomeAnalysisforIlluminamRNA-seqlibrarypreparationandsequencing.For
allbuttwosamples,polyAselectionwasusedduringlibraryconstruction,whilefortheABRsampleofS.
conicaandthesingleS.macrodontasample,mRNAselectionwasperformedusingaRibo-ZeroPlant
LeafrRNARemovalKit(Illumina)inanefforttocapturemoreorganellartranscripts(aspartofan
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unrelatedproject).Resultingstrand-specificIlluminalibrariesweresequencedontwolanesofan
IlluminaHiSeq2500togeneratepaired-end151bp(2´151)reads.Raw(i.e.,non-normalizedand
untrimmed)readswerethenassembledusingTrinityr20140717withdefaultparameters(notethatthe
strand-specificityofthereadswasnottakenintoaccountduringassembly).Transcriptomeassembly
statisticsandnumbersofreadsweresimilaramongthe20samples,exceptforanapproximately50%
reductionintheaverageandtotallengthofassembledtranscriptsforsampleswhereRibo-Zerowas
used(TableS2).
ExtractionandAlignmentofOrthologousSequencesfromSileneaeSpecies
Thefocusofourstudywasthenuclear-encodedcomponentsoftheplastidCLPandACCasecomplexes
(TableS3).Inaddition,setsofgenesequenceswereobtainedfromphotosystemI(PSI)andthe
mitochondrial-targetedCLPprotease(mtCLP)toserveasabasisforcomparison.PSIwasselected
becauseitcontainssubunitsfromboththenuclearandplastidgenomesbut,unlikeCLPandACCase,the
plastid-encodedsubunitshavebeenhighlyconservedeveninSilenespecieswithacceleratedratesof
evolutioninotherplastidgenes(Sloanetal.2012a,2014a).ThemtCLPcomplexwaschosenbecauseitis
homologoustotheplastidCLPbutconsistsentirelyofnuclear-encodedsubunitsandistargetedtoa
differentcellularcompartment(themitochondria).ThemtCLPcomplexhasahomotetradecamercore
consistingentirelyofCLPP2subunitsthatinteractswiththechaperonesCLPX1,2,3(vanWijk2015).
Additionally,50geneswithaminimumcodingsequencelengthof600bpwereselectedatrandomfrom
apublishedlistofsingle-copynucleargenesinangiospermsinordertotestforglobalincreasesin
evolutionaryrateswithinthenucleargenome(TableS4;Duarteetal.2010).Genesthatwereannotated
asbeingtargetedtothemitochondriaorplastidswereexcludedfromthisrandomset.TheArabidopsis
thalianasequencesforselectedgeneswereobtainedthroughtheTAIRdatabase
(https://www.arabidopsis.org/)withaccessionnumbersfromtheliterature(TablesS3andS4;Peltieret
al.2004;Duarteetal.2010;Olinaresetal.2011;vanWijk2015).
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BLAST+2.2.31(Camachoetal.2009)wasusedtoruntblastnsearches(defaultsettings)withthe
selectedArabidopsisthalianaaminoacidsequencesasqueriesagainsteachoftheassembledSileneae
transcriptomes.ThetophitineachtranscriptomewasretrievedwithacustomPerlscriptusingBioPerl
modules(Stajichetal.2002).Incaseswherethereweremultipleparalogousgenes,manualcuration
aidedbyexploratorytree-buildingwasperformedtoidentifyorthologs.Genesthatwereabsentfrom
thetranscriptomesorforwhichorthologscouldnotbeconfidentlyidentifiedwereexcludedfrom
furtheranalysis.Fortherandomset,geneswereexcludedandreplacedwithanotherrandomlyselected
geneifoneormorespecieslackedorthologoussequenceorhadapartiallyassembledtranscriptthat
waslessthantwo-thirdsthelengthofthecodingsequence.Extractedsequenceswerealignedby
nucleotideusingtheMUSCLEalgorithmembeddedwithinMEGAv6.0(Tamuraetal.2013).Thelongest
openreadingframe(ORF)intheAgrostemmagithagosequencewaspredictedwiththeNCBIORFFinder
(http://www.ncbi.nlm.nih.gov/gorf/gorf.html),andothersequencesweretrimmedaccordingly.
SequenceswerethenrealignedbycodonusingMUSCLE.TargetPv1.1(Emanuelssonetal.2000)
wasusedwithtranslatedORFsfromA.githagotopredictthelengthoftheN-terminalsignalpeptide(for
plastid-andmitochondrial-targetedproteins),whichwasthenremovedfromallsequences.Inafew
cases,TargetPwasunabletopredictatargetingpeptidebasedonA.githagosequence,soS.Iatifolia,S.
vulgaris,orA.thalianawasusedinsteadtoidentifyandremovesignalpeptides.Concatenated
sequencesforsetsofgenesineachcomplexweregeneratedfromfinalalignmentswithacustomPerl
scriptusingBioPerlmodules.
PhylogeneticAnalysisofRatesofSequenceEvolution
Foreachgeneindividuallyandfortheconcatenatedsetsofnuclear-encodedgenesofeachcomplex,we
conductedmultipleanalysesofratesofsynonymousandnonsynonymoussubstitutionsbyusingthe
codemlprogramwithinPAMLversion4.8(Yang2007).AnF1´4codonfrequencymodelwasappliedin
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eachanalysis,andaconstrainedtreetopologywasusedwiththespeciesinSilenesubgenusBehenantha
(S.conica,S.latifolia,S.noctiflora,andS.vulgaris)collapsedasapolytomy.First,weimplementedafree
branchmodel(model=1inPAML)toestimatedN/dSforeachbranchindependently.Branchesthatwere
identifiedashavingdN/dSvaluesgreaterthanonewerethentestedforsignificanceusingalikelihood
ratiotest(LRT)thatcomparedthefreebranchmodeltoamodelthatconstraineddN/dSforthe
individualbranchinquestiontoavalueofone.Finally,weclassifiedspecies/branchesintotwogroups–
“fast”and“slow”–basedonknownratesofplastidgenomeevolution(Sloanetal.2012a,2014a)and
estimatedseparatedN/dSvaluesforeachgroup(model=2inPAML).TheterminalbranchesforSilene
conica,S.noctifloraandS.paradoxawereassignedtothefastgroup,whilethoseforS.latifolia,S.
vulgaris,andA.githagowereassignedtotheslowgroup.Theinternalbranchconnectingthecommon
ancestorofSilenetothebaseofSilenesubgenusBehenanthawasalsoincludedintheslowgroup.As
above,weusedanLRTtotestforsignificanceincasesinwhichindividualgenesorconcatenated
sequencesforentirecomplexeshadanestimateddN/dSvaluegreaterthanoneforthefastgroup(no
suchcaseswhereidentifiedfortheslowgroup).FortheconstrainedmodelintheseLRTcomparisons,
thedN/dSvalueforthefastgroupwassettoone.
McDonald-KreitmanTests
McDonald-Kreitman(MK)tests(McDonaldandKreitman1991)wereperformedusingsequencesfrom
theSileneconicapopulationgeneticdataset.Sequenceswereextracted,alignedandtrimmedfollowing
thesamemethodologydescribedaboveforthephylogeneticanalysis.Thetestswereimplementedwith
thewebserverdescribedbyEgeaetal.(2008).Foreachgene,theneutralityindex(NI)wascalculatedby
dividingtheratioofnonsynonymoustosynonymouspolymorphismswithinS.conica(Pn/Ps)bytheratio
ofnonsynonymoustosynonymousdivergencefromacloselyrelatedoutgroup(seebelow)species
(Dn/Ds)(RandandKann1996).NIvalueslessthanoneareindicativeofpositiveselection,withstatistical
significanceassessedbyastandardcontingency-tablec2analysis.Wealsocalculatedthedirectionof
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selection(DoS)foreachgene(StoletzkiandEyre-Walker2011).PositiveDoSvaluesareindicativeof
positiveselectionandanexcessofnonsynonymoussubstitutions.Welookedforevidenceofselectionin
setsofrelatedgenesbysummingpolymorphismanddivergencecountsforgenesbelongingtothe
plastidCLP,ACCase,PSI,ormtCLPcomplexesaswellasforthesetofrandomgenes.Becausesumming
acrosscontingencytablescanintroducestatisticalbias,wealsocalculatedNITGforeachcombinedsetof
relatedgenes,whichisanunbiasedestimatorofNI(StoletzkiandEyre-Walker2011).Twoseparatesets
ofanalyseswerecarriedout,usingeitherS.latifoliaorS.macrodontaastheoutgroup.Extracted
sequencesfromtheS.conicaandS.macrodontatranscriptomeassembliesthatcouldnotbeconfidently
identifiedasorthologouswereremovedfromtheanalysis.Specifically,CLPR2,CLPX1,andonerandomly
selectedgene(AXS2:AT1G08200)showedevidenceofrecentduplicationsintheS.conicalineage,
leadingtoapparentchimericassemblyartifacts.Therefore,thesegeneswerenotusedforMKtests.In
addition,allthreeCLPXgenesand32oftherandomlyselectednucleargeneshadlowcoverageand
fragmentedassembliesintheS.macrodontadataset,soMKtestsforthesegeneswereonlyperformed
withS.latifoliaasanoutgroup.ThelowcoverageformanynucleargenesintheS.macrodontaassembly
waslikelyrelatedtotheuseofRibo-Zeroinconstructionofthatlibrary(TableS2).
AnalysisofProteinStructureandPositionofSubstitutions
Togaininsightintothefunctionalconsequencesofaminoacidchangesobservedinfast-evolvingSilene
species,wemappedsubstitutionsontoplastidCLPandACCaseproteinstructures.AncestralSilene
sequenceswereinferredusingcodemlinPAMLwiththeguidetreecontainingthefiveSilenespecies
encodedasapolytomy,withAgrostemmagithagoandEscherichiacoliasoutgroups.Partialsequences
wereexcludedwheninferringancestralsequences.ForeachCLPPandCLPRsubunit(includingthe
plastid-encodedClpP1),changesthatwereinferredtohaveoccurredinS.conica,S.paradoxa,orS.
noctiflorafromtheancestralSilenesequenceweremappedontothestructureofanindividualE.coli
CLPPsubunit(PDBaccession1YG6;Bewleyetal.2006;YuandHoury2007).Likewise,changesinACCase
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subunitswerealsomappedontosolvedE.colistructures(PDBaccessions4HR7and2F9Y:Bilderetal.
2006;Broussardetal.2013).TemplatestructuresfromE.coliwereusedbecausenoplantCLPorACCase
structureshavebeensolved.Whileitislikelytherehavebeenstructuralchangeswithinthese
complexesbetweenbacteriaandplants,mostofthesesubunitsareancientlyconservedandcanbe
reliablyaligned(averageaminoacididentity=43.3%).
DataAvailability
RawIlluminareadsandassembledtranscriptomesequencesareavailableviatheNCBISRAand
TranscriptomeShotgunAssembly(TSA)database,respectively.AccessionnumbersareprovidedinTable
S2.SequencealignmentsusedinPAML,MK,andphylogeneticanalysesareprovidedinSupplementary
FileS1.
RESULTS
CLPandACCaseGeneContentintheTribeSileneae
WewereabletorecovermostoftheexpectedgenesfromtheSileneaetranscriptomes,asthegene
contentinthesespecieswaslargelysimilartothatofArabidopsisthaliana.However,wedidfindthat
somegeneshadexperiencedrecentduplicationsorlosses.Weidentifiedorthologsofalleightofthe
nuclear-encodedCLPPandCLPRgenesthataretargetedtotheplastidinArabidopsis(FigureS1),andwe
foundCLPP5hasbeenduplicated(withtheresultingcopiesdesignatedasCLPP5aandCLPP5b).The
duplicationappearstohaveoccurredpriortothedivergencebetweenAgrostemmaandSilene,butonly
oneofthesecopies(CLPP5b)wasrecoveredfromtheAgrostemmatranscriptome(FigureS1).As
describedintheMethods,theremayalsohavebeenmorerecentduplicationsofgenessuchasCLPR2in
individualspecies.InadditiontothesubunitsthatmakeupthecoreproteolyticringsoftheplastidCLP
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complex,wealsoidentifiedorthologsoftheassociatedchaperones,adapters,andaccessoryproteins
thathavebeendescribedinArabidopsis(Table1;NishimuraandvanWijk2015).Thenewlydiscovered
CLPFadapterwasalsoidentifiedinourdatasetbutwasnotincludedinthepresentanalysis(Nishimura
etal.2015).ArabidopsiscontainsthreeparalogouschaperonegenesthatcontributetotheplastidCLP
complex(CLPC1,CLPC2,andCLPD).WefoundevidencethatmultiplecopiesofCLPC/Dalsoexistinthe
Sileneae,buttheassembliesoftheselonggeneswereoftenfragmented,andwewereonlyableto
successfullyextractonesetoforthologs,whichwerefertoasCLPC.Inadditiontotheseplastid-targeted
subunits,wealsofoundorthologsofthemitochondrial-targetedCLPgenesthathavebeenidentifiedin
Arabidopsis(CLPP2,CLPX1,CLPX2,andCLPX3;vanWijk2015).
Sileneaegenesweresuccessfullyidentifiedfromeachofthethreeclassesofnuclear-encoded
subunitsoftheheteromericplastidACCase(ACCA,ACCB,andACCC),includingtwodivergentcopiesof
ACCB.TwocopiesofthisgenealsoexistinArabidopsis(Fukudaetal.2013),butitwasnotreadily
apparentfromphylogeneticanalysisifthereisanorthologousrelationshipbetweenSileneaeand
Arabidopsiscopiesoriftheyaretheproductofindependentduplicationevents(datanotshown).
AlthoughalloftheheteromericACCasegeneswereidentifiedinthisclade,wefoundevidenceofrecent
genelossinsomeoftheSilenespecies,whichisdescribedindetailbelow(see“GeneLossand
AcceleratedEvolutionofSomeSubunitsintheACCaseComplex”).
WithrespecttothehomomericACCasethatistypicallytargetedtothecytosol,our
transcriptomedataindicatedthatSileneaespeciesexpresstwodistinctcopiesofthisgene(FiguresS2
andS3)andthatoneoftheresultingproteinshasanN-terminalextensionthatisstronglypredictedto
actasaplastid-targetingpeptide(withaspecificity>0.95basedonTargetPanalysis).Duplicationofthe
homomericACCaseandre-targetingtotheplastidshasoccurredrepeatedlyandindependentlyduring
angiospermevolution(FigureS3).Theobservedduplicationinourdatasetprecededthedivergence
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betweenAgrostemmaandSilene,butitwasindependentfromsimilarduplicationsingrasses(Konishi
andSasaki1994)andtheBrassicaceae(Schulteetal.1997;Babiychuketal.2011;Parkeretal.2014).
Nuclear-EncodedComponentsofthePlastidCLPComplexShowElevatedRatesofAminoAcid
SubstitutioninSpecieswithRapidlyEvolvingPlastidGenomes
Wefoundthatnuclear-encodedCLPgeneshavedramaticallyelevateddN/dSvaluesinSilenespecieswith
recentaccelerationsintheevolutionaryratesofplastid-encodedclpP1(Tables1and2).When
concatenated,all13nuclear-encodedCLPgeneshadadN/dSvaluesignificantlygreaterthanoneforboth
S.conicaandS.noctifloraandnearlyequaltooneforS.paradoxa(Table1).Incontrast,concatenated
CLPgeneshaddN/dSvaluesbetween0.05and0.16forcloselyrelatedspecieswithtypicalratesofclpP1
evolution(Table1).TheextremevarianceindN/dSestimatesresultedfromelevatednonsynonymous
substitutionrates,whereassynonymoussubstitutionrateswereverysimilaracrossspecies(Figure1).
RatedifferencesweremostpronouncedforCLPRsubunits,whichoccupythesamestructuralringasthe
plastid-encodedCLPP1subunit(vanWijk2015).All12dN/dSestimatesforCLPRgeneswithinthe“fast”
speciesweregreaterthanone,witheightfoundtobesignificantlygreaterthanone(Table1).ThedN/dS
estimatesfortheCLPPgeneswithinthesefastspecieswerealsohighlyelevated,butonlyeightofthe15
weregreaterthanone,andonlyonewassignificantlyso(Table1).TheadaptorgeneCLPSwasa
noticeableoutlier,beinggenerallyconservedinspeciesregardlessoftheirratesofplastidgenome
evolution(Figure2).
GeneLossandAcceleratedEvolutionofSomeSubunitsintheACCaseComplex
Thethreespecieswithhighratesofplastid-encodedaccDevolutionexhibitedvariedpatternswith
respecttonuclear-encodedACCasegenes,includingsomecasesofacceleratedevolutionandother
examplesofoutrightgeneloss.Notably,noneofthenuclear-encodedACCasegeneswereidentifiedin
theassembledS.noctifloratranscriptome.Weconfirmedthelossofthesegenesbysearchingadraft
15
assemblyoftheS.noctifloranucleargenome(DBS,unpublisheddata).Thenucleargenomeassembly
containedonlypseudogenizedfragmentsofACCA,andnoneoftheotherACCasegenesweredetected.
TheS.paradoxatranscriptomealsoappearedtolackafullcomplementoffunctionalnuclear-encoded
ACCasegenes.MostofthespeciescontainedtwoACCBparalogs,butwedidnotdetectacopyofACCB1
intheS.paradoxatranscriptome.Inaddition,theassemblyoftheS.paradoxaACCAtranscriptwas
incomplete,coveringonly543ntof2133-ntalignmentandexhibitingadN/dSof0.99(Table1).This
partialACCAtranscriptwasaberrantlyspliced,resultingina9-ntinsertionthatintroducedapremature
in-framestopcodon(FigureS4).Therefore,despitebeingtranscribed,ACCAislikelyapseudogeneinS.
paradoxa.Incontrast,ACCB2andACCCwerebothintactwithverylowdN/dSvaluesinS.paradoxa.
Finally,unlikeinS.noctifloraandS.paradoxa,allfournuclear-encodedACCasegeneswerepresentand
intactinthetranscriptomesofS.conicaandallthree“slow”species.
TheACCAsubunit,whichinteractsdirectlywiththeplastid-encodedACCDsubunit,washighlydivergent
inaminoacidsequenceinS.conica.WeinitiallyestimatedanelevateddN/dSvalueof0.49forACCAinS.
conica,andthatvalueincreasedto0.94whenweanalyzedthefull-lengthofthegenebyexcludingthe
partialS.paradoxasequencefromthealignment(PAMLwasrunwiththe“cleandata”option,which
ignoresalignmentpositionsforwhichgapsarepresentinanyofthesequences).Therewasastriking
differencebetweenthiselevateddN/dSinS.conicaandtheverylowvalues(£0.06)forACCAintheslow
groupspecies(Table1).TheS.conicaACCB1genealsoexhibitedsubstantiallyhigherdN/dSvaluesthanin
anyoftheotherspecies,whereasdN/dSwasverylowinACCB2andACCC(Table1).Ingeneral,ratesof
nonsynonymoussubstitutionintheslowspecieswereverylow(dN/dS<0.2)forACCasegenes,withthe
exceptionofACCB2.Interestingly,thedN/dSvaluesforthisgeneshowedaconversepattern,inwhich
dN/dSwaselevatedintheslowspeciesrelativetoS.conicaandS.paradoxa(Table1).LowRatesof
NonsynonymousSubstitutionsinmtCLP,PSI,andRandomlySelectedGenes
16
ForboththePSIandmtCLPconcatenatedgenesets,therewassignificantlyhigherdN/dSinthefast
group(Table2).However,dN/dSvaluesforbothPSIandmtCLPweregenerallylowinallspecies,andthe
differencesbetweenthetwospeciesgroupswasverysmall,especiallyincomparisontothedifferences
observedfortheplastidCLPcomplexandsomeACCasegenes(Table1;FigureS5).Theloweroverall
dN/dSestimatefortheslowspeciesgroupappearedtobelargelydrivenbythelowvaluesfortheA.
githagobranch,whichhasadisproportionateeffectontheestimationofdN/dSinthisgroupbecauseit
representsmoredivergencetimeandalargefractionoftotalobservedsubstitutions.Therandomly
selectednucleargenesshowednosignificantdifferenceindN/dSbetweenthefastandslowspecies
groups(Tables1,2andS5).Thus,itdoesnotappearthatthereisaglobalelevationofdN/dSinthe
nucleargenomesofspecieswithrapidlyevolvingplastidgenomes.
McDonald-KreitmanTestsRevealExcessofNonsynonymousDivergenceBetweenSpecies
DespitehighlevelsofobservednonsynonymousdivergenceinACCAandthemajorityofthenuclear
genesthatencodecomponentsoftheplastidCLPcomplex,mostofthesegregatingvariantswithinS.
conicaaresynonymous.Thus,inACCAandtheconcatenatedsetofnuclear-encodedCLPgenes,there
wasalargeandhighlysignificantexcessofnonsynonymousdivergencefromtheoutgroupS.latifolia
relativetolevelsofnonsynonymousandsynonymouspolymorphismwithinS.conica(Table3).ThePSI
geneshadextremelylowDn/Dsvalues,butthePn/Psvalueswereevenlower,againresultingina
significantexcessofnonsynonymousdivergencefortheconcatenatedgeneset(Table3).Incontrast,
therewerenoindicationsofasimilarexcessinthemtCLPgenesorthesetofrandomlyselectednuclear
genes,astheirconcatenatedsequenceshadNIvaluesveryclosetoone(Tables3andS6).Repeatingthe
MKanalysiswithamorecloselyrelatedoutgroup(S.macrodonta)producedsimilarresults(TablesS6
andS7).
SubstitutionsinNuclear-EncodedSubunitsPreferentiallyOccuratInterfaceswithPlastid-Encoded
SubunitswithintheCLPComplexbutnotwithintheACCaseComplex
17
ToinvestigatetheeffectofphysicalinteractionsbetweenCLPcomplexsubunitsonsubstitution
patterns,wemappedobservedchangesinCLPPandCLPRsubunitsontothesolvedstructureofthe
representativeClpPsubunitfromE.coli(Figure3).Thisanalysisshowedthatnumeroussubstitutions
haveoccurredthroughouttheentiretyoftheplastid-encodedClpP1subunitinfast-evolvingSilene
species.Theseincludesubstitutionsin1)thehandledomainwhichphysicallyinterconnectsthetwo
heptamericringsandlikelystabilizesring-ringinteractions,2)theheaddomainwhichlikelystabilizes
interactionsbetweensubunitswithinasinglering,and3)theaxialloopregionswhichformtheaxial
poresandmediateinteractionswithassociatedchaperones(Figure3C)(YuandHoury2007).Several
individualresiduesthathavebeenimplicatedinringstabilityorsubstrateinteractions(Wangetal.
1997)werealsoobservedtohaveundergonechangesinClpP1(Figure3).
Giventheextremelevelsofdivergenceintheplastid-encodedClpP1subunit,wereasonedthat
substitutionsmightbenon-randomlydistributedwithininteractingnuclear-encodedsubunits.
Specifically,wepredictedthatthenuclear-encodedCLPRsubunitswouldhaveanabundanceofchanges
intheheaddomainbecausetheClpP1subunitassembleswithnuclear-encodedCLPRsubunitstoform
theR-ring(NishimuraandvanWijk2015),andtheheaddomaincontainsresiduesthatarelikelyto
maintainintra-ringinteractions(YuandHoury2007).Similarly,wepredictedthatnuclear-encodedCLPP
subunitswouldhaveadisproportionatenumberofsubstitutionsintheirhandledomainsbecausethese
subunitsformtheP-ring,andtheirhandledomainsarelikelyinvolvedininteractionswiththehighly
divergentcopiesofClpP1intheR-ring.Aspredicted,amino-acidsubstitutionsweresignificantly
overrepresentedintheheaddomainsofCLPRsubunitsandthehandledomainsofCLPPsubunitsinall
threefast-evolvingspecies(Table4).
Inmostplants,boththenuclear-andplastid-encodedCLPPsubunits(butnottheCLPRsubunits)
retaintheSer-His-Asptriadthatconfersproteaseactivity(Peltieretal.2004).However,wefoundthatin
fast-evolvingSilenespecies,manyofthesesubunitshaveexperiencedsubstitutionsattheHisorAsp
18
positionswithinthishighlyconservedtriad(whereasthecatalyticSerisuniversallyconservedinour
dataset;TableS8).
RelativetotheCLPcomplex,therewerefewerchangesintheACCasesubunits,andonlya
fractionofthesecouldbeanalyzedinastructuralcontextbecauseseveralregionsofACCaseproteins
lackstructuralinformation(Broussardetal.2013).Inparticular,thereisalargeN-terminalextensionof
AccDthatishighlyvariableamongangiosperms(Greineretal.2008a)andabsententirelyfromE.coli.
Likewise,theC-terminalhalfofACCAisalsouniquetoplants.Incontrasttothepatternobservedinthe
CLPcomplex,theACCasechangesthatwereabletobemappedtotheE.colistructureoccurredaway
fromprotein-proteininterfacesandgenerallydidnotinvolvefunctionallyimportantresidues(Figure4).
ThiswasalsotrueforasiteatwhichlargeinsertionsarepresentintheAccDsubunitinbothS.conica
andS.paradoxa(Figure4B).
DISCUSSION
Innumerousangiospermlineages,asubsetofplastidgenes,includingclpP1andaccD,display
acceleratedevolutionaryrates,butthecausesofthisrecurringphenomenonhaveremainedunclear
(Jansenetal.2007;ErixonandOxelman2008;Greineretal.2008b;Guisingeretal.2008,2010,2011;
Straubetal.2011;Sloanetal.2014a,2012a;Barnard-Kubowetal.2014;Wengetal.2014;Williamset
al.2015;Dugasetal.2015;Blazieretal.2016;Zhangetal.2016).Weinvestigatedthenucleargenes
thatcontributetothemultisubunitcomplexesthatincludeClpP1andAccDandincorporatedpopulation
geneticandstructuraldatatodistinguishbetweenrelaxedpurifyingselectionandpositiveselectionas
driversofelevateddN/dSvalues.
Ouranalysisrevealeddifferentpatternsofselectiononthenuclear-encodedCLPandACCase
genes,whichmayreflectthecontrastingevolutionaryhistoriesoftheplastid-encodedsubunitsinthese
twocomplexes.ThepatternsofclpP1sequencedivergenceinsomelineagesaretrulyremarkableand
19
includebothstructuralchanges(i.e.,indelsandlossofintrons)andextremeincreasesin
nonsynonymoussubstitutionrates(e.g.,ErixonandOxelman2008;Sloanetal.2012a).Forexample,the
amino-acididentitybetweentheclpP1copiesinthefast-evolvingspeciesSileneconicaandS.noctiflora
isonly34%,andmanySilenespecieshaveelevateddN/dSvalues(upto5.9inS.fruticosa;Erixonand
Oxelman2008).Incontrast,slow-evolvingspeciesfromthesamegenussuchasS.latifoliaretainupto
58%identitywithfree-livingcyanobacteria,sotheserecentaccelerationshaveledtofarmore
divergenceinthelastfewmillionsyearsthanhastypicallyaccumulatedsincetheendosymbioticorigins
ofphotosyntheticeukaryotesroughlyonebillionyearsago.Thecontrastsbetweenfastandslow
lineagesforaccDarefarlessstark.Theincreasedratesofaminoacidsubstitutioninfastlineagesare
onlymodest,andmostofthesequencechangeiscausedbyindels(Sloanetal.2012a,2014a).
Furthermore,eveninspecieswithtypical,slow-evolvingplastomes,itisprimarilythecatalyticCterminaldomainofAccDthatishighlyconserved,whereastheN-terminaldomain,whichisplantspecificandhasanunknownfunction,accumulatessubstantialstructuraldivergence(Greineretal.
2008a).TheevolutionofaccDisfurthercomplicatedinsomeangiospermlineagesbyfunctionaltransfer
tothenucleus(Mageeetal.2010;Rousseau-Gueutinetal.2013)orbyfunctionalreplacementwitha
duplicatedandre-targetedcopyofthehomomericACCase(KonishiandSasaki1994).Incontrast,there
isnoevidencetoourknowledgeoffunctionaltransferofclpP1tothenucleusingreenplants.
ThecomplexhistoryoftheplastidaccDgeneinangiospermsismirroredbythevaried
evolutionaryhistoriesthatweobservedwithinSileneforthenuclear-encodedACCasesubunits.One
lineage(S.noctiflora)hasexperiencedtheoutrightlossoftheheteromericACCasecomplex,and
anotherlineage(S.paradoxa)appearstobeundergoinggeneloss/pseudogenizationwithsignaturesof
relaxedselection.However,inathirdfastspecies(S.conica),alltheACCasesubunitsareretainedand
onegeneshowsclearevidenceofpositiveselection.Incontrasttothisheterogeneityintheevolutionof
ACCasegenes,wefoundaconsistentsignalofpositiveselectionthroughoutnearlyallthesubunitsin
20
theplastidCLPcomplexinallthreefastspecies.ItwasespeciallystrikingtofinddN/dSvaluessignificantly
greaterthanonewhenaveragedovermorethanadozennuclear-encodedCLPgenes.
LossofPlastidHeteromericACCase
ThefindingthatSilenenoctiflorahascompletelylostthenuclear-encodedheteromericACCasegenesis
consistentwithpreviousobservationsthatthecopyoftheplastid-encodedaccDmaybeapseudogene
inthisspecies(Sloanetal.2012a).AlthoughtheaccDreadingframeisintactinS.paradoxa,thegeneis
highlydivergentandcontainsmultiplelargeinsertions,raisingquestionsastoitsfunctionality(Sloanet
al.2014a).ThesequestionsextendtotheentireACCasecomplex,aswefoundevidenceofgene
loss/decayinnuclear-encodedS.paradoxaACCasegenes(i.e.,theapparentlossofACCB1and
pseudogenizationofACCA).Inatleasttwoangiospermlineages,theplastidaccDgenehasbeen
transferredtothenucleus(Mageeetal.2010;Rousseau-Gueutinetal.2013).However,wefoundno
evidenceofafunctionalnuclearcopyofaccDinanySilenespeciesexamined.Instead,thepresenceofa
duplicatedhomomericACCasethatispredictedtobetargetedtotheplastidsmaybecompensatingfor
thelostoralteredfunctionoftheheteromericACCase,asshowningrasses(KonishiandSasaki1994).
BecausetheduplicationofthehomomericACCaseappearstohavehappenedlongbeforethe
divergenceofAgrostemmaandSilene(FigureS3),theheteromericandduplicatedhomomericACCases
haveremainedconservedandexpressedformorethan20millionyears(Sloanetal.2009)inmany
lineageswithinthisclade.Thisraisesintriguingquestionsforfutureinvestigationabouttherespective
rolesoftheseenzymesandwhyfunctionallossoftheheteromericACCasehasoccurredinsome
lineages,whileothershaveretainedallofthesubunitsandevenexhibitevidenceofstrongpositive
selectioninonecase(seebelow).
PositiveSelectionActingonNuclear-PlastidEnzymeComplexes
21
WefoundsignaturesofintensepositiveselectionactingontheplastidCLPcomplex.Inmanygenes
withinthefastspecies,dN/dSisgreaterthanone(oftensignificantlyso;Table1),whichisanespecially
powerfulsignatureofpositiveselectionbecauseanyeffectsareaveragedacrosstheentirelengthof
eachgeneandlikelydampenedbypurifyingselectionactingonmanyresidues.Althoughanincreasein
therateofnonsynonymoussubstitutionscanalsobeindicativeofreducedfunctionalimportanceor
evenapseudogene,wecanrejectthathypothesisbasedonthepopulationgeneticdata.Thevast
majorityoftheCLPsequencepolymorphismthatissegregatingwithinS.conicaissynonymous(Table3),
meaningthatthesegenesarestillfunctionallyconstrainedandthatpurifyingselectionispurgingmost
newnonsynonymousmutationsfromthepopulation.Instead,thelargeobservedexcessof
nonsynonymousdivergencebetweenspecies(relativetointraspecificpolymorphism)isanindication
thataspecificsubsetofaminoacidsubstitutionshavebeenpreferentiallydriventofixationdueto
positiveselection(McDonaldandKreitman1991).
Interestingly,someoftheobservedsequencechangesaffectedresiduesinthecatalytictriadin
SileneCLPPsubunits(TableS8).Substitutionsinthecatalytictriadoftheplastid-encodedClpP1subunit
inAcaciahavepreviouslybeeninterpretedasevidenceofpseudogenization(Williamsetal.2015).
However,thestrongselectionactingontheplastidCLPcomplexinSilenesuggeststhatbothplastid-and
nuclear-encodedCLPPsubunitsmayretainanimportantfunctionalroledespitechangesinthecatalytic
triad(TableS8).AlthoughtheSer-His-Aspcatalyticsiteisawidelyconservedfeatureacrossthediversity
oflife,someofthesamesubstitutionsinthistriadhavebeenobservedinotheratypicalserine
proteasesandfunctionallyrelatedenzymes(Schragetal.1991;Ekicietal.2008;Zeileretal.2013).
Notably,substitutionsinthecatalytictriadwereonlyobservedinonenuclear-encodedCLPPgenein
eachofthefastspecies.Itispossiblethatsuchchangesaretolerableaslongassomeofthesubunitsin
theP-ringretainthecanonicalcatalyticresidues.
22
GiventheevidenceofgenelossandrelaxedselectionontheheteromericACCaseinS.noctiflora
andS.paradoxa,weinitiallysuspectedthattheelevateddN/dSforACCAinS.conica(0.94forthefulllengthgene)wasalsoduetorelaxedselection.However,thepopulationgeneticdatademonstratedthat
ACCAandtheheteromericACCasecomplexarestillfunctionallyconstrainedinS.conica,asmostof
intraspecificpolymorphismsweresynonymous(Table3).Therefore,theunusuallyhighrateoffixed
ACCAaminoacidsubstitutionsinthislineagearemostlikelytheresultofpositiveselectionandadaptive
evolution.
Werandomlyselectedasetofnucleargenesthatarenottargetedtotheplastidsor
mitochondriaaswellasgenesfromthemtCLPandPSIcomplexes.Thesewerechosenwiththeapriori
expectationthattheywouldbesimilarbetweenthefastandslowspecies,becausetheyeitherdonot
interactwithorganelle-encodedsubunits(randomgenesandmtCLP),ortheyinteractwithplastidencodedsubunitsthatshowtypical,slowratesofevolutioninallofthespecies(PSI).ThemtCLP
complex,whichiscomprisedsolelyofnuclearsubunits,andthesetofrandomnucleargeneslargely
supportedthisexpectation.TheconcatenatedmtCLPgenesinfastspeciesexhibitedverysimilar(albeit
slightlyhigher)dN/dSlevelscomparedtoslowspecies,andtherewasnosignificantdifferenceforthe
randomgenes(Tables1and2).Furthermore,theMKtestsfoundnoevidenceofpositiveselectionfor
eitherofthesedatasets(Tables3,S6,andS7).Incontrast,thenuclearPSIgenesdidexhibitasignificant
excessofnonsynonymousdivergence(Tables3andS7)eventhoughtheabsoluterateofaminoacid
substitutionswasextremelylow.Thisevidencesuggeststhat,althoughrare,someofthe
nonsynonymoussubstitutionsinPSIareadaptivechangesthatspreadunderpositiveselectionrather
thanfixingbydrift.
Analternateinterpretationfortheobservedexcessofnonsynonymousdivergencebetween
speciesisthattherewasanancestralbottleneck,whichcouldhaveledtoanincreasedfrequencyof
weaklydeleteriousallelesspreadingtofixationbecauseofthereducedefficiencyofselectioninsmall
23
populations(Hughes2007).However,weidentifiedthreelinesofevidencethatleadustorejectthis
possibilityandconcludethatourresultsare,infact,indicativeofstrongpositiveselection.First,ifa
demographicbottleneckhadoccurred,wewouldexpectanexcessofnonsynonymousdivergenceacross
allgenes,butwedidnotobservethisinthemtCLPgenesortherandomlychosennucleargenes.
PreviousstudiesinSilenealsosupporttheconclusionthattheobservedchangesarenottheresultof
genome-widedemographiceffects;analysisof140cytosolicribosomalproteinsandseven
mitochondrial-targetedcomplexIIgenesinS.conicaandS.noctifloradidnotshowelevateddN/dS
relativetootherSilenespecies(Sloanetal.2014b;Havirdetal.2015).Second,weobservedelevated
levelsofnonsynonymousdivergenceacrossdifferenttimescalesinourMKtests(Tables3andS7)by
usingtwodifferentoutgroups:S.latifolia(~5.7Myrdivergencetime)andS.macrodonta(~1.8Myr
divergencetime;Rautenbergetal.2012).Therefore,separatebottlenecksataminimumoftwo
differenthistoricalpointswouldhavetohavetakenplace.Third,themagnitudeoftheobservedeffects
isinconsistentwithabottleneck.TherelaxedselectionassociatedwithareducedNe,shouldnot
increasedN/dStovaluessignificantlyaboveone,whichwerefrequentlyobservedinourdataset(Table
1).Thus,weconcludethat,althoughitispossiblethatanancestralbottleneckinS.conicamighthave
contributedtosomeminorincreasesinaminoacidsubstitutionrates,themassiverateincreases(e.g.,in
CLPgenes)aremorelikelytohavebeendrivenbypositiveselectionthanatemporaryreductioninNe.
AntagonisticCo-EvolutionandPlastid-NuclearConflict
Theacceleratedaminoacidsubstitutionratesinboththenuclear-andplastid-encodedcomponentsof
CLPandACCaseareveryunusualforancientlyconservedenzymecomplexes,butaresimilarinmany
waystothepatternsthatresultfromantagonisticco-evolutionbetweenpathogensandhostimmune
genes(HughesandNei1988;Borghansetal.2004).Selfishinteractionsand“armsraces”canoccur
withinacell(i.e.,intragenomicconflict)whenthereisopportunityforgeneticelementstoenhancetheir
owntransmissionattheexpenseoforganismal-levelfitness(BurtandTrivers2006).Suchconflictsare
24
commonbetweenthenucleusandcytoplasmicgenomes.Forexample,copiesofmitochondrial
genomeswithlargedeletionscanconferareplicationadvantagewithinthecelleveniftheyharm
overallfitnessbyreducingoreliminatingthecell’sabilitytorespire(Tayloretal.2002;Clarketal.2012;
Phillipsetal.2015).Inaddition,becausemostcytoplasmicgenomesareinheritedmaternally,theycan
benefitfrommanipulatingsexualreproductiontoincreasefemalereproductionandfitness(Perlmanet
al.2015).ExamplesofthisphenomenonincludechimericORFsinplantmitochondrialgenomesthat
inducecytoplasmicmalesterility(CMS;IngvarssonandTaylor2002;TouzetandBudar2004;Fujiietal.
2011)andnumerousbacterialendosymbiontsthatmanipulatesexualreproductioninanimalhosts
(Werrenetal.2008).
Interestingly,someoftheearliesthypothesesaboutcytonuclearconflictweredevelopedbased
onobservationsofdifferentialratesofreplicationofplastidgenomesinheteroplasmicplants(Grun
1976;reviewedinGreineretal.2015).Sincethatpoint,however,researchoncytonuclearconflictin
plantshasoverwhelminglyfocusedonmitochondria,particularlytheirroleinCMS.Althoughplastidsare
oftenviewedasbeingrelativelybenign,inprinciple,thesameevolutionarypressurescouldapplyto
thesematernallyinheritedorganelles.Onepossibilityisthattherearelimitedpathwaysavailablefor
plastidstoexploitinaselfishfashion.Forexample,themajorroleofplastids(specificallychloroplasts)is
inphotosynthesis,andmalereproductivetissuesaregenerallynon-photosynthetic.However,plastids
alsoperformotherimportantprocesses(includingCLPandACCaseactivity).Recentstudieshave
providedsupportforthepossibilityofselfishplastid-nuclearinteractionswithincomplexessuchasthe
heteromericACCaseandtheCLPcomplex.Inparticular,reproductiveincompatibilities(includingmale
sterility)betweenwildanddomesticatedlinesofpeaswererecentlyattributedtovariationinnuclear-
andplastid-encodedcomponentsoftheheteromericACCase(Bogdanovaetal.2015).Plastid-nuclear
incompatibilitieshavealsobeenimplicatedinmalesterilityinOenothera(StubbeandSteiner1999).
Disruptingsynthesisoffattyacidsandtheirderivativessuchasjasmonicacidhasalsobeenassociated
25
withsterilityphenotypes(Parketal.2002).Inaddition,arecentproteomicanalysisinwheatfoundthat
plastid-encodedclpP1wasoneofthemostupregulatedgenesintheanthersofmale-sterileindividuals,
suggestingthatitmayplayimportantfunctionalrolesinmalereproductivetissues(Lietal.2015).
Althoughthecorrelatedincreasesinevolutionaryratesandsignaturesofpositiveselectionin
Sileneplastid-nuclearcomplexescouldindicateahistoryofgenomicconflict,theyarenotconclusive
evidencethatplastidandnucleargenesarelockedinanarmsrace,oreventhattheyareco-evolvingin
anyfashion(LovellandRobertson2010).GeneralchangesinselectionforCLPorACCasefunctioncould
simultaneouslyaffectallsubunits,withoutinteractionsamongthesubunitsbeingamajorsourceof
selection.ItisalsopossiblethattheoverallstructureorsubunitcompositionoftheCLPcomplexhas
beenradicallydisruptedorreorganized.Furthermore,evenifco-evolutionarydynamicsareatplay,they
involvemutuallybeneficialchangesratherthanantagonisticinteractions(Randetal.2004).Adaptive
changesinonegenomemayalterfitnesslandscapesandfacilitatesubsequentadaptivechangesinthe
othergenome,thoughitisunclearwhatforcesmighttriggersuchrunawayadaptiveevolutioninthese
systems.Amoreconventionalmodelofcompensatorychangeinthenucleusinresponseto
accumulationofdeleteriouschangesinasexualorganellegenomes(Randetal.2004;OsadaandAkashi
2012)seemslesslikely–particularlyfortheCLPcomplex–giventheevidencethatacceleratedratesof
sequenceevolutionintheplastid-encodedclpP1evolutionaredrivenlargelybypositiveselection
(ErixonandOxelman2008;Sloanetal.2012a;Barnard-Kubowetal.2014).
Itisworthnotingthatthelargestincreasesinsubstitutionrateswerefoundinthenuclearencodedsubunitsthatinteractmostdirectlywithplastid-encodedsubunits.Specifically,thegreatest
elevationofdN/dSintheCLPcomplexoccurredintheCLPRsubunits(Table1),whichassemblewith
plastid-encodedClpP1subunitstoformtheproteolyticR-ring(NishimuraandvanWijk2015).More
detailedstructuralanalysisoftheCLPcomplex(Figure3;Table4)showedthat,evenwithinsubunits,
therewasanenrichmentforsubstitutionsindomainsthathaveintimateinteractionswithClpP1(i.e.,
26
theheaddomainsofCLPRproteinsandhandledomainsofCLPPproteins;YuandHoury2007;Nishimura
andvanWijk2015).FortheheteromericACCase,wefoundevidenceofpositiveselection(Table3)only
onthenuclear-encodedACCAsubunitinS.conica,whichinteractsdirectlywiththeplastid-encoded
ACCDsubunittomakeupthecarboxyltransferase(SasakiandNagano2004).However,ourstructural
analysisdidnotdetectanenrichmentofsubstitutionsattheinterfacebetweenACCAandAccD(Figure
4),anditisdifficulttodrawfirmconclusionsaboutACCasestructuregiventhelackofknowledgeabout
thefunctionsandinteractionsoftheplant-specificportionsofACCAandAccD.
Therefore,itappearsmorelikelythatplastid-nuclearinteractionsandco-evolutionhaveplayed
aroleingeneratingpositiveselectionandtheobservedaccelerationsinratesofsequenceevolutionin
theCLPcomplexthanintheheteromericACCase.Wespeculatethatthemysteriousrateaccelerations
thathaveoccurredrepeatedlyinclpP1inSileneandthroughoutthediversificationoffloweringplants
(andpossiblythosethathaveoccurredinotherplastidgenesaswell)aretheresultofantagonisticcoevolutionbetweentheplastidandnucleus.Animportanttestofthishypothesiswillbetofunctionally
characterizethesequencechangesfromrapidlyevolvinglineages,particularlywithrespecttotheir
phenotypiceffectsonplastidreplicationwithincellsandplantallocationtomalevs.female
reproductiveoutput.
ACKNOWLEDGEMENTS
WethankAndreaBerardi,RollandDouzet,PeterFields,MichaelHood,AndreasKönig,ArneSaatkamp,
theKewMilleniumSeedBank,theOrnamentalPlantGerplasmCenter,andtheVilledeNantesJardin
Botaniqueforcollecting/providingseeds.WealsothankCodyKalousandJessicaHurleyforperforming
RNAextractionsandqualitycontrol.WearegratefulforvaluablecommentsfromStephenWrightand
twoanonymousreviewersonanearlierversionofthismanuscript.Thisresearchwassupportedby
27
grantsfromtheNationalScienceFoundation(NSFMCB-1412260andMCB-1022128).KRissupportedby
aGAANNgraduatefellowshipfromtheU.S.DepartmentofEducation(P200A140008)andisa
participantintheNSF-fundedGAUSSIgraduatetrainingprogram(DGE-1450032).JCHissupportedbya
NationalInstitutesofHealthPostdoctoralFellowship(F32GM116361).
28
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Table 1. Summary of dN/dS estimates. Values greater than one are highlighted in bold, with underlined
text indicating statistical significance based on likelihood ratio tests. Cells containing "--" indicate that the
particular gene was not found in the corresponding transcriptome. Only concatenated results are reported
for the set of 50 random genes. Individual gene results for this random set are available in Table S5. Note
that CLPP5a and CLPP5b are recently duplicated paralogs and that only one of these copies was
recovered from the Agrostemma transcriptome. Therefore, this Agrostemma sequence was used in both
CLPP5 analyses.
Random
MT CLP
Photosystem I
ACC
Plastid CLP
Gene
A. githago
S. paradoxa
S. conica
S. noctiflora
S. latifolia
S. vulgaris
CLPP3
0.09
0.88
2.89
2.31
0.52
0.00
CLPP4
0.02
0.36
0.62
1.24
0.21
0.00
CLPP5a
0.03
0.52
1.55
0.53
0.00
0.03
CLPP5b
0.02
0.94
2.03
1.77
0.13
0.17
CLPP6
0.10
0.57
2.84
1.23
0.35
0.09
CLPR1
0.04
1.79
8.86
2.40
0.46
0.10
CLPR2
0.02
3.65
1.16
2.41
0.06
0.00
CLPR3
0.03
8.64
1.24
2.29
0.22
0.07
CLPR4
0.01
1.83
2.37
2.02
0.08
0.06
CLPC
0.02
0.22
0.47
0.75
0.02
0.03
CLPT1
0.10
1.66
1.31
7.31
0.27
0.10
CLPT2
0.17
0.67
0.80
1.24
0.31
0.36
CLPS
0.02
0.03
0.05
0.39
0.00
0.04
Concatenated
0.05
0.87
1.30
1.55
0.16
0.06
ACCA
0.01
0.99
0.49
--
0.06
0.05
ACCB1
0.17
--
0.45
--
0.07
0.14
ACCB2
0.67
0.18
0.11
--
0.42
0.59
ACCC
0.01
0.08
0.08
--
0.08
0.06
Concatenated
0.13
0.33
0.21
--
0.16
0.13
LHCA2
0.03
0.00
0.04
0.04
0.09
0.02
LHCA3
0.02
0.05
0.13
0.06
0.02
0.07
PSAK
0.00
0.07
0.28
0.45
0.00
0.00
PSAL
0.02
0.08
0.18
0.11
0.00
0.00
PSAN
0.07
0.14
0.09
0.05
0.09
0.00
PSAO
0.09
0.00
0.20
0.13
0.21
0.05
PSAP
0.18
0.12
0.03
0.14
0.18
0.16
Concatenated
0.05
0.05
0.14
0.11
0.08
0.04
CLPP2
0.05
0.48
0.09
0.00
0.12
0.21
CLPX1
0.06
0.21
0.08
0.12
0.07
0.02
CLPX2
0.16
0.13
0.26
0.20
0.22
0.18
CLPX3
0.12
0.52
0.32
0.08
0.31
0.30
Concatenated
0.10
0.28
0.18
0.11
0.16
0.15
Concatenated
0.12
0.15
0.12
0.15
0.13
0.15
35
Table 2. Summary of dN/dS for constrained sets of fast and slow lineages based on concatenations of
genes in each functional complex. The p-values indicate significant differences in rate between the two
sets of lineages based on LRTs.
dN/dS
Genes
Slow Lineages
Fast Lineages
Plastid CLP
0.07
1.25
0.000
ACC
0.14
0.26
0.014
Photosystem I
0.05
0.10
0.002
MT CLP
0.11
0.20
0.002
Random
0.13
0.13
0.894
p-value
36
Random
MT CLP
Photosystem I
ACC
Plastid CLP
Table 3. Summary of MK tests using intraspecific polymorphism within S. conica relative to interspecific
divergence between S. conica and S. latifolia. For each gene, the neutrality index (NI) and the direction of
selection (DoS) were calculated, with NI values less than 1 and DoS values greater than 0 indicative of
positive selection (i.e., an excess of nonsynonymous divergence). For concatenations of genes within each
complex, NITG (an unbiased estimator of NI) is reported in parentheses. Only concatenated results are
reported for the set of random genes. Individual gene results for this random set are available in Table S6.
Gene
Ps
Pn
Ds
Dn
NI
DoS
p-value
CLPP3
8
3
16
83
0.07
0.57
0.000
CLPP4
18
0
31
51
0.00
0.62
0.000
CLPP5a
5
2
4
19
0.08
0.54
0.006
CLPP5b
0
0
11
25
--
--
--
CLPP6
0
2
11
59
--
-0.16
0.543
CLPR1
1
1
22
144
0.15
0.37
0.256
CLPR3
3
2
48
120
0.27
0.31
0.128
CLPR4
1
1
22
80
0.28
0.28
0.337
CLPC
4
1
55
50
0.28
0.28
0.226
CLPT1
4
0
10
34
0.00
0.77
0.001
CLPT2
7
1
19
39
0.07
0.55
0.002
CLPS
3
0
11
2
0.00
0.15
0.467
Concatenated
54
13
260
706
0.09 (0.11)
0.54
0.000
ACCA
13
7
42
111
0.20
0.38
0.001
ACCB1
0
1
14
11
--
-0.56
0.270
ACCB2
4
3
10
7
1.07
-0.02
0.939
ACCC
10
3
22
18
0.37
0.22
0.160
Concatenated
27
14
88
147
0.31 (0.31)
0.28
0.001
LHCA2
4
0
19
6
0.00
0.24
0.271
LHCA3
13
1
28
9
0.24
0.17
0.167
PSAK
1
0
13
7
0.00
0.35
0.468
PSAL
5
0
15
5
0.00
0.25
0.211
PSAN
11
2
18
10
0.33
0.20
0.183
Concatenated
34
3
93
37
0.22 (0.20)
0.20
0.011
CLPP2
1
1
21
4
5.25
-0.34
0.233
CLPX2
4
5
26
23
1.41
-0.09
0.634
CLPX3
9
3
12
8
0.50
0.15
0.387
Concatenated
14
9
59
35
1.08 (1.05)
-0.02
0.866
Concatenated
368
178
1297
570
1.10 (1.07)
-0.02
0.371
37
Table 4. The ratio of amino acid substitutions in the head vs. handle regions is higher in CLPR subunits
than CLPP subunits. Values represent counts of substitutions summed across all nuclear-encoded genes in
each category.
Head:Handle Ratio
Fisher's Exact Test
Species
CLPP
CLPR
p-value
Silene conica
85:63
136:61
0.031
Silene noctiflora
50:37
109:36
0.006
Silene paradoxa
49:40
127:22
0.000
38
Figure 1. Rates of sequence evolution in nuclear genes coding for subunits of the plastid and
mitochondrial CLP complexes. Branch lengths are scaled to the amount of nonsynonymous (dN) and
synonymous (dS) divergence per site. Species with rapid rates of plastid genome evolution (Sloan et al.
2014a) are highlighted in red.
39
Figure 2. Gene-by-gene comparison of dN/dS in fast vs. slow sets of lineages. Points are color-coded by
functional complex, and the diameter of each point is proportional to gene length. All points represent
individual genes except the “random” point, which is based on the concatenation of all 50 genes in that
set. The size of that point is scaled to the average length of each gene rather than the total concatenation
length. ACCA and CLPS, which exhibit distinct patterns from the other ACCase and CLP subunits are
labeled individually.
40
Figure 3. A) ClpP protease structure from E. coli (PDB 1YG6; Yu and Houry 2007) oriented with the
two heptameric rings stacked on top of each other (left) or in front and behind (right). One of the 14
(identical) E. coli subunits is highlighted in blue. B) A single E. coli ClpP subunit (as in part A), with the
head, handle, and axial-loop domains highlighted. The surface of the catalytic triad is indicated in mesh
and the individual residues (Ser, His, and Asp) are labeled. Other important residues are numbered and
indicated with stick models, including those that interact with substrates (Phe-17 and Phe-49) or
contribute to heptamer stability by forming hydrophobic bonds (Asn-41/Tyr-20, Asn-41/Thr-32, Asp78/Asn-116, and Asp-171/Try-128) or ion pairs (Arg-118/Glu-141, Arg-170/Glu-134, and Asp-171/His138). C) Locations of substitutions in Silene species with fast-evolving CLP sequences (S. conica, S.
noctiflora, and S. paradoxa). Residues with changes (relative to the inferred ancestral Silene sequence) in
one, two, or three species are indicated in yellow, orange, and red, respectively. The black triangle
highlights the ClpP1 site at which large insertions have occurred in S. conica (39 amino acids) and S.
noctiflora (7 amino acids).
41
Figure 4. A) Structure of the carboxyltransferase component from the E. coli ACCase (PDB 2F9Y),
oriented as in Figure 2C from Bilder et al. (2006). Residues of active sites (Gly-204, Gly-205, Gly-206,
Gly-207) are shown as spheres, the four cysteines that form cysteinyl zinc ligands (Cys-27, Cys-30, Cys46, Cys-49) are shown as stick models, and the helices and sheets that compose the catalytic platform are
indicated. B) The same structure as in part A with inferred Silene changes mapped as described in Figure
3. The red arrow indicates the location of an AccD site with large insertions in both S. conica (26 amino
acids) and S. paradoxa (58 amino acids). Importantly, this model contains only the portions of ACCA and
AccD that can be aligned and mapped to E. coli, as the copies of these genes in plants are roughly twice
the length of their counterparts in E. coli owing to large N- or C-terminal extensions of unknown
function. C) Structure of the biotin carboxylase (ACCC)-biotin carboxyl carrier protein (ACCB) complex
from the E. coli ACCase (PDB 4HR7) as in Figure 2A from Broussard et al. (2013). D) An ACCC
monomer complexed with two ACCB monomers as in Figure 2D in Broussard et al. (2013), indicating
functional domains and ACCB-ACCC interfaces. The Lys-122 residues that bind biotin and the active site
of ACCC (Arg-338) are shown as spheres, while residues that play important roles in ACCB-ACCC
interactions are shown as sticks and highlighted with arrows. E) The same structure as part D with
inferred Silene changes mapped as described in Figure 3. F) Two ACCC dimers and a single ACCB
monomer, showing ACCC-ACCC interfaces as in Figure 4A from Broussard et al. (2013). Residues that
play important roles in ACCC-ACCC interactions are shown as sticks and highlighted with arrows. G)
The same structure as part F with inferred Silene changes mapped as described in Figure 3.
42