University of Dundee

Progressive and biased divergent evolution underpins the origin and diversification of peridinin plastids Dorrell, Richard G.; Klinger, Christen M.; Newby, Robert J.; Butterfield, Erin R.; Richardson, Elisabeth; Dacks, Joel B. Published in: Molecular Biology and Evolution

DOI: 10.1093/molbev/msw235

Publication date: 2016

Licence: CC BY-NC

Document Version Peer reviewed version

Link to publication in Discovery Research Portal

Citation for published version (APA): Dorrell, R. G., Klinger, C. M., Newby, R. J., Butterfield, E. R., Richardson, E., Dacks, J. B., Howe, C. J., Nisbet, R. E. R., & Bowler, C. (2016). Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids. Molecular Biology and Evolution, 34(2), 361-379. https://doi.org/10.1093/molbev/msw235

General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

• Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Download date: 28. Sep. 2021 MBE Advance Access published November 4, 2016

Articles:*Discoveries* * Progressive*and*biased*divergent*evolution*underpins*the*origin*and*diversification*of* peridinin*dinoflagellate*plastids* * Richard(G.(Dorrell1+,(Christen(M.(Klinger*2,(Robert(J.(Newby*3,(Erin(R.(Butterfield4,(5,(Elisabeth( Richardson2,(Joel(B.(Dacks2,(Christopher(J.(Howe6,(R.(Ellen(R.(Nisbet6,(and(Chris(Bowler1( ( 1(Ecole(Normale(Supérieure,(PSL(Research(University,(Institut(de(Biologie(de(l’Ecole(Normale( Supérieure((IBENS),(CNRS(UMR(8197,(INSERM(U1024,(46(rue(d’Ulm,(F[75005(Paris,(France( 2(Department(of(Cell(Biology,(University(of(Alberta( 3(Department(of(Biology,(Middle(Tennessee(State(University(( 4(Department(of(Biochemistry,(Pennsylvania(State(University(

5(School(of(Life(Sciences,(University(of(Dundee( Downloaded from 6(Department(of(Biochemistry,(University(of(Cambridge( *(Contributed(equally(to(this(manuscript( +(To(whom(correspondence(should(be(addressed:([email protected](( ( * http://mbe.oxfordjournals.org/ * * * * * * * at University of Dundee on November 11, 2016 * * * * * * * * * * * * * * * * * * * * *

©* The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This* is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/),* which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]*

! 1! * Abstract* * *are*algae*of*tremendous*importance*to*ecosystems*and*to*public*health.* The*cell*biology*and*genome*organisation*of*dinoflagellate*species*is*highly*unusual.*For* example,*the*plastid*genomes*of*peridinin?containing*dinoflagellates*encode*only*a* minimal*number*of*genes*arranged*on*small*elements*termed*“minicircles”.*Previous* studies*of*peridinin*plastid*genes*have*found*evidence*for*divergent*sequence*evolution,* including*extensive*substitutions,*novel*insertions*and*deletions,*and*use*of*alternative* translation*initiation*codons.*Understanding*the*extent*of*this*divergent*evolution*has* been*hampered*by*the*lack*of*characterised*peridinin*plastid*sequences.*We*have* identified*over*300*previously*unannotated*peridinin*plastid*mRNAs*from*published* transcriptome*projects,*vastly*increasing*the*number*of*sequences*available.*Using*these* data,*we*have*produced*a*well?resolved*phylogeny*of*peridinin*plastid*lineages,*which* Downloaded from uncovers*several*novel*relationships*within*the*dinoflagellates.*This*enables*us*to*define* changes*to*plastid*sequences*that*occurred*early*in*dinoflagellate*evolution,*and*that*have* contributed*to*the*subsequent*diversification*of*individual*dinoflagellate*clades.*We*find* that*the*origin*of*the*peridinin*dinoflagellates*was*specifically*accompanied*by*elevations* both*in*the*overall*number*of*substitutions*that*occurred*on*plastid*sequences,*and*in*the* http://mbe.oxfordjournals.org/ Ka/Ks*ratio*associated*with*plastid*sequences,*consistent*with*changes*in*selective* pressure.**These*substitutions,*alongside*other*changes,*have*accumulated*progressively* in*individual*peridinin*plastid*lineages.*Throughout*our*entire*dataset,*we*identify*a* persistent*bias*towards*non?synonymous*substitutions*occurring*on*sequences*encoding* photosystem*I*subunits*and*stromal*regions*of*peridinin*plastid*proteins,*which*may*have* underpinned*the*evolution*of*this*unusual*organelle.* ( at University of Dundee on November 11, 2016 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

! 2! ( Introduction* ( Dinoflagellates(are((with(immense(ecological(and(evolutionary(importance.(They( include(photosynthetic,(heterotrophic,(mixotrophic,(and(parasitic(representatives((Dorrell( and(Howe(2015).(The(photosynthetic(species(are(a(major(component(of(plankton( communities(in(marine(and(freshwater(environments((Leterme,(et(al.(;(de(Vargas,(et(al.( 2015),(and(include(causative(agents(of(harmful(algal(blooms((Protoperidinium,-)( (Hallegraeff(2010;(Hinder,(et(al.(2012),(symbionts(of(corals(()(and(marine( protozoa((Pelagodinium,-Brandtodinium)((Siano,(et(al.(2010;(Probert,(et(al.(2014).(The(non[ photosynthetic(species(include(parasites(of(marine(invertebrates((,- ,(and(ellobiopsids)((Gornik,(et(al.(2012),(and(bioluminescent(phagotrophs( (Noctiluca)((Nakamura(1998).(Dinoflagellates(are(members(of(the(,(a(group(that( additionally(contains(important(laboratory(model(species(((such(as(-and( Downloaded from ),(pathogens(of(terrestrial(and(marine(animals((e.g.,(the(apicomplexan(parasites( -and(Toxoplasma,(and(the(mollusc(pathogen(),(ecologically(significant( heterotrophs((ciliates(and(gregarines).(Two(other(photosynthetic(alveolates(have(been( described((the(“chromerids”(Chromera-and(Vitrella),(both(of(which(are(closely(related(to(the( apicomplexans,(and(serve(as(model(organisms(for(understanding(the(origins(of(parasitism(in( http://mbe.oxfordjournals.org/ this(lineage((Janouskovec,(et(al.(2010;(Dorrell(and(Howe(2015).( ( Dinoflagellates(are(renowned(for(their(unusual(and(distinctive(cell(biology((Lin(2011;( Wisecaver(and(Hackett(2011;(Dorrell(and(Howe(2015).(Unlike(those(of(all(other(studied( eukaryotes,(dinoflagellate(chromosomes(are(maintained(in(a(permanently(condensed(state,( and(do(not(principally(utilise(histones(for(DNA(packaging((Gornik,(et(al.(2012).(The( mitochondrial(genomes(of(dinoflagellates(and(apicomplexans(are(highly(reduced,(and( at University of Dundee on November 11, 2016 several(otherwise(well[conserved(subunits(of(the(ATP(synthase(complex(have(not(been( documented(in(any(dinoflagellates((Butterfield,(et(al.(2013;(Janouškovec,(et(al.(2013).( ( One(of(the(oddest(traits(of(dinoflagellates(is(their(possession(of(unusual(plastids.(While(all( other(major(eukaryotic(groups(are(either(non[photosynthetic,(or(contain(only(one(plastid( lineage,(at(least(four(and(potentially(as(many(as(seven(phylogenetically(distinct(plastid( lineages(have(been(documented(across(the(dinoflagellates((Janouskovec,(et(al.(2010;(Dorrell( and(Howe(2015).(The(majority(of(photosynthetic(dinoflagellates(possess(plastids(that( contain(the(light(harvesting(pigment(peridinin((Haxo,(et(al.(1976).(These("peridinin(plastids"( are(most(likely(of(red(algal(origin,(although(the(exact(endosymbiotic(events(through(which( they(originated(remain(debated((Keeling(2010;(Ševčíková,(et(al.(2015).(Phylogenetic(studies( have(indicated(that(the(peridinin(plastid(was(present(in(a(common(ancestor(of( dinoflagellates,(chromerids,(and(apicomplexans,(and(that(the(alternative(plastids(found(in( some(dinoflagellates(have(originated(through(serial(endosymbiosis((Janouskovec,(et(al.( 2010;(Dorrell(and(Howe(2015).(( ( The(genome(of(the(peridinin(plastid(is(the(smallest(known(from(a(photosynthetic(plastid,( retaining(only(twelve(protein[coding(genes((Barbrook,(et(al.(2014;(Mungpakdee,(et(al.(2014),( plus(ribosomal(RNA(and(some(transfer(RNA(genes((Barbrook,(et(al.(2006;(Nelson,(et(al.( 2007).(The(protein(coding(genes(solely(encode(core(subunits(of(the(photosynthetic(electron( transport(machinery,(comprising(genes(encoding(six(subunits(of(photosystem(II((psbA,-psbB,- psbC,-psbD,-psbE,-psbI),(and(two(subunits(each(of(photosystem(I((psaA,-psaB),(cytochrome( b6/f((petB,-petD)(and(plastid(ATP(synthase((atpA,-atpB).(These(genes(are(located(on(small( elements(termed(“minicircles”((of(1600[6600(bp(length)((Zhang,(et(al.(1999;(Nelson(and( Green(2005)(and("microcircles"((of(400[(600bp(length)((Nisbet,(et(al.(2004).(Minicircles(

! 3! typically(contain(single(genes,(although(minicircles(that(contain(no(genes((Nisbet,(et(al.(2004;( Nelson,(et(al.(2007),(or(combinations(of(multiple(genes(have(also(been(identified(in(multiple( dinoflagellate(species((Hiller(2001;(Nisbet,(et(al.(2004;(Nelson,(et(al.(2007).(All(other(genes(of( plastid(origin(that(have(been(documented(in(peridinin(dinoflagellates(are(located(in(the( nucleus((Morse,(et(al.(1995;(Bachvaroff,(et(al.(2004;(Mungpakdee,(et(al.(2014).(Some( minicircles(contain(sizeable(open(reading(frames(of(unknown(function(that(are(unique(to( dinoflagellates((Barbrook,(et(al.(2001;(Nisbet,(et(al.(2004;(Barbrook,(et(al.(2006).(It(has(been( proposed(that(some(peridinin(plastids(contain(genes(encoding(ribosomal(proteins((rpl28,- rpl33)(and(iron[sulfur(cluster(biogenesis(factors((ycf16,-ycf24),(which(were(acquired(by( horizontal(gene(transfer(from(non[photosynthetic(bacteria,(although(whether(these( sequences(are(genuinely(plastid[encoded(remains(controversial((Moszczynski,(et(al.(2012;( Dorrell(and(Howe(2015).(( (

Alongside(the(extreme(level(of(reduction(observed(in(the(peridinin(plastid(genome,(the( Downloaded from transcript(sequences(produced(in(peridinin(plastids(are(highly(unusual.(This(is(in(part(due(to( the(unusual(transcript(processing(machinery(associated(with(peridinin(plastids,(which( includes((in(some(species)(extensive(in[frame(sequence(editing((Zauner,(et(al.(2004;(Dorrell( and(Howe(2015)(and((in(all(documented(species)(the(addition(of(a(3’(poly(U)(tail(to(plastid( mRNAs((Wang(and(Morse(2006;(Barbrook,(et(al.(2012),(which(contrast(with(the(3'(poly(A)(tail( http://mbe.oxfordjournals.org/ and(5'(spliced[leader(sequences(added(to(transcripts(in(dinoflagellate(nuclei((Zhang,(et(al.( 2007).(In(addition,(individual(genes(within(peridinin(plastids(are(highly(divergent(from( orthologues(from(other(plastid(lineages((Shalchian[Tabrizi,(et(al.(2006;(Pochon,(et(al.(2014).( Genes(encoded(located(in(peridinin(plastids(frequently(contain(extensive(sequence( substitutions((Barbrook,(et(al.(2014)(and(in[frame(insertions(and(deletions((Barbrook,(et(al.( 2006;(Barbrook,(et(al.(2014),(have(unusual(codon(usage(preferences((Inagaki,(et(al.(2004;( Bachvaroff,(et(al.(2006)(and(use(a(range(of(alternative(translation(initiation(codons(in( at University of Dundee on November 11, 2016 addition(to(ATG((ATA,(ATT,(GTA,(TTG)((Zhang,(et(al.(1999;(Barbrook,(et(al.(2014).(( ( This(project(was(conceived(to(investigate(the(timing(and(extent(of(the(divergent(sequence( evolution(in(peridinin(plastids.(It(is(broadly(agreed(that(application(of(poly(U)(tails(to(plastid( transcripts(is(an(ancestral(feature(of(peridinin(dinoflagellates,(and(it(has(been(proposed(that( the(fragmentation(of(the(peridinin(plastid(genome(into(minicircles,(and(the(reduction(of(the( plastid(genome(to(a(minimal(protein[coding(gene(set,(are(likewise(ancestral((Janouskovec,(et( al.(2010;(Dorrell,(et(al.(2014;(Dorrell(and(Howe(2015).(However,(it(is(not(known(when(other( divergent(evolutionary(events(occurred(in(peridinin(plastids.(This(has(in(part(been(due(to(the( lack(of(available(sequence(information,(with(essentially(complete(plastid(coding(sequences( previously(available(only(for(-carterae-(Nisbet,(et(al.(2004),(and(for(two(strains( of(Symbiodinium-(Clade(C3,(and(Clade(Mf)(-(Barbrook,(et(al.(2014;(Mungpakdee,(et(al.(2014).( In(addition,(the(phylogenetic(relationships(within(the(peridinin(dinoflagellates(remain(poorly( resolved.(While(Amphidinium-is(agreed(to(diverge(at(the(base(of(the(peridinin( dinoflagellates,(the(branching(order(of(other(lineages(remains(debated((Hoppenrath(and( Leander(2010;(Bachvaroff,(et(al.(2014;(Gavelis,(et(al.(2015).(We(wished(to(generate(a(robust( phylogeny(of(extant(peridinin(dinoflagellate(lineages,(and(use(this(phylogeny(to(answer( three(questions:((1)(which(of(the(divergent(features(associated(with(peridinin(plastid( sequences(probably(have(arisen(in(an(ancestor(to(all(species(studied;((2)(to(what(extent(and( in(which(dinoflagellate(lineages(have(divergent(plastid(evolution(events(occurred(more( recently,(and((3)(whether(there(are(any(consistent(trends(across(the(dinoflagellates(in(terms( of(which(plastid[encoded(proteins,(or(regions(of(plastid[encoded(proteins,(are(the(most( divergent,(extending(from(their(common(ancestor(through(to(extant(species.(( (

! 4! We(present(a(taxonomically(detailed(reconstruction(of(the(evolution(of(peridinin(plastid( sequences.(We(have(focused(on(identifying(novel(plastid(transcripts,(which(allows(us(to( assess(the(composite(effects(of(divergent(gene(evolution(and(transcript(editing(on(peridinin( plastid(sequences((Zauner,(et(al.(2004;(Dorrell(and(Howe(2015).(We(have(annotated(over( 300(new(peridinin(plastid(sequences(from(published(transcriptome(resources,(and( demonstrate(that(the(twelve(photosystem(genes(previously(identified(in(peridinin(plastids( (Mungpakdee,(et(al.(2014;(Dorrell(and(Howe(2015)(probably(represent(the(complete( protein[coding(component(of(the(plastid(genome(of(a(common(ancestor(of(all(extant( photosynthetic(dinoflagellates.(We(have(used(these(sequences(to(generate(a(well[resolved( phylogeny(of(peridinin(plastids,(uncovering(novel(evolutionary(relationships(between(among( the(major(dinoflagellate(lineages,(and(have(used(this(phylogeny(to(determine(when(different( divergent(evolutionary(events(are(have(occurred(in(peridinin(plastids.(To(disentangle(the( different(factors(that(have(underpinned(this(unusual(sequence(evolution,(we(have(calculated( substitution(rates,(and(Ka/Ks(ratios((also(referred(to(as(dN/dS,(and(defined(as(the(relative( Downloaded from enrichment(in(non[synonymous(substitutions,(Ka,(to(synonymous(substitutions,(Ks,(over(a( particular(sequence,(which(provides(an(indicator(of(the(strength(of(selective(pressure((Yang( and(Bielawski(2000;(Hurst(2002))(for(each(dinoflagellate(species(and(each(residue(of(each( plastid[encoded(protein(studied.( ( http://mbe.oxfordjournals.org/ We(show(that(the(origin(of(the(peridinin(plastid(was(specifically(marked(by(an(elevation(in( substitution(rates(and(in(Ka/Ks(ratios,(consistent(with(changes(in(selection(pressure(in(the( dinoflagellate(common(ancestor.(We(additionally(show(that(these(and(other(divergent( features,(such(as(in[frame(insertions(and(alternative(translation(initiation(codons,(have( continued(to(evolve(progressively(in(individual(dinoflagellate(clades.(Finally,(we(show(that(in( both(the(common(ancestor(of(all(studied(peridinin(dinoflagellates(and(in(extant(species,( elevated(Ka/Ks(ratios(are(concentrated(on(genes(encoding(photosystem(I(subunits,(and( at University of Dundee on November 11, 2016 codons(encoding(stromal[facing(residues(of(plastid(proteins.(This(may(suggest(that(specific( changes(to(dinoflagellate(physiology(have(driven(the(divergent(evolution(of(the(peridinin( plastid.(Ultimately,(our(study(provides(valuable(insights(into(the(evolutionary(history(of(this( unusual(plastid(lineage,(from(its(very(origins(to(subsequent(diversification.( ( Results* * 1.*Annotation*of*new*peridinin*plastid*sequences*from*transcriptome*data* * Identification-of-plastid-sequences* * New(dinoflagellate(orthologues(of(the(twelve(protein[coding(genes((atpA,-atpB,-petB,-petD,- psaA,-psaB,-psbA,-psbB,-psbC,-psbD,-psbE-and(psbI)(previously(found(to(be(retained(in( peridinin(plastids((Howe,(et(al.(2008;(Barbrook,(et(al.(2014;(Mungpakdee,(et(al.(2014)(were( identified(in(the(NCBI(EST(library,(and(transcriptome(libraries(within(the(Marine( Microeukaryote(Transcriptome(Sequencing(Project((MMETSP)((Keeling,(et(al.(2014).(A(total( of(381(sequences(were(identified,(364(of(which(had(no(previously(annotated(equivalents( (Fig.(1;(Table(S1).(The(majority((348)(of(the(novel(sequences(were(identified(from(previously( unannotated(transcripts(within(the(MMETSP(libraries,(with(only(a(few(sequences((16)( identifiable(from(EST(libraries(located(in(NCBI((Fig.(1).(Sequences(of(plausible(plastid(origin( were(identified(in(all(but(a(few(peridinin(dinoflagellate(MMETSP(libraries,(with(a(minimum(of( 15(novel(orthologues(found(for(each(gene(studied((Table(S1),(and(complete(sets(of(protein[ coding(plastid(genes(identified(for(13(new(dinoflagellate(species(in(addition(to(the(three( already(characterised((Howe,(et(al.(2008;(Barbrook,(et(al.(2014;(Mungpakdee,(et(al.(2014).(( *

! 5! Novel-sequences-are-of-probable-plastid-origin- ( Uninterrupted(poly(T)(stretches(of(4(bp(or(longer(were(detected(on(the(3’(end(of(half( (191/381)(of(the(novel(dinoflagellate(sequences,(consistent(with(the(presence(of(the(poly(U)( tails(associated(with(dinoflagellate(plastid(transcripts((Fig.(S1;(Table(S1).(Across(the(entire( dataset,(only(one(sequence(was(found(that(terminated(in(a(possible(3’(poly(A)(tail,(and(none( contained(evidence(of(5’(spliced[leader(sequences,(or(plausible(tripartite(targeting( sequences((consisting(of(a(signal(peptide,(ASAFAP[delimited(transit(peptide,(and(a( downstream(hydrophobic(region),(as(are(associated(with(nucleus[encoded,(plastid[targeted( proteins(in(peridinin(dinoflagellates((Nassoury(and(Morse(2005;(Zhang,(et(al.(2007)((Table( S1).(( ( Limited-additional-coding-sequences-in-peridinin-plastids-

* Downloaded from The(entire(assembled(transcriptome(dataset(was(screened(for(further(transcripts(that(might( originate(from(peridinin(plastids.(We(could(not(find(convincing(evidence(for(any(further( genes(that(are(plastid[encoded(in(other(non[dinoflagellate(lineages,(and(might(still(be( plastid[encoded(within(individual(dinoflagellates,(beyond(the(twelve(photosystem(genes( previously(documented((Fig.(S2;(Supplementary(Results,(Section(1).(The(entire(dataset(was( http://mbe.oxfordjournals.org/ additionally(searched(for(homologues(of(the(four(plastid(genes((rpl28,-rpl33,-ycf16,-ycf24)( proposed(to(have(been(gained(by(horizontal(transfer(from(bacteria(into(specific(peridinin( plastids((Moszczynski,(et(al.(2012).(A(total(of(148(new(sequences(were(identified(and( inspected(using(single[gene(phylogenies.(While(the(original(Pyrocystis-lunula-and(Ceratium- horridum-sequences(grouped(with(Bacteroidetes,(none(of(the(homologues(within(this( dataset(did:(instead,(the(majority(resolved(as(a(monophyletic(group(and(all(grouped(either( within,(or(as(sister[groups(to,(other(plastid(or(cyanobacterial(lineages,(with(robust((>90%)( at University of Dundee on November 11, 2016 bootstrap(support((Fig.(S3[S6).(None(of(the(rpl28,-rpl33,-ycf16-or(ycf24-transcript(sequences( identified(in(this(study(possessed(a(3’(poly(U)(tail((Table(S1);(however,(many(contained(3'( poly(A)(tails,(spliced[leader(sequences((Table(S1),(and(tripartite(plastid(targeting(sequences( (Fig.(S7),(consistent(with(a(nuclear(origin.(( ( Homologues(of(the(four(novel(open(reading(frames(previously(identified(on(minicircles( located(in(the(Amphidinium-carterae(plastid((Barbrook(and(Howe(2000;(Dorrell(and(Howe( 2015)(were(searched(for(across(the(entire(transcript(dataset.(Only(equivalents(of(A.-carterae- ORF1,(ORF2-and(ORF3-were(detected,(and(these(were(limited(to(the(related(species( Amphidinium-massartii((Fig.(S8,(panel(A).(The(ORF[like(sequences(identified(in(A.-massartii- were(highly(divergent(from(those(of(A.-carterae,(with(only-46%((ORF1),(32%((ORF2)(and(33%( (ORF3)((between(the(two(sequences,(compared(to((for(example)(97%(sequence(conservation( between(the(psbD-sequences(from(each(species((Fig.(S8,(panel(A).(We(could(find(only(limited( evidence(for(the(presence(of(additional(conserved(polyuridylylated(transcripts(that(might( correspond(to(novel(plastid(ORFs(within(the(dataset((Fig.(S8;(Supplementary(Results,(Section( 1).( ( 2.*Reconstruction*of*phylogenetic*relationships*between*peridinin*dinoflagellates* ( A(concatenated(protein(alignment((3,410(amino(acids,(average(72.7%(pairwise(identities)( was(generated,(consisting(of(the(twelve(plastid(sequences(studied,(for(each(of(the( dinoflagellates(present(in(MMETSP,(and(a(reference(set(of(fifteen(non[dinoflagellates((Table( S3).(Bayesian(and(Maximum(Likelihood(trees(were(generated(from(this(alignment((Fig.(2;( Table(S4).(Two(phylogenetically(distinct(sets(of(plastid(sequences(were(identified(for(clade(A( Symbiodinium,(one(of(which(resolved(with(other(Symbiodinium-species,(and(the(other(as(a(

! 6! sister(group(to(A.-carterae,(which(presumably(represents(a(contamination(within(the( Symbiodinium-A(MMETSP(library((Fig.(2;(Table(S1).(( ( Consistent(with(previous(data,(Amphidinium-was(identified(as(the(earliest(diverging(peridinin( dinoflagellate(genus((Fig.(2)((Dorrell(and(Howe(2015;(Gavelis,(et(al.(2015).((Following( Amphidinium,-the(next(most(basal(dinoflagellates(were(Togula-jolla,(and(a(clade(consisting(of( ,(,(and(the((previously(Suessialean)(species(Pelagodinium-beii,( which(diverged(from(a(clade(consisting(of(,(,(and(the((previously( Peridinialean)(genus(Heterocapsa,(with(moderate(to(robust(support(((in(Bayesian(analysis,( >60%(in(ML(trees)((Fig.(2).(Identical((Fig.(S9)(or(nearly(identical((Fig.(S10)(topologies(to(the( original(tree(were(obtained(in(trees(calculated(from(alignments(from(which(long(branches,( fast(evolving(sites,(or(individual(plastid(genes(had(been(removed((Supplementary(Results,( Section(2),(suggesting(that(the(initial(tree(topology(was(largely(accurate.(

( Downloaded from 3.*Dinoflagellate?wide*changes*in*plastid*sequence*composition* * Changes-in-plastid-GC-content-may-have-occurred-before-the-radiation-of-the-dinoflagellates- ( The(first,(second(and(third(position(GC(content(were(compared(across(a(4,478(nt(gap[free( http://mbe.oxfordjournals.org/ alignment(of(six(plastid(genes((psaA,-psaB,-psbA,-psbB,-psbC,-psbD),(for(each(of(the( dinoflagellates(studied,(and(all(of(the(non[dinoflagellate(sequences(previously(used(for(the( multigene(phylogeny((Fig.(2).(Elevated(GC(contents(were(observed(at(third(codon(positions( in(many(of(the(dinoflagellates(compared(to(non[dinoflagellate(species((Fig.(S11).(These( included(third(position(GC(content(values(of(>(35%(in(three(of(the(earliest(diverging( dinoflagellate(clades((Fig.(S11;(Amphidinium(GC[3]=(39.6[49.6%,(Peridiniales(GC[3]=(29.8[ 40.9%,(Prorocentrales((GC[3]=(31.7[39.5%).(However,(the(chromerid(Vitrella-brassicaformis,( at University of Dundee on November 11, 2016 which(forms(the(closest(sister[group(to(the(dinoflagellates(within(the(multigene(tree((Fig.(2),( also(has(a(high(third(position(GC(content((Fig.(S11;(46.9%),(so(it(is(possible(that(this(GC( enrichment(is(not(specific(to(dinoflagellates.(( ( Limited-changes-to-plastid-translation-in-the-dinoflagellate-ancestor-- - Across(the(entire(4,478(nt(gap[free(plastid(alignment,(18(codons(occurred(with(lower( frequency((and(19(codons(with(higher(frequency)(in(dinoflagellates(compared(to(non[ dinoflagellate(species((one[way(ANOVA,(P(<(0.05;(Table(S5;(Fig.(S12).(Ten(amino(acids(were( likewise(found(to(occur(with(lower(frequency,(and(5(with(higher(frequency,(in(dinoflagellates( (one[way(ANOVA,(P(<(0.05;(Table(S5;(Fig.(S12)(over(this(alignment,(as(inferred(by(a(standard( translation(table.(We(could(not(identify(any(convincing(evidence(for(changes(to(plastid( translation(tables(within(the(dinoflagellates((Table(S6;(Fig.(S13;(Supplementary(Results,( Section(3).( ( A(much(smaller(number(of(codons(were(found(to(have(undergone(specific(de[enrichments( (6)(or(enrichments((8;(chi[squared(test,(P<(0.05;(Table(S5,(Fig.(S12)(in(a(common(ancestor(of( all(studied(dinoflagellates((inferred(by(comparing(the(regressed(ancestral(sequence(of(all( dinoflagellates(studied(to(that(of(the(regressed(ancestral(sequence(of(the(common(ancestor( of(all(studied(dinoflagellates(and(Vitrella-brassicaformis,(which(was(the(closest(sister[group( to(dinoflagellates(included(in(the(alignment-(Janouskovec,(et(al.(2010)).(Comparing(the(two( datasets,(only(four(codons((ATA[Ile,(AGA[Arg,(ACC[Thr,(and(TAT[Tyr)(were(found(both(to( occur(at(significantly(higher(frequencies(in(dinoflagellates(than(non[dinoflagellates,(and(to( have(undergone(a(specific(enrichment(in(the(common(ancestor(of(all(studied(dinoflagellates( (Table(S5;(Fig.(S12).(Similarly,(only(one(codon((CGT[Arg)(was(found(to(occur(at(a(significantly(

! 7! lower(frequency(in(dinoflagellates,(and(to(have(undergone(a(specific(de[enrichment(in(the( common(ancestor(of(all(studied(dinoflagellates((Table(S5;(Fig.(S12).(Finally,(only(one(amino( acid((Tyrosine)(was(found(to(have(undergone(a(significant(change(in(frequency(in(the( common(ancestor(of(all(studied(dinoflagellates((Table(S5;(Fig.(S12),(suggesting(overall(that( relatively(limited(changes(to(plastid(codon(usage(are(associated(with(dinoflagellate(origins.( ( 4.*Dinoflagellate?wide*changes*to*plastid*sequence*evolution** * Elevated-pairwise-substitutions-at-the-origin-of-dinoflagellates- - Total(numbers(of(pairwise(substitutions(were(calculated(for(every(species(used(in(the( multigene(phylogeny,(over(the(4,478(nucleotide(gap[free(alignment((Fig.(3;(Table(S7).(The( dinoflagellate(sequences(were(highly(divergent(from(the(non[dinoflagellate(species((Fig.(3;( compare(top(left(hand(corner(of(figure(to(remainder).(On(average,(dinoflagellate(and(non[ Downloaded from dinoflagellate(species(pairs(were(separated(by(1,549(nucleotide(substitutions,(while(pairs(of( non[dinoflagellate(species(were(separated(by(an(average(of(994(substitutions,(which(was( significantly(lower((Table(S7;(Fig.(S14,(panel(A;(one[way(ANOVA,(P=(1.86(x(10[179).(Even(the( related((lineage(Vitrella-brassicaformis,(which(was(separated(from(other(non[ dinoflagellate(species(by(a(much(larger(number(of(substitutions((average(value(1276)(was( http://mbe.oxfordjournals.org/ significantly(less(divergent(than(dinoflagellate(species(studied((Table(S7;(Fig.(S14,(panel(A;(P=( 1.2(x(10[18),(suggesting(that(this(elevated(substitution(rate(is(specifically(associated(with( dinoflagellate(species.( ( We(tested(whether(the(elevated(numbers(of(substitutions(observed(between(dinoflagellate( and(non[dinoflagellate(species(were(related(either(to(plastid(GC(content,(codon(usage,(or( amino(acid(composition((Figs.(S14,(S15;(Supplementary(Results,(Section(4).(While(changes(to( at University of Dundee on November 11, 2016 plastid(GC(content(and(codon(usage(were(correlated(to(the(total(numbers(of(pairwise( substitutions(observed((Fig.(S15),(alignment(recoding(to(remove(these(effects(did(not( eliminate(the(elevated(substitution(rates(associated(with(dinoflagellate(species((Fig.(S14,( panel(B).( ( Elevated-Ka/Ks-ratios-at-the-origin-of-dinoflagellates- - Pairwise(Ka/Ks(ratios((which(provide(information(regarding(the(strength(of(selective( pressure(acting(on(individual(sequences,(as(expressed(by(the(ratio(of(non[synonymous(to( synonymous(substitutions)(were(additionally(calculated(for(each(species(pair((Fig.(3;(Table( S7).(Similar(to(the(situation(observed(for(total(substitution(rates,(much(higher(pairwise(Ka/Ks( ratios(were(observed(between(dinoflagellate(and(non[dinoflagellate(species(pairs((average( value(0.0592)(than(within(non[dinoflagellate(species(pairs((Fig.(S16,(panel(A;(average(value( 0.0183;(P=(2.75(x(10[111).(A(dramatic(difference(was(also(observed(for(the(Ka/Ks(ratios( calculated(between(dinoflagellates(and(non[dinoflagellate(species,(and(Vitrella-and(all(other( non[dinoflagellate(species((Fig.(S16,(panel(A;(average(value(0.0271;(P=(2.87(x(10[18).(( ( We(tested(whether(the(elevated(Ka/Ks(ratios(observed(in(dinoflagellates(might(be(related(to( the(extremely(high(total(numbers(of(pairwise(substitutions(observed,(for(example(due(to(a( saturating(substitution(rate(at(codon(third(positions(leading(to(an(underestimate(of(the(total( synonymous(substitutions(between(dinoflagellate(and(non[dinoflagellate(species((Figs.(S17[ S18;(Supplementary(Results,(Section(5).(The(third(position(substitution(rates(between( dinoflagellate(and(non[dinoflagellate(species,(while(high,(were(not(at(saturation(rate((Figs.( S17,(S18;(Supplementary(Results,(Section(5).(Other(variables(tested(were(either(not( correlated(to(the(pairwise(Ka/Ks(ratios(obtained((in(the(case(of(amino(acid(composition),(or(

! 8! were(not(sufficient((judged(by(alignment(recoding)(to(explain(the(differences(in(Ka/Ks(ratios( observed((in(the(case(of(third(position(GC(content,(and(codon(usage;(Figs.(S16,(S19;( Supplementary(Results,(Section(6).(( ( 5.*Lineage?specific*changes*to*peridinin*plastid*sequences* * Extremely-elevated-Ka/Ks-ratios-within-dinoflagellates- * Some(dinoflagellate(species(were(found(to(have(extremely(elevated(pairwise(Ka/Ks(ratios( calculated(relative(to(other(dinoflagellates(in(the(alignment((Fig.(3).(For(example,( Pelagodinium-beii(was(separated(from(all(other(dinoflagellates(by(a(minimum(number(of( 1,400(substitutions,(and(Brandtodinium-nutriculum-was(separate(from(all(other( dinoflagellates(by(a(minimum(number(of(1,399(substitutions((Fig.(3;(Table(S7),(both(of(which( were(far(larger(than(the(average(minimum(number(of(total(pairwise(substitutions((416)( Downloaded from calculated(for(other(dinoflagellate(species((Z(test,(P(<(0.05).(These(separations(were(found(to( be(independent(of(plastid(GC(content(and(codon(usage(patterns(in(both(species((Fig.(S20;( Supplementary(Results,(Section(8).(Both(species(were(also(found(to(have(elevated(Ka/Ks( ratios((minimum(P.-beii-Ka/Ks(0.0618;(minimum(B.-nutriculum(Ka/Ks(0.0408;(average( dinoflagellate(minimum(Ka/Ks(0.0174;(Fig.(3),(but(these(differences(were(eliminated(by( http://mbe.oxfordjournals.org/ removing(codon(third(positions(from(Ka/Ks(calculations,(suggesting(that(they(are(the(result( of(saturating(mutation(rates(in(each(species((Fig.(S20;(Supplementary(Results,(Section(8).( ( A(dramatic(evolutionary(divergence(was(observed(within(members(of(the(Gonyaulacales(and( Suessiales((Fig.(3,(bottom(right(hand(sector;(Fig.(S21).(Species(within(these(lineages(had( extremely(high(pairwise(Ka/Ks(ratios,(with(a(maximum(value(of(0.284(between(the( Gonyaulacales(Protoceratium-reticulatum-and(Pyrodinium-bahamense,(which(is(significantly( at University of Dundee on November 11, 2016 greater(than(the(average(maximum(pairwise(Ka/Ks(ratio((0.140)(observed(for(all( dinoflagellates-(Fig.(3;(Table(S7;(Z(test,(P<(0.05).(The(average(pairwise(Ka/Ks(ratio(between( Gonyaulacalean(and(Suessialean(dinoflagellates((0.110)(was(significantly(higher(than(the( average(pairwise(Ka/Ks(ratio(between(other(dinoflagellate(pairs((0.043;(Fig.(S21,(panel(A;( P=1.57(x(10[09).(This(was(found(to(be(independent(of(both(third(position(substitutions,(and( changes(to(codon(usage(in(these(species((Supplementary(Results,(Section(8).(In(contrast,(the( average(number(of(pairwise(substitutions(between(Gonyaulacalean(and(Suessialean(species( (711)(was(significantly(lower((P=0)(than(the(average(pairwise(substitution(frequencies( observed(between(other(dinoflagellates((1417;(Fig.(3;(Fig.(S21,(panel(B),(indicating(that(the( rapid(divergent(evolution(within(this(lineage(is(specifically(due(to(an(elevated(Ka/Ks(ratio.( * Widespread-evolution-of-alternative-translation-initiation-codons-in-peridinin-plastids- ( 44%(of(all(the(sequences(identified(lacked(a(plausible(ATG(initiation(codon,(hence(probably( use(alternative(translation(initiation(codons((Table(S8).(Eight(different(codons(were( identified(as(probable(alternative(initiation(sites(for(individual(peridinin(plastid(transcripts,( with(TTG(and(ATT(occurring(the(most(frequently((Fig.4;(Table(S8).(None(of(the(alternative( translation(initiation(codons(identified(was(conserved(across(all(dinoflagellates,(and(the( majority(were(species[specific((Fig.(4,(panel(A).(However,(twenty(alternative(initiation( codons(were(conserved(across(multiple(dinoflagellate(species((Fig.(4,(panel(B,(square(labels),( such(as(the(adoption(of(a(TTG(alternative(initiation(codon(in-psbA-in(a(common(ancestor(of( Alexandrium,-,-Pyrodinium-and(-sp.((Fig.(4,(panel(B,(label(I;(Fig.(S22).( Some(lineages(utilise(alternative(initiation(codons(more(frequently(than(others,(with(six( alternative(initiation(codons((petB[GTG,(petD[(KTG,(psaB[ATW,(psbC[ATC,(psbD[ATY,(psbE[

! 9! ATT)(found(in(Scrippsiella-hangoei-and(its(sister(species(-aciculiferum-(Fig.(4,(panel( B,(labels(C,(D),(compared(to(only(one((atpA[TTG)(in(Amphidinium-sp.((Fig.(4,(panel(B,(label(A).(( (( Multiple-discrete-changes-to-plastid-protein-sequences-within-dinoflagellates* ( A(total(of(111(insertions(and(48(deletions(distributed(across(80(positions(were(identified( within(dinoflagellates((Fig.(4,(Panel(A;(Table(S9).(This(contrasts(to(the(situation(for(the(non[ dinoflagellate(species(in(the(alignment,(for(which(only(19(insertions(and(22(deletions(were( identified-(Table(S9).(Twelve(insertions(and(five(deletions(were(conserved(across(all( dinoflagellates((Fig.(4,(panel(A;(Fig.(S23,(panel(A),(although(many(of(these(indels(have( undergone(substantial(expansions(or(contractions(in(individual(species((Fig.(S23,(panel(B).( The(majority(of(indels,(however,(were(restricted(to(individual(dinoflagellate(species((77( insertions,(25(deletions)(or(clades((22(insertions,(18(deletions)((Fig.(4,(panel(A;(Fig.(S23,( panel(C).(These(included(two(insertions((in(PsaB,(insertion(starting(at(consensus(residue(41;( Downloaded from and(PsbC,(residue(197)(and(one(deletion((in(PsbB,(starting(at(consensus(residue(291)(that( evolved(in(a(common(ancestor(of(all(species(studied,(except(for(the(basally(divergent( Amphidinium,(and(one(insertion((PsaB,(residue(168)(and(one(deletion((PsaB,(residue(602)(in( a(common(ancestor(of(Gonyaulacales(and(Suessiales((Fig.(4,(panel(B;(triangular(labels).(- * http://mbe.oxfordjournals.org/ Instances(in(which(a(residue(that(is(conserved(in(all(other(plastid(species(studied(was(lost(in( dinoflagellates(were(tabulated(for(one(representative(protein,(the(ATP(synthase(subunit( AtpA.(200(such(changes(were(found,(of(which(only(15(were(ancestral((Fig.(4,(panel(A;(Table( S10).(Of(the(remaining(185(substitutions,(93(were(species[specific,(while(92(were(shared( across(specific(dinoflagellate(clades.(These(clade[specific(changes(included(the(loss(of( thirteen(otherwise(conserved(residues(in(a(common(ancestor(of(all(genera(except( Amphidinium,-and(two(in(a(common(ancestor(of(Gonyaulacales(and(Suessiales((Fig.(4,(panel( at University of Dundee on November 11, 2016 B,(circular(labels;(Table(S10).(( ( 6.*Identification*of*consistent*trends*in*peridinin*plastid*evolution* ( Photosystem-I-sequences-in-peridinin-plastids-have-elevated-Ka/Ks-ratios- - An(elevated(dinoflagellate(Ka/Ks(ratio(was(observed(in(the(two(photosystem(I(subunit(genes( (psaA-and(psaB)(retained-in(dinoflagellate(plastids.(For(psaA,(the-dinoflagellate(Ka/Ks((0.481)( was(4.89(times(that(of(the(non[dinoflagellate(value((0.098);(whereas(for(psaB-the( dinoflagellate(Ka/Ks((0.446)(was(4.47(times(that(of(the(non[dinoflagellate(value((0.099;(Fig.( 5,(panel(A).(Both(the(Ka/Ks(ratios(observed(were(substantially(greater(than(those(observed( for(dinoflagellate(sequences(across(the(entire(dataset,(both(in(terms(of(the(raw(Ka/Ks(ratio( (0.296)(and(in(terms(of(the(relative(enrichment((2.41)(compared(to(the(non[dinoflagellate( Ka/Ks(ratio((0.123;(Fig.(5,(panel(A).(Elevated(Ka/Ks(values(were(also(obtained(for( dinoflagellate(psaA-and(psaB-genes(in(alignments(recoded(to(eliminate(third(position( substitutions(and(codon(usage(bias((Fig.(S24;(Tables(S11,(S12[S15;(Supplementary(Results,( Section(8).( ( Photosystem-I-sequences-in-the-dinoflagellate-ancestor-also-had-elevated-Ka/Ks-ratios( ( Individual(Ka/Ks(ratios(were(also(calculated(for(each(sequence,(solely(between(the( hypothetical(sequences(calculated(for(the(common(ancestor(of(all(studied(dinoflagellates,( and(the(common(ancestor(of(dinoflagellates(and(Vitrella,(which(should(correspond(to(the( substitutions(that(most(probably(occurred(in(dinoflagellates(immediately(following(their( divergence(from(other(plastid(lineages((Table(S11).(Elevated(Ka/Ks(ratios(were(found(in(both(

! 10! the(dinoflagellate(ancestor(psaA-(ancestral(Ka/Ks(0.425;(dinoflagellate(ancestor/non[ dinoflagellate(ratio(4.32)(and(psaB-sequences((ancestral(Ka/Ks(0.593;(dinoflagellate( ancestor/non[dinoflagellate(ratio(5.95),(compared(to(all(other(genes((ancestral(Ka/Ks(0.376;( dinoflagellate(ancestor/non[dinoflagellate(ratio(3.07;(Fig.(5,(panel(A;(Tables(S11,(S12).(The( Ka/Ks(ratios(associated(with(the(psaB-dinoflagellate(ancestor(sequence(were(confirmed( using(alignment(recoding(to(be(genuine,(rather(than(a(consequence(of(a(highly(elevated( mutation(rate(or(change(in(codon(usage(preference(specific(to(dinoflagellate(photosystem(I( genes((Fig.(S24;(Supplementary(Results,(Section(8).( ( Elevated-Ka/Ks-ratios-are-specific-to-photosystem-I-genes- - Only(one(other(gene,(psbI,(was(found(to(have(a(greater(than(average(Ka/Ks(ratio(between( dinoflagellates(and(non[dinoflagellates((dinoflagellate(Ka/Ks(0.451;(non[dinoflagellate(Ka/Ks(

0.132;(dinoflagellate/non[dinoflagellate(ratio(3.39)(and(between(the(dinoflagellate(ancestor( Downloaded from and(non[dinoflagellates((dinoflagellate(ancestor(Ka/Ks(0.512;(dinoflagellate(ancestor/non[ dinoflagellate(ratio(3.84;(Fig.(5,(panel(A;(Table(S11).(However,(the(elevated(psbI-Ka/Ks(ratio( was(eliminated(in(alignments(recoded(to(eliminate(third(position(substitutions((Fig.(S24;( Supplementary(Results,(Section(8),(suggesting(that(it(is(the(result(of(due(a(saturating( mutation(rate(at(dinoflagellate(psbI-third(codon(positions.(No(other(genes((except(for(psaA- http://mbe.oxfordjournals.org/ and(psaB)(were(found(to(have(elevated(associated(Ka/Ks(ratios.( ( Stromal-domains-of-peridinin-plastid-proteins-have-elevated-Ka/Ks-ratios- ( Individual(Ka/Ks(ratios(were(calculated(for(each(residue(within(each(plastid(gene,(for( dinoflagellates,(non[dinoflagellates,(and(the(common(ancestor(of(all(studied(dinoflagellates( (Table(S11).(The(distribution(of(residues(with(elevated(Ka/Ks(was(biased(towards(specific( at University of Dundee on November 11, 2016 regions(of(individual(proteins.(For(example,(within(psbB,(59(codons(have(elevated(associated( Ka/Ks(ratios(either(in(the(common(ancestor(of(all(studied(dinoflagellates((positions(at(the( centre(of(a(block(of(ten(residues(with(aggregate(Ka/Ks(>1,(and(Ka/Ks(significantly(greater( than(that(calculated(for(the(equivalent(non[dinoflagellate(residue;(Z[test,(P<(0.05),(or(within( the(dinoflagellates((same(criteria,(but(with(Ka/Ks(>(0.5;(Fig.(S25).((All(of(these(codons(are( predicted(to(encode(stromal[(or(luminal[facing(psbB-residues((Fig.(S25).( ( Across(the(entire(dataset,(171(of(the(331(residues(identified(to(have(elevated(Ka/Ks(ratios(in( the(common(ancestor(of(all(studied(dinoflagellates(were(located(on(predicted(stromal(faces( of(plastid(proteins((Fig.(5,(panel(B;(Fig.(S26,(panel(A;(Tables(S11,(S12).(This(is(significantly( greater(than(the(number((123)(expected(through(a(random(distribution(of(residues((chi[ squared,(P=(6.43(x(10[10;(Table(S11).(The(elevated(Ka/Ks(ratios(on(stromal(residues(were(also( identified(in(calculations(performed(with(alignments(recoded(to(eliminate(third(position( substitutions(and(codon(usage(bias((Fig.(S26;(Supplementary(Results,(Section(9).( ( The(same(trends(were(not(directly(observed(within(the(dinoflagellates,(where(the(number(of( stromal(residues(with(elevated(Ka/Ks(was(in(fact(slightly(fewer(than(expected((202/582( residues;(expected(number(216;(Fig.(S26,(panel(B;(Tables(S11,(S12).(However,(this(may(be( influenced(by(the(low(Ka/Ks(ratios(observed(in(dinoflagellates(for(AtpA((0.251)(and(AtpB( (0.254;(Fig.(5,(panel(A;(Table(S11),(which(are(the(only(two(proteins(encoded(in(peridinin( dinoflagellate(plastids(to(be(entirely(extrinsic(to(the(thylakoid(membrane,(hence(entirely( stromal[facing((Walker(2013).(Excluding(AtpA(and(AtpB,(the(number(of(residues(with( elevated(Ka/Ks(in(dinoflagellates(that(are(predicted(to(face(into(the(plastid(stroma(was( extremely(greater(than(expected((172/(552(residues(with(elevated(Ka/Ks;(expected(number( of(residues(29;(P=0;(Fig.(S26,(panel(B;(Tables(S11,(S12).(Similar(to(the(dinoflagellate(ancestor,(

! 11! the(enrichment(in(dinoflagellate(stromal(Ka/Ks(was(confirmed(through(alignment(recoding( to(be(genuine,(rather(than(a(result(of(mutation(rate(saturation(or(codon(usage(bias( (Supplementary(Results,(Section(9).( ( Discussion* ( We(have(identified(and(analysed(previously(unannotated(sequences(for(plastid[encoded( transcripts(in(peridinin(dinoflagellates.(These(data(constitute(over(three(times(the(number(of( previously(annotated(peridinin(dinoflagellate(plastid(sequences,(and(increases(the(number( of(complete(peridinin(plastid(protein[coding(datasets(fivefold((Fig.(1).(Particularly(large( numbers(of(plastid(transcript(sequences(were(identified(from(the(MMETSP(transcriptome( datasets((Fig.(1).(This(may(be(due(to(the(presence(of(the(3'(poly(U)(tail(on(dinoflagellate( plastid(transcripts,(which(has(previously(been(speculated(to(enable(the(enrichment(of(plastid( transcript(sequences(in(poly(A)(enriched(RNA(libraries((Wang(and(Morse(2006)(such(as(those( Downloaded from used(for(generation(of(the(MMETSP(libraries((Keeling,(et(al.(2014),(and(is(corroborated(by( the(presence(of(poly(U)(tails(on(many(of(the(transcripts(we(identified((Fig.(S1;(Table(S1).(( ( Almost(all(of(the(dinoflagellates(investigated(appear(to(possess(the(same(twelve(plastid[ encoded(protein[coding(genes(previously(identified((Howe,(et(al.(2008;(Barbrook,(et(al.( http://mbe.oxfordjournals.org/ 2014;(Mungpakdee,(et(al.(2014).(We(found(no(evidence(for(relocation(of(any(of(these( sequences(to(the(nucleus(in(any(species((Fig.(S1;(Table(S1)(or(the(retention(of(other(plastid[ derived(sequences(in(any(peridinin(plastid((Fig.(S2;(Table(S2).(We(additionally(found(only( limited(evidence(for(the(presence(of(laterally(acquired(genes(in(the(plastids(of(peridinin( dinoflagellates,(or(for(the(conservation(of(other(novel(ORFs(previously(identified(in(individual( peridinin(plastid(lineages(across(multiple(species((Fig.(S8;(Table(S2).(While(we(cannot( formally(exclude(that(other(plastid(transcripts((transcripts(that(do(not(receive(a(3'(poly(U)( at University of Dundee on November 11, 2016 tail(hence(are(unlikely(to(be(present(in(poly(A)[enriched(libraries)(are(produced,(our(data( indicates(that(the(twelve(previously(identified(protein[coding(genes(represents(the(ancestral( protein[coding(component(of(dinoflagellate(plastids.(( ( These(data(have(allowed(us(to(produce(a(well[resolved(reference(tree(for(the(branching( relationships(between(major(clades(of(peridinin(dinoflagellates((Fig.(2;(Fig.(S9;(Fig.(S10).( Several(novel(phylogenetic(relationships(were(uncovered.(For(example,(Pelagodinium-beii- (previously(-beii)-grouped(with(the(Prorocentrales(with(reasonable(support( (100/64%)(whereas(previous(studies(based(on(single[gene(phylogenies(placed(it(within(the( Suessiales((Siano,(et(al.(2010;(Decelle,(et(al.(2014).(Similarly,(sister[group(relationships( between(Prorocentrum(and(the(Peridiniales,(and(the(monophyletic(clade(of(Gonyaulacales,( Suessiales(and(Heterocapsa-(Fig.(2),(have(not(to(our(knowledge(been(previously(described( (Hoppenrath(and(Leander(2010;(Bachvaroff,(et(al.(2014;(Gavelis,(et(al.(2015).(Each(of(these( novel(relationships(were(also(recovered(using(modified(alignments(from(which(long( branches,(individual(genes,(gapped(positions,(or(fast[evolving(sites(were(removed((Fig.(S9;( Fig.(S10).(We(accordingly(conclude(that(the(relationships(obtained(within(our(dataset(are( probably(genuine,(and(not(the(artifact(of(fast(sequence(evolution(or(recent(discrete(changes( (to(plastid(translation(tables,(which(might(bias(the(conceptual(translations(obtained,(but( presumably(should(be(removed(in(the(fast(site(analysis)(in(individual(dinoflagellate(plastids.(( ( We(have(correlated(divergent(changes(to(peridinin(plastid(transcript(sequences(to(the( branching(relationships(from(the(multigene(phylogeny,(allowing(us(to(infer(when(these( changes(occurred.(For(these(analyses,(we(have(focused(exclusively(on(the(sequences(and( conceptual(translations(of(plastid(transcripts,(which(provide(an(understanding(of(the( aggregate(consquences(of(divergent(gene(evolution(and(transcript(editing(on(peridinin(

! 12! plastids((Zauner,(et(al.(2004;(Bachvaroff,(et(al.(2006;(Dorrell(and(Howe(2015).(As(many(of(the( sequences(identified(from(MMETSP(possess(poly(U)(tails((Fig.(S1;(Table(1),(and(the(presence( of(the(poly(U)(tail(is(associated(with(the(completion(of(transcript(editing(in(dinoflagellate( species((Dang(and(Green(2009;(Dorrell,(et(al.(2016),(we(presume(that(the(majority(of(the( sequences(identified(in(this(study(probably(have(been(edited(to(completion,(as(opposed(to( representing(unedited(precursor(transcripts.( ( First,(we(have(identified(changes(to(peridinin(plastid(sequence(composition(across(the( dinoflagellates.(These(include(changes(to(plastid(sequence(GC(content(and(codon(usage((Fig.( S11;(Fig.(S12),(although(many(of(these(changes(are(either(shared(with(relatives((such(as(the( elevated(third(position(GC(content(in(the(chromerid(Vitrella;(Fig.(S11),(or(appear(largely(to( consist(of(changes(specific(to(individual(dinoflagellate(lineages((such(as(the(changes(to( plastid(codon(usage(frequencies,(which(are(much(more(numerous(in(extant(dinoflagellate( species(than(in(the(inferred(sequences(of(their(last(common(ancestor;(Fig.(S12).(We(could( Downloaded from not(find(convincing(evidence(for(changes(to(the(plastid(translation(table(across(the( dinoflagellates((Fig.(S13).(While(we(cannot(exclude(alternative(hypotheses((for(example,( independent(increases(in(third(position(GC(content(in(the(lineages(giving(rise(to(Vitrella,(and( in(a(common(ancestor(of(all(dinoflagellates),(we(cannot(find(sufficient(evidence(for(major( changes(to(plastid(sequence(composition,(or(translation,(occurring(in(the(dinoflagellate( http://mbe.oxfordjournals.org/ common(ancestor.( ( Next,(we(have(identified(widespread(changes(to(the(translation(products(of(plastid( sequences(in(dinoflagellates.(These(include(large(numbers(of(synonymous(substitutions,(and( greatly(elevated(Ka/Ks(ratios(in(the(dinoflagellates((Fig.(3;(Fig.(S14;(Fig.(S16)).(The(elevated( Ka/Ks(ratios(substitutions(are(unlikely(to(be(explained(by(changes(in(codon(preference((Fig.( S11;(Fig.(S16),(or(saturation(of(synonymous(substitution(rates(at(third[position(sites(in( at University of Dundee on November 11, 2016 dinoflagellate(plastids((Fig.(S17;(Fig.(S18;(Fig.(S19).((It(is(possible(that(other(factors(related(to( sequence(composition((e.g.,(transitory(changes(in(codon(preference(in(individual( dinoflagellate(lineages,(or(saturation(of(synonymous(substitution(rates(at(first[(and(second[ position(sites)(may(have(contributed(to(the(elevated(Ka/Ks(ratios(observed;(however,(we( suggest(that(the(most(parsimonious(explanation(for(the(remaining(elevated(Ka/Ks(ratios( observed(in(dinoflagellate(plastids(are(changes(in(plastid(selection(pressure(throughout(their( evolution,(following(their(divergence(from(other(plastid(lineages,(and(prior(to(the(radiation( of(extant(species.(Although(previous(studies(have(posited(changes(in(selection(pressure(on( certain(peridinin(plastid(sequences((Shalchian[Tabrizi,(et(al.(2006),(this(is(to(our(knowledge( the(first(evidence(that(indicates(that(selective(events(have(played(a(widespread(role(in( dinoflagellate(plastid(evolution.( ( We(have(additionally(identified(changes(that(have(occurred(in(individual(dinoflagellate( lineages(since(their(radiation((Fig.(3;(Fig.(4;(Figs.(S20[S23).(We(have(found(extremely( elevated(Ka/Ks(ratios(in(pairwise(comparisons(between(dinoflagellates((Fig.(3;(Figs.(S20;( S21),(and(map(multiple(acquisitions(of(alternative(translation(initiation(codons((Fig.(S22),(in[ frame(insertion(and(deletions((Fig.(S23),(and(the(loss(of(otherwise(conserved(atpA-residues( to(the(dinoflagellate(tree((Fig.(4).(These(events(have(occurred(progressively,(with(the( majority(of(the(dinoflagellate(clades(being(marked(by(discrete(changes(to(plastid(sequence( (Fig.(4),(and(contrasts(with(the(much(more(conservative(evolution(observed(in(other(plastid( lineages((Tables(S8,(S9).(The(divergent(evolutionary(events(observed(in(this(study(await( detailed(biochemical(characterisation.(For(example,(it(will(be(interesting(to(determine( experimentally(whether(peridinin(dinoflagellates(utilise(the(alternative(translation(initiation( codons(identified(in(this(study,(or(whether(there(are(further(translation(initiation(codons( with(weaker(similarity(to(ATG(in(peridinin(plastids.(This(could(be(accomplished,(for(example,(

! 13! by(proteomic(characterisation(of(the(N[termini(of(peridinin(plastid(proteins((Huesgen,(et(al.( 2013).(Regardless,(our(data(show(that(peridinin(plastid(sequences(have(not(remained(static,( but(have(continued(to(diverge(from(one(another(to(a(remarkable(extent.( ( We(have(found(evidence(that(different(peridinin(plastid(lineages(have(evolved(in(different( manners(since(their(radiation.(For(example,(we(observe(elevated(minimum(pairwise( substitution(rates(in(some(Peridinialean/(Prorocentralean(species((e.g.,(Brandtodinium,- Pelagodinium;(Fig.(3),(which(is(corroborated(by(the(rather(long(branch(lengths(associated( with(these(species(in(the(multigene(tree((Fig.(2;(Fig.(S9),(and(appears(to(explain(at(least( partially(explain(the(high(Ka/Ks(ratios(observed(for(these(species((Fig.(S20).(We(also(observe( extremely(high(maximum(pairwise(Ka/Ks(ratios(within(members(of(the(Gonyaulacales(and( Suessiales,(but(in(contrast(these(occur(alongside(relatively(low(pairwise(substitution(rates,( suggesting(that(they(have(resulted(from(a(change(in(plastid(selective(pressure(in(these( lineages((Fig.(3;(Fig.(S21).(Similarly,(We(have(found(that(specific(nodes(on(the(peridinin( Downloaded from plastid(tree(are(marked(by(the(origins(of(large(numbers(of(alternative(initiation(codons((the( common(ancestor(of(Scrippsiella-hangoei-and(Peridinium-aciculiferum;(Fig.(4),(indels((the( common(ancestor(of(Amphidinium-and(Heterocapsa;(Fig.(4),(or(changes(to(conserved(atpA- residues((the(common(ancestor(of(Symbiodinium;(Fig.(4).(It(remains(to(be(determined(why( the(plastids(of(specific(dinoflagellate(lineages(and(not(others(are(unusual,(although(we(note( http://mbe.oxfordjournals.org/ that(many(of(the(most(divergent(species(within(our(dataset(have(symbiotic(life(strategies( (for(example,(Pelagodinium-is(an(endobiont(of(foraminiferans((Siano,(et(al.(2010),(whereas( Brandtodinium(is(a(radiolarian(symbiont((Probert,(et(al.(2014)).(More(taxonomically(detailed( comparisons(of(plastid(evolution(in(endosymbiotic(dinoflagellates(to(their(free[living( relatives(may(provide(insights(into(whether(symbiosis(has(driven(divergent(plastid(evolution( within(the(dinoflagellates.( ( at University of Dundee on November 11, 2016 More(globally,(it(remains(to(be(resolved(why(the(peridinin(dinoflagellates(have(undergone( such(divergent(plastid(evolution(compared(to(other(lineages.(Notably,(we(show(that(residues( with(elevated(Ka/Ks(ratios(in(peridinin(plastids(are(concentrated,(both(in(extant( dinoflagellates(and(in(the(dinoflagellate(common(ancestor,(on(photosystem(I(subunits,(and( stromal(regions(of(plastid(proteins,(which(cannot(be(explained(by(changes(to(codon(usage(or( third(position(substitution(rate((Fig.(5;(Fig.(S24;(Fig.(S25;(Fig.(S26).(While(divergent(evolution( has(previously(been(reported(in(the(photosystem(I(sequences(in(individual(peridinin(plastid( lineages((Bachvaroff,(et(al.(2006;(Shalchian[Tabrizi,(et(al.(2006;(Pochon,(et(al.(2014),(this(is(to( our(knowledge(the(first(evidence(that(this(bias(in(evolutionary(events(is(conserved( throughout(peridinin(plastids,(from(the(origin(of(dinoflagellates(to(extant(species.(( ( Similar,(albeit(less(extreme(divergent(evolution(events(to(those(described(in(this(study(have( been(observed(in(nuclear(genes(encoding(plastid[targeted(proteins((Bachvaroff,(et(al.(2006;( Mungpakdee,(et(al.(2014)(and(non[plastid(proteins((Kim,(et(al.(2011)(in(peridinin( dinoflagellates.(It(will(be(interesting(to(determine(if(the(divergent(evolution(events(observed( in(nucleus[encoded(and(plastid[encoded(genes(are(linked;(for(example(if(the(nucleus[ encoded(components(of(dinoflagellate(photosystem(I,(or(nucleus[encoded(proteins(likely(to( interact(with(the(stromal(faces(of(plastid[encoded(dinoflagellate(proteins,(likewise(possess( specifically(elevated(Ka/Ks(ratios.(Conserved(trends(in(the(evolution(of(nucleus(and(plastid[ encoded(proteins(in(dinoflagellates(might(arise(as(a(result(of(compensatory(evolution(events( to(maintain(plastid(physiology((for(example,(maintaining(plastid(redox(state(for(plastid( physiology(and(gene(regulation((Allen(1993;(Puthiyaveetil,(et(al.(2008),(or(balancing(cyclic( and(linear(electron(flow(to(meet(the(physiological(requirements(of(individual(lineages( (Reynolds,(et(al.(2008),(or(as(a(result(of(divergent(selection(on(plastid(proteins(to( accommodate(some(of(the(more(unusual(nucleus[encoded(proteins(and(structures(present(

! 14! in(peridinin(plastids((for(example,(the(dinoflagellate(pyrenoid((Nassoury,(et(al.(2005;(Siano,( et(al.(2010),(or(the(peridinin(pigment[binding(protein(complexes((Haxo,(et(al.(1976)).( Ultimately,(understanding(the(relationships(between(peridinin(plastid(sequences,(evolution( and(physiology(may(provide(valuable(insights(into(the(biology(of(this(unusual(and( ecologically(important(lineage.( ( Materials*and*Methods* * Assembly*and*annotation*of*previously*known*peridinin*plastid*sequences* * Nucleotide(sequences(corresponding(to(the(twelve(protein[coding(genes((atpA,-atpB,-petB,- petD,-psaA,-psaB,-psbA,-psbB,-psbC,-psbD,-psbE,-psbI)(that(are(found(on(the(three(effectively( complete(peridinin(plastid(genomes((Amphidinium-carterae-CCAP1102/6;(Symbiodinium-sp.,(

Clade(C3,(and(Clade(Mf)((Nisbet,(et(al.(2004;(Barbrook,(et(al.(2014;(Mungpakdee,(et(al.( Downloaded from 2014),(were(assembled(from(peridinin(dinoflagellate(EST(libraries(within(NCBI(and(the( Marine(Microeukaryote(Transcriptome(Sequencing(Project((MMETSP)((Keeling,(et(al.(2014).( Sequences(were(identified(by(tBLASTn(searches,(using(annotated(plastid(protein(sequences( from(peridinin(dinoflagellates(and(their(close(relatives((listed(in(Table(S1).(Where(possible,( the(predicted(translation(products(of(plastid(transcript(sequences(were(used,(instead(of(the( http://mbe.oxfordjournals.org/ translation(products(of(plastid(gDNA(sequences,(due(to(the(presence(of(plastid(transcript( editing(in(some(dinoflagellates((Zauner,(et(al.(2004;(Dorrell(and(Howe(2015).(( ( Sequences(that(matched(the(queries(with(expect(values(of(below(1x10[5(were(selected,(and( were(searched(using(BLASTx(against(the(entire(NCBI(database.((Sequences(that(yielded(a(top( hit(against(another(peridinin(dinoflagellate(sequence(were(retained(for(subsequent(analysis.( Sequences(that(were(excluded(on(the(basis(of(being(of(probable(non[dinoflagellate(origin( at University of Dundee on November 11, 2016 are(listed(in(Table(S1.(In(the(case(of(psbI,(for(which(peridinin(dinoflagellate(sequences(are( known(to(be(highly(divergent((Nisbet,(et(al.(2004),(an(initial(expect(value(threshold(<1.0(was( used(to(identify(possible(dinoflagellate(orthologues,(and(these(orthologues(were(used(in( turn(as(query(sequences(for(a(second(round(of(reciprocal(tBLASTn/BLASTx(searches(against( each(dataset,(to(identify(even(the(most(divergent(sequences(present.(( ( To(investigate(whether(novel(plastid(ORFs,(and(genes(proposed(to(have(been(acquired(by( lateral(transfer(from(Bacteroidetes(are(conserved(across(peridinin(plastids,(similar(BLAST( searches(were(performed(with(translation(products(of(the(four(predicted(ORFs((ORF1,-ORF2,- ORF3,-and(ORF4)(described(in(the(Amphidinium-carterae-plastid(genome((Barbrook,(et(al.( 2001;(Nisbet,(et(al.(2004;(Barbrook,(et(al.(2006),(and(the(proposed(plastid(rpl28-and(rpl23( genes(from(Pyrocystis-lunula-and(ycf24-and(ycf16-sequences(from(Ceratium-horridum- (Moszczynski,(et(al.(2012).(For(these(genes,(sequences(were(retained(if(the(top(reciprocal( BLAST(hit(was(homologous(to(the(genes(in(question,(regardless(of(evolutionary(affinity.(- ( Sequences(that(passed(the(reciprocal(BLASTx(search(were(assembled(into(contigs(using( GeneIOUS(v(4.76((Kearse,(et(al.(2012).(Each(nucleotide(sequence(was(searched(manually(for( possible(5'(spliced(leader(sequences((Zhang,(et(al.(2007),(and(uninterrupted(3’(poly(T)(and( poly(A)(tracts(of(more(than(3(bp(length,(which(might(respectively(correspond(to(transcript( poly(U)(and(poly(A)(tails((Wang(and(Morse(2006).(The(predicted(translation(products(of(each( contig(were(inspected(for(the(presence(of(possible(plastid[targeting(sequences(using(SignalP( version(3.0((Bendtsen,(et(al.(2004)(and(ASAFind((Gruber,(et(al.(2015),(and(for(mitochondria[ targeting(sequences(using(TargetP(version(1.1((Emanuelsson,(et(al.(2007).(Annotated(copies( of(each(novel(peridinin(plastid(transcript((including(information(on(the(presence(of(possible(

! 15! poly(U)(tails,(poly(A)(tails,(spliced[leader(sequences,(and(plastid(–targeting(sequences)(are( included(in(Table(S1.( ( Global*identification*and*annotation*of*peridinin*plastid*transcripts* ( To(determine(whether(further,(previously(undocumented(ORFs(are(conserved(across( peridinin(plastids,(an(independent,(top[down(search(was(performed(for(the(entire( dinoflagellate(sequence(library.(We(focused(on(transcripts(possessing(a(3'(poly(U)(tail,(as(this( feature(is(believed(to(be(uniquely(associated(with(plastid(transcripts(in(dinoflagellates( (Dorrell(and(Howe(2012),(and(polyuridylylated(transcripts(have(previously(been(indicated(to( be(enriched(by(poly(A)(selection,(hence(may(be(present(in(poly(A)[selected(transcriptome( datasets(such(as(MMETSP((Wang(and(Morse(2006;(Keeling,(et(al.(2014).( (

For(this(analysis,(all(sequences(that(terminated(at(either(end(in(a(potential(poly(T)(sequence( Downloaded from (>3bp)(from(all(RNA(libraries(were(extracted,(and(filtered(to(remove(sequences(that(either( contained(a(more(plausible(reverse(complement(of(a(poly(A)(sequence((length(of(poly(A)( sequence(≥(poly(T)(sequence),(or(a(spliced[leader((≥(6(bp(spliced[leader(sequence)(on(the( other(transcript(end.(Parallel(libraries(were(constructed,(containing(all(dinoflagellate( sequences(that(by(the(same(criteria(possessed(either(a(poly(A)(tail,(or(spliced[leader( http://mbe.oxfordjournals.org/ sequences,(and(the(remaining(poly(T)[containing(sequences(were(searched(against(these( libraries(using(tBLASTx.(Poly(T)(sequences(that(were(contiguous(with(other(poly(A)(or( spliced[leader(containing(sequences((as(judged(by(BLAST(best(hit;(expect(value(<(1x10[5,(>( 90%(similarities)(were(likewise(removed(from(the(dataset.( ( Next,(to(identify(sequences(encoding(proteins(of(probable(plastid(function,(the(filtered( poly(T)[containing(sequences(were(oriented(such(that(the(poly(T)(sequence(was(located(on( at University of Dundee on November 11, 2016 the(transcript(3'(end,(and(searched(by(BLASTx(against(a(composite(protein(dataset,( consisting(of(the(complete(protein(sequences(encoded(in(the(nuclear(and(plastid(genomes(of( the(model(diatom(Phaeodactylum-tricornutum((Oudot[Le(Secq,(et(al.(2007;(Bowler,(et(al.( 2008),(and(all(dinoflagellate(plastid(sequences(previously(identified(by(the(BLAST(searches( detailed(above.(Sequences(that(yielded(a(top(hit(in(a(reverse(orientation(reading(frame((i.e.( such(that(the(poly(T)(sequence(could(not(be(located(on(a(transcript(3'(end),(or(were( identified(to(correspond(to(an(internal(region(of(a(transcript((judged(if(the(3'(end(of(the( transcript(alignment(corresponded(to(<90%(the(full(length(of(the(subject(length),(that( yielded(a(top(hit(against(a(known(dinoflagellate(plastid(protein,(or(that(yielded(a(top(hit( against(a(Phaeodactylum-nucleus[encoded(protein((expect(value(<(1x10[5)(were(removed.(( ( The(longest(ORFs(from(sequences(that(were(found(to(possess(plausible(3'(ends,(and(were( homologous(to(plastid[encoded(proteins(in(Phaeodactylum-that(have(not(previously(been( identified(in(peridinin(dinoflagellate(plastids(were(extracted,(and(searched(for(the(presence( of(plastid[(and(mitochondria[targeting(sequences(as(above.(The(sequences(for(which(no( plausible(BLAST(hit(was(found((and(thus(may(contain(novel(plastid(ORFs),(were(searched( against(one(another(using(reciprocal(tBLASTx/tBLASTx(searches.(Sequences(that(were(found( to(match(one(another(with(a(reciprocal(BLAST(hit(expect(value(of(<(1x10[5((and(thus(might( correspond(to(conserved(novel(plastid(proteins)(were(retained,(assembled(into(contigs,(and( aligned(using(GeneIOUS(v(4.76,(as(above.(The(final(annotations(for(each(poly(T)[containing( sequence,(as(well(as(full(nucleotide(and(protein(sequences(for(the(possible(novel(plastid( ORFs(identified,(are(presented(in(Table(S2.( ( Phylogenetic*analysis( (

! 16! To(assess(the(phylogenetic(relationships(between(the(different(dinoflagellates(studied,( conceptual(translations(were(generated(for(each(novel(peridinin(plastid(sequence(identified( from(the(MMETSP(libraries((Keeling,(et(al.(2014),(using(a(standard(translation(table.(This(and( all(subsequent(analyses(conducted(throughout(the(remainder(of(the(study(were(restricted(to( proteins(generated(through(the(conceptual(translations(of(plastid(transcript(sequences,(to( minimise(complications(resulting(from(the(comparison(of((edited)(transcript(sequences(and( (unedited)(gene(sequences,(and(to(reflect(most(accurately(the(probable(eventual(translation( products(of(each(plastid(sequence.(( ( The(conceptual(dinoflagellate(protein(sequences(for(each(gene(were(aligned,(using(twenty( iterations(of(MUSCLE(v(3.8((Edgar(2004),(to(a(reference(set(of(orthologous(sequences(from( fifteen(other(plastid(lineages,(including(a(chromerid((Vitrella-brassicaformis)(that(represents( the(closest(documented(plastid[containing(relative(of(the(peridinin(dinoflagellates(

(Janouskovec,(et(al.(2010).(Orthologues(from(other(alveolate(plastids((those(of( Downloaded from apicomplexans,(and(the(chromerid(Chromera-velia)(were(not(included,(due(to(the(loss(of( photosynthesis(genes((in(the(case(of(apicomplexans),(or(the(exceptionally(divergent(nature( of(the(plastid(genome,(including(changes(in(the(plastid(translation(table((in(the(case(of(C.- velia)((Moore,(et(al.(2008;(Janouskovec,(et(al.(2013).(The(petD-sequence(from(Ostreococcus- tauri-was(excluded(from(this(and(all(subsequent(analyses,(as(it(is(not(present(within(the( http://mbe.oxfordjournals.org/ plastid(genome((Derelle,(et(al.(2006;(Robbens,(et(al.(2007).( ( The(alignments(were(manually(corrected,(trimmed(to(remove(all(positions(at(which(the(most( common(non[ambiguous(identity(was(a(gapped(position,(and(concatenated.(For(single[gene( trees(of(rpl28,-rpl33,-ycf16-and(ycf24(sequences,(similar(alignments(were(generated,(in(this( case(containing(all(of(the(novel(sequences(identified,(the(previously(annotated(Pyrocystis- lunula-and(Ceratium-horridum-sequences((Moszczynski,(et(al.(2012),(and(a(representative( at University of Dundee on November 11, 2016 sample(of(sequences(from(different(plastid(and(bacterial(lineages,(and(trimmed(as(before.( All(of(the(multiple[(and(single[gene(alignments(generated(for(inference(of(phylogenetic(trees( are(provided(in(Table(S3.(( ( Bayesian(inference(of(single(gene(alignments(was(carried(out(using(PhyloBayes(v3.3( (Lartillot,(et(al.(2009),(with(default(settings(and(the(LG(+(!(model.((Two(chains(were(run(in( parallel,(using(the(automatic(stopping(rule(such(that(sampling(was(conducted(every(100( points(until(the(maximum(difference(was(≤(0.1(and(the(effective(size(≥(100.(Bayesian( analyses(of(concatenated(alignments(were(performed(using(MrBayes(3.2.6(hosted(on(the( CIPRES(Science(Gateway(webportal((Ronquist,(et(al.(2012;(Miller,(et(al.(2015).(For(each( dataset,(two(independent(runs(were(performed,(each(comprising(four(chains(for(1,000,000( MCMC(generations,(sampling(every(1,000(points(and(selecting(the(first(quarter(as(burn[in(for( the(consensus.(The(burn[in(was(selected(such(that(the(resulting(standard(deviation(of(split( frequencies(was(<(0.06(for(all(datasets,(and(that(the(log(likelihood(of(the(cold(chain(had( reached(a(stable(plateau.(Analyses(were(run(with(model(mixing(and(!(distributed(rate( categories(with(an(additional(invariant(category.( ( All(Maximum(Likelihood(analyses(were(carried(out(using(RAxML(v8.1.17((Stamatakis(2014),( under(the(LG(+(!(model,(as(hosted(on(CIPRES.(For(analyses(of(both(Bayesian(and(Maximum( Likelihood(inference,(the(–b(option(was(used(to(conduct(500(non[parametric(bootstraps.( These(were(then(subsequently(assembled(into(a(consensus(tree(using(the(consense(program( of(the(PHYLIP(package((v3.695)(on(default(settings.(Bipartitions(present(in(both(trees(were( mapped(onto(the(Bayesian(topology.(Trees(based(solely(on(Maximum(Likelihood(were(run( using(the(–f(a(option,(such(that(bootstraps(were(automatically(mapped(onto(the(Maximum( Likelihood(estimate(of(the(tree(from(the(same(run.(In(all(cases,(gamma(distributed(rates(

! 17! were(modeled(under(a(discrete(model(with(four(rate(categories.(Newick(format(tree(outputs( for(each(alignment,(under(each(condition(tested,(are(provided(in(Table(S4.( ( Alignments(from(which(individual(genes(or(all(gapped(positions(were(removed(were( constructed(using(GeneIOUS.(Ten(further(alignments(were(constructed(from(which(the( species(corresponding(to(the(ten(longest(branches(obtained(in(the(initial(Bayesian(consensus( tree(analysis(were(serially(removed.(Finally,(the(evolutionary(rate(associated(with(each( position(within(the(alignment(was(calculated(using(TIGER,(with(the([b(100(option((Cummins( and(McInerney(2011).(Fifteen(alignments(were(constructed,(from(which(the(twenty[five( fastest(evolving(categories(were(serially(removed.(All(modified(alignments(were(tested(using( RAxML(using(the(same(conditions(as(previously(defined.(Modified(alignments(and(TIGER( output(data(for(fast(site(identification(are(supplied(in(Table(S3.( (

Codon*usage*comparisons* Downloaded from * Changes(in(nucleotide(sequence(composition(across(the(peridinin(dinoflagellates(were( assessed(over(the(six(genes((psaA,-psaB,-psbA,-psbB,-psbC,-and(psbD)(for(which(sequences( had(been(identified(in(each(dinoflagellate(MMETSP(library(studied.(Due(to(the(probable( sequence(contamination(present,(the(Symbiodinium-Clade(A(library(was(excluded(from(this( http://mbe.oxfordjournals.org/ and(all(subsequent(analyses((Fig.(2).(First,(nucleotide(alignments(of(each(gene(were( generated,(and(manually(trimmed(to(remove(insertions(specific(to(dinoflagellate(lineages;( these(alignments(were(concatenated(to(produce(an(alignment(of(all(six(genes((Table(S3).(GC( content(and(codon(usage(frequencies(of(each(sequence(within(the(alignment(were( calculated(using(PAML(v4.8((Yang(2007),(using(a(standard(genetic(code.(Amino(acid( composition(of(each(species(was(quantified(by(summing(the(different(codon(frequencies( obtained.(To(assess(the(relative(codon(usage(amino(acid(composition(biases(associated(with( at University of Dundee on November 11, 2016 each(species,(the(average(frequency(of(each(codon,(and(each(amino(acid,(were(calculated( for(all(species(in(the(alignment.(The(sum(of(squares(of(difference(between(the(frequencies(of( each(codon,(or(amino(acid(for(each(species,(and(the(alignment[wide(mean(values,(were(( calculated,(and(then(ranked(in(ascending(order.(All(statistics(pertaining(to(codon(and(amino( acid(frequency(are(provided(in(Table(S5.( ( To(assess(the(distribution(and(form(of(translationally(invariant(sites(in(dinoflagellate(plastids,( untrimmed(nucleotide(alignments(were(generated(for(each(gene.(dN(and(dS(ratios(were( calculated(for(each(site,(within(each(gene,(for(dinoflagellate(sequences(only,(using(PAML( v4.8(under(a(standard(genetic(code.(All(codon(positions(within(each(alignment(with(an( observed(Ka(of(0((i.e.(no(non[synonymous(substitutions)(within(the(dinoflagellates(were( extracted,(and(are(shown(in(Table(S6.(( ( Finally,(to(assess(which(of(the(trends(identified(might(represent(ancestral(changes(to( dinoflagellate(plastid(translation,(predicted(ancestral(sequences(of(each(plastid(sequence( were(generated(by(regression(using(PAML(as(before((Table(S3).(Two(sequences(were( generated,(one(corresponding(to(the(common(ancestor(of(all(studied(dinoflagellates( included(in(this(study,(and(one(corresponding(to(the(common(ancestor(of(dinoflagellates( and(Vitrella(brassicaformis,(the(closest(sister[group(to(dinoflagellates(within(the(alignment.( Codon(frequencies(associated(with(each(ancestral(sequence(were(calculated(using(PAML( v4.8(as(before,(and(compared(to(one(another.( ( Nucleotide*sequence*substitutions* ( Pairwise(Ka/Ks(ratios(and(total(number(of(pairwise(substitutions(were(calculated(using(the(

! 18! gap[free(concatenated(nucleotide(alignment,(for(each(possible(combination(of(species,(with( KaKs(Calculator(version(2.0,(using(the(Model(Averaging(method((which(performs(weighted( calculations(using(eight(different(substitution(matrices,(based(on(the(relative(likelihood(of( each(matrix(to(explain(the(observed(sequences)(and(a(standard(genetic(code((Wang,(et(al.( 2010).(Pairwise(Ka/Ks(calculations(and(the(total(number(of(substitutions(for(each(species( pair(are(provided(in(Table(S7.( ( To(investigate(the(different(factors(underpinning(substitution(rates(within(the(alignment,( total(numbers(of(pairwise(substitutions(and(Ka/Ks(ratios(were(correlated(against((1)(the( third[position(GC(content,((2)(the(codon(usage(bias((defined(as(above)(and((3)(the(amino( acid(composition(bias(for(each(species(pair.(Correlation(calculations(were(performed(for( both(the(average(and(modular(differences(in(each(variable(for(each(species(pair,(and(were( performed(for(both(the(absolute(substitution(rates(and(Ka/Ks(ratios(observed,(and(the(rank( of(each(value(within(the(entire(dataset.(Third[position(GC,(amino(acid(composition(bias(and( Downloaded from codon(usage(bias(values(for(each(species(are(provided(in(Table(S5,(and(values(for(each( species(pair(are(provided(in(Table(S7.( ( Mutation(rate(saturation(was(assessed(using(the(DAMBE(software(package((Xia,(et(al.(2003).( The(concatenated(gap[free(alignment(was(separated(into(first[,(second[,(and(third[codon( http://mbe.oxfordjournals.org/ positions(only,(and(separate(calculations(were(performed(for(each(codon(position.(A( separate(series(of(calculations(were(performed(for(each(codon(position(using(alignments( consisting(only(of(the(dinoflagellate(sequences(from(the(first[,(second[(or(third[position( alignments.(For(each(alignment(and(codon(position,(the(proportion(of(invariant(sites(was( first(calculated,(using(the(tree(topology(obtained(using(the(multigene(phylogeny((Table(S4),( under(otherwise(default(conditions.(Substitution(rates(were(then(assessed(at(each(codon( position(through(each(alignment(using(Xia's(test,(with(the(empirically(verified(invariant(site( at University of Dundee on November 11, 2016 frequencies((Xia,(et(al.(2003;(Xia(2013),(and(otherwise(under(the(default(conditions.(( (( To(determine(whether(any(of(the(above(factors(have(had(significant(effects(on(the(total( numbers(of(pairwise(substitutions(observed,(the(total(number(of(pairwise(substitutions(and( Ka/Ks(ratios(were(calculated(for(alignments(that(were(manually(recoded(in(several(ways((all( modified(alignments(provided(in(Table(S3).(To(investigate(the(affect(of(GC(content(shifting( on(substitution(rates,(total(number(of(pairwise(substitutions(were(calculated(for(alignments( that(had(been(RY[recoded((Ishikawa,(et(al.(2012).(Two(RY[recoded(alignments(were( produced:(one(alignment(in(which(all(adenosines(were(replaced(by(guanosines(and(all( thymidines(were(replaced(by(cytosines,(and(one(for(which(the(converse(substitutions(were( performed.(To(investigate(the(different(roles(of(each(codon(positions(on(substitution(rates,( total(number(of(pairwise(substitutions(were(calculated(for(the(separate(first,(second(and( third(position(alignments(previously(generated(for(inspection(with(DAMBE,(and(two(further( alignments(consisting(of(third(codon(positions(only(that(had(been(manually(RY[recoded(as( above((Table(S3).(To(investigate(the(significance(of(third(position(mutations(on(Ka/Ks,(ratios( were(calculated(for(two(alignments(that(had(been(manually(recoded(so(that(all(third(codon( positions(were(replaced(either(by(adenosines,(or(by(guanosines((Table(S3).(These(two( substitutions(were(performed(as(they(preserve(the(possibility(for(synonymous(first[position( substitutions(associated(with(leucine((YTR)(and(arginine(codons((RGR),(whereas(manual( recoding(of(the(third(position(to(pyrimidine(nucleotides(would(eliminate(all(synonymous( single(substitutions(associated(with(six[fold(degenerate(codons(under(a(standard(genetic( code((Crick(1968;(Inagaki,(et(al.(2004).(Finally,(to(investigate(the(effects(of(changes(in( dinoflagellate(plastid(codon(preference(on(Ka/Ks(and(substitution(rates,(calculations(were( performed(for(an(alignment(which(was(globally(recoded(so(that(19(codons,(which(occur(at( significantly(lower(frequencies(in(dinoflagellates(than(in(non[dinoflagellates((one[way(

! 19! ANOVA,(P<0.05),(were(universally(replaced(with(19(synonymous(codons(that(occur(at( elevated(frequencies(in(dinoflagellates((Table(S5).(Pairwise(substitution(value(calculations( for(all(manually(recoded(alignments(are(provided(in(Table(S7.( ( Translation*initiation*site*identification* * Possible(alternative(translation(initiation(codons(in(peridinin(plastids(were(identified(from( nucleotide(sequence(alignments(of(each(sequence(that(contained(all(of(the(dinoflagellate( sequences(studied,(and(orthologues(from(each(of(the(non[dinoflagellate(reference( sequences(used(for(construction(of(the(multigene(phylogeny.(The(5’(end(of(each(nucleotide( sequence(was(trimmed(so(that(it(started(from(the(first(in[frame(termination(codon(within( the(5'(UTR,(or((if(no(such(codon(existed)(so(that(the(coding(sequence(was(located(within( reading(frame(position(+1.((

( Downloaded from To(determine(which(alternative(translation(initiation(codons(are(utilised(in(peridinin(plastids,( the(most(probable(initiation(codon(was(identified(in(the(N[terminal(coding(region(of(each( dinoflagellate(sequence(using(a(custom[built(automated(pipeline((Text(S1).(As(the(presence( of(Shine[Dalgarno(sequences(on(peridinin(plastid(genes(remains(unclear((Zhang,(et(al.(1999;( Dang(and(Green(2009),(initiation(codons(were(searched(for(in(the(entire(possible(5’(coding( http://mbe.oxfordjournals.org/ region(of(each(sequence.(This(was(defined(as(the(region(extending(upstream(from(the(first( residue(that(was(identical(in(75%(of(the(non[dinoflagellate(reference(sequences.(The(most( probable(initiation(codon(was(taken(to(be(ATG,(if(such(a(codon(were(present.(If(not,(the( sequence(was(screened(for(codons(that(shared(two(bases(in(common(with(ATG,(which(has( been(shown(experimentally(in(other(plastid(lineages(to(be(sufficient(to(permit(translation( initiation((Chen,(et(al.(1995),(alongside(GTA,(which(has(been(documented(to(function(as(an( alternative(initiation(codon(in(Proteobacteria((Kim,(et(al.(2008),(and(has(previously(been( at University of Dundee on November 11, 2016 proposed(to(be(used(as(an(alternative(translation(initiation(codon(in(Amphidinium-(Barbrook( and(Howe(2000;(Barbrook,(et(al.(2001).(The(nearest(codon(to(the(consensus(initiation(site(in( the(non[dinoflagellate(reference(species((hence(would(produce(the(ORF(with(the(greatest( homology(to(the(expected(protein(sequence)(was(then(taken(as(the(most(probable( translation(initiation(site.(These(data,(including(full(lists(of(all(codons(deemed(to(be(possible( initiation(codons(for(each(gene(and(in(each(species,(as(well(as(the(most(probable(initiation( codons,(are(provided(in(Table(S8.( ( Indel*analysis* ( Indels(within(peridinin(plastid(sequences(were(identified(using(untrimmed(protein(sequence( alignments(generated(for(each(gene,(as(above.(Pairwise(comparisons(were(performed( between(each(dinoflagellate(sequence(and(non[dinoflagellate(sequence(previously(used(for( the(construction(of(the(multigene(alignment,(using(a(custom(built(Python(script(that( automatically(detects(and(reports(both(insertions(and(deletions((Text(S2).(Sequences(were( counted(as(insertions(only(if(they(occurred(in(at(least(one(dinoflagellate(but(were(absent( from(all(of(the(non[dinoflagellate(reference(sequences,(or(as(deletions(if(they(were(absent( from(at(least(one(dinoflagellate,(but(present(in(every(reference(sequence(examined.( ( Each(predicted(indel(was(confirmed(by(visual(inspection(of(the(alignment.(Positions(that( were(within(5(amino(acids(of(sequence(N[(or(C[termini,(and(patterns(of(alternating( insertions(and(deletions(that(indicated(poor(alignment(of(the(sequence(were(rejected.( Tabulated(indels(identified(for(each(species,(and(the(inferred(phylogenetic(origin(point((as( defined(using(the(multigene(tree(topology)(of(each(indel(is(shown(in(Table(S9.( (

! 20! Changes*to*conserved*atpA*residues* * Changes(to(residues(that(were(conserved(in(all(other(plastid(lineages(were(identified(by( visual(inspection(of(the(untrimmed(atpA(protein(sequence(alignment(containing(all(of(the( dinoflagellate(sequences(studied,(and(orthologues(from(each(of(the(non[dinoflagellate( reference(sequences(used(for(generation(of(the(multigene(phylogeny.(Residues(were( deemed(to(be(conserved(in(non[dinoflagellate(lineages(only(if(they(were(found(in(all,(or(all( but(one,(of(the(species(studied,(including(the(closest(relative(to(the(dinoflagellates(within( the(alignment((Vitrella-brassicaformis).-The(full(list(of(substitutions(to(conserved(AtpA( residues(for(each(species,(alongside(the(inferred(origin(points(for(each(substitution,(is( provided(in(Table(S10.(( ( Site?specific*substitution*rates*

* Downloaded from Substitution(rates(for(each(residue(within(each(sequence(were(calculated(using(the( previously(constructed(single[gene(nucleotide(alignments.(Each(sequence(was(trimmed(to( remove(indels(that(were(specific(to(dinoflagellates,(and(Ka/Ks(ratios(were(calculated(for(each( site(using(PAML(v(4.8,(site(model(constraint(2,(and(a(standard(genetic(code((Yang(2007).(The( consensus(multigene(tree(generated(above(was(used(as(the(reference(tree(topology.( http://mbe.oxfordjournals.org/ Separate(alignments(were(constructed(for(each(sequence(in(which(the(codon(third(positions( were(either(manually(recoded(to(adenosines(or(to(guanosines,(or(in(which(all(codon( positions(were(manually(altered(to(reflect(the(predominant(codon(usage(patterns(observed( in(dinoflagellates,(as(detailed(above.(Raw(and(modified(alignments(for(each(sequence(are( provided(in(Table(S3,(and(the(Newick(format(trees(used(for(each(analysis(in(Table(S4.( ( To(allow(comparisons(between(the(evolutionary(rates(observed(for(dinoflagellate(and(non[ at University of Dundee on November 11, 2016 dinoflagellate(species,(separate(values(were(calculated(for(each(site,(using(only( dinoflagellates,(and(using(only(non[dinoflagellate(sequences.(To(identify(substitution(events( that(occurred(at(the(divergence(of(peridinin(dinoflagellates(from(other(plastids,(values(were( also(calculated(for(each(gene(for(the(previously(inferred(sequence(for(the(dinoflagellate( common(ancestor,(when(compared(directly(to(the(sequence(for(the(common(ancestor(of( dinoflagellates(and(Vitrella((Table(S3).(Individual(rate(calculations(for(each(gene,(including( gene(length,(are(provided(in(Table(S11.( ( Averaged(Ka/Ks(ratios(were(then(calculated(over(a(sliding(window(of(eleven(residues(in(each( sequence.(For(regions(of(sequence(which(through(this(approach(contained(no(synonymous( substitutions,(the(sliding(window(was(expanded(to(include(the(nearest(codon(upstream,(and( the(nearest(codon(downstream,(to(have(undergone(synonymous(substitutions.(The(residues( with(Ka/Ks(>1(in(the(dinoflagellate(common(ancestor,(or(Ka/Ks(>(0.5(within(dinoflagellates( were(inspected(for(localisation((transmembrane,(stromal(or(luminal[facing),(and(function( (interaction(with(other(plastid[encoded(subunits,(or(with(nucleus[encoded(subunits(and( cofactors),(as(inferred(by(alignment(to(annotated(PDB(sequences(from(the(plastid(of( Arabidopsis-thaliana((Sato,(et(al.(1999),(and(the(model(cyanobacterium( Thermosynechococcus-elongatus((Kamiya(and(Shen(2003).(Separate(Ka/Ks(calculations(were( also(performed(and(annotated(for(the(previously(generated(codon(re[optimised,(third[ position(adenosine[(and(third[position(guanosine(recoded(alignments((Table(S3),(with( substitution(values(for(each(alignment(provided(in(Tables(S12[S15.( ( Data*Deposition* *

! 21! All(sequences(and(supplementary(tables(referenced(in(this(study(are(publically(accessible( from(the(University(of(Cambridge(dserve(server( (https://www.repository.cam.ac.uk/handle/1810/252774).* ( Acknowledgments* * This(work(was(supported(by(a("Research(in(Paris"(post[doctoral(fellowship([to(RGD](from(the( Mairie(de(Paris((grant(number(2014/89).(Work(in(the(Bowler(lab(is(supported(by(the(ERC( Advanced(Award(Diatomite.( * References* * Allen(JF.(1993.(Control(of(gene(expression(by(redox(potential(and(the(requirement(for( chloroplast(and(mitochondrial(genomes.(J(Theor(Biol(165:609[631.( Downloaded from Bachvaroff(TR,(Concepcion(GT,(Rogers(CR,(Herman(EM,(Delwiche(CF.(2004.(Dinoflagellate( expressed(sequence(tag(data(indicate(massive(transfer(to(the(nuclear(genome(sequence.( Protist(155:65[78.( Bachvaroff(TR,(Gornik(SG,(Concepcion(GT,(Waller(RF,(Mendez(GS,(Lippmeier(JC,(Delwiche(CF.( 2014.(Dinoflagellate(phylogeny(revisited:(Using(ribosomal(proteins(to(resolve(deep( http://mbe.oxfordjournals.org/ branching(dinoflagellate(clades.(Mol(Phyl(Evol(70:314[322.( Bachvaroff(TR,(Sanchez[Puerta(MV,(Delwiche(CF.(2006.(Rate(variation(as(a(function(of(gene( origin(in(plastid[derived(genes(of(peridinin[containing(dinoflagellates.(J(Mol(Evol(62:42[52.( Barbrook(AC,(Dorrell(RG,(Burrows(J,(Plenderleith(LJ,(Nisbet(RER,(Howe(CJ.(2012.( Polyuridylylation(and(processing(of(transcripts(from(multiple(gene(minicircles(in(chloroplasts( of(the(dinoflagellate(Amphidinium-carterae.(Plant(Mol(Biol(79:347[357.( Barbrook(AC,(Howe(CJ.(2000.(Minicircular(plastid(DNA(in(the(dinoflagellate(Amphidinium( at University of Dundee on November 11, 2016 operculatum.(Molecular(and(General(Genetics(263:152[158.( Barbrook(AC,(Santucci(N,(Plenderleith(LJ,(Hiller(RG,(Howe(CJ.(2006.(Comparative(analysis(of( dinoflagellate(chloroplast(genomes(reveals(rRNA(and(tRNA(genes.(BMC(Genom(7:(297.( Barbrook(AC,(Symington(H,(Nisbet(RER,(Larkum(A,(Howe(CJ.(2001.(Organisation(and( expression(of(the(plastid(genome(of(the(dinoflagellate(Amphidinium-operculatum.(Mol( Genet(Genom(266:632[638.( Barbrook(AC,(Voolstra(CR,(Howe(CJ.(2014.(The(chloroplast(genome(of(a(Symbiodinium(sp( clade(C3(isolate.(Protist(165:1[13.( Bendtsen(JD,(Nielsen(H,(von(Heijne(G,(Brunak(S.(2004.(Improved(prediction(of(signal( peptides:(SignalP(3.0.(J(Mol(Biol(340:783[795.( Bowler(C,(Allen(AE,(Badger(JH,(Grimwood(J,(Jabbari(K,(Kuo(A,(Maheswari(U,(Martens(C,( Maumus(F,(Otillar(RP,(et(al.(2008.(The(Phaeodactylum(genome(reveals(the(evolutionary( history(of(diatom(genomes.(Nature(456:239[244.( Butterfield(ER,(Howe(CJ,(Nisbet(RER.(2013.(An(analysis(of(dinoflagellate(metabolism(using( EST(data.(Protist(164:218[236.( Chen(X,(Kindle(KL,(Stern(DB.(1995.(The(initiation(codon(determines(the(efficiency(but(not(the( site(of(translation(initiation(in(Chlamydomonas(chloroplasts.(Plant(Cell(7:1295[1305.( Crick(FH.(1968.(The(origin(of(the(genetic(code.(J(Mol(Biol(38:367[379.( Cummins(CA,(McInerney(JO.(2011.(A(method(for(inferring(the(rate(of(evolution(of( homologous(characters(that(can(potentially(improve(phylogenetic(inference,(resolve(deep( divergence(and(correct(systematic(biases.(Syst(Biol(60:833[844.( Dang(Y,(Green(BR.(2009.(Substitutional(editing(of(Heterocapsa-triquetra-chloroplast( transcripts(and(a(folding(model(for(its(divergent(chloroplast(16S(rRNA.(Gene(442:73[80.(

! 22! de(Vargas(C,(Audic(S,(Henry(N,(Decelle(J,(Mahé(F,(Logares(R,(Lara(E,(Berney(C,(Le(Bescot(N,( Probert(I,(et(al.(2015.(Ocean(plankton.(Eukaryotic(plankton(diversity(in(the(sunlit(ocean.( Science(348:1261605.( Decelle(J,(Siano(R,(Probert(I,(Poirier(C,(Not(F.(2014.(Multiple(microalgal(partners(in(symbiosis( with(the(acantharian(Acanthochiasma(sp.((Radiolaria).(Symbiosis(58:233[244.( Derelle(E,(Ferraz(C,(Rombauts(S,(Rouzé(P,(Worden(AZ,(Robbens(S,(Partensky(F,(Degroeve(S,( Echeynié(S,(Cooke(R,(et(al.(2006.(Genome(analysis(of(the(smallest(free[living(( Ostreococcus-tauri(unveils(many(unique(features.(Proc(Natl(Acad(Sci(USA(103:11647[11652.( Dorrell(RG,(Drew(J,(Nisbet(RE,(Howe(CJ.(2014.(Evolution(of(chloroplast(transcript(processing( in(Plasmodium(and(its(chromerid(algal(relatives.(PLoS(Genet(10:1004008.( Dorrell(RG,(Hinksman(GA,(Howe(CJ.(2016.(Diversity(of(transcripts(and(transcript(processing( forms(in(plastids(of(the(dinoflagellate(alga(-mikimotoi.(Plant(Mol(Biol(90:233[247.( Dorrell(RG,(Howe(CJ.(2012.(Functional(remodeling(of(RNA(processing(in(replacement( chloroplasts(by(pathways(retained(from(their(predecessors.(Proc(Natl(Acad(Sci(USA(109:( Downloaded from 18879[18884.( Dorrell(RG,(Howe(CJ.(2015.(Integration(of(plastids(with(their(hosts:(lessons(learnt(from( dinoflagellates(Proc(Natl(Acad(Sci(USA(112:(10247–10254.( Edgar(RC.(2004.(MUSCLE:(multiple(sequence(alignment(with(high(accuracy(and(high( throughput.(Nucl(Acids(Res(32:1792[1797.( http://mbe.oxfordjournals.org/ Emanuelsson(O,(Brunak(S,(von(Heijne(G,(Nielsen(H.(2007.(Locating(proteins(in(the(cell(using( TargetP,(SignalP(and(related(tools.(Nat(Protocol(2:953[971.( Gavelis(GS,(White(RA,(Suttle(CA,(Keeling(PJ,(Leander(BS.(2015.(Single[cell(transcriptomics( using(spliced(leader(PCR:(evidence(for(multiple(losses(of(photosynthesis(in(polykrikoid( dinoflagellates.(BMC(Genom(16:528.( Gornik(SG,(Ford(KL,(Mulhern(TD,(Bacic(A,(McFadden(GI,(Waller(RF.(2012.(Loss(of(nucleosomal( DNA(condensation(coincides(with(appearance(of(a(novel(nuclear(protein(in(dinoflagellates.( at University of Dundee on November 11, 2016 Curr(Biol(22:2303[2312.( Gruber(A,(Rocap(G,(Kroth(PG,(Armbrust(EV,(Mock(T.(2015.(Plastid(proteome(prediction(for( diatoms(and(other(algae(with(secondary(plastids(of(the(red(lineage.(Plant(J(81:519[528.( Hallegraeff(GM.(2010.(Ocean(climate(change,(phytoplankton(community(responses,(and( harmful(algal(blooms:(a(formidable(predictive(challenge.(J(Phycol(46:220[235.( Haxo(FT,(Kycia(JH,(Somers(GF,(Bennett(A,(Siegelman(HW.(1976.(Peridinin(chlorophyll(a( proteins(of(dinoflagellate(Amphidinium-carterae-(Plymouth(450).(Plant(Physiol(57:297[303.( Hiller(RG.(2001.('Empty'(minicircles(and(petB/atpA(and(psbD/psbE((cytb(559)(alpha)(genes(in( tandem(in(Amphidinium-carterae(plastid(DNA.(FEBS(Lett(505:449[452.( Hinder(SL,(Hays(GC,(Edwards(M,(Roberts(EC,(Waine(AW,(Gravenor(MB.(2012.(Changes(in( marine(dinoflagellate(and(diatom(abundance(under(climate(change.(Nat(Climate(Change.(2:( 271[275.( Hoppenrath(M,(Leander(BS.(2010.(Dinoflagellate(phylogeny(as(inferred(from(heat(shock( protein(90(and(ribosomal(gene(sequences.(PLoS(One(5:(13220.( Howe(CJ,(Nisbet(RER,(Barbrook(AC.(2008.(The(remarkable(chloroplast(genome(of( dinoflagellates.(J(Exp(Bot(59:1035[1045.( Huesgen(PF,(Alami(M,(Lange(PF,(Foster(LJ,(Schröder(WP,(Overall(CM,(Green(BR.(2013.( Proteomic(amino[termini(profiling(reveals(targeting(information(for(protein(import(into( complex(plastids.(PLoS(One(8:74483.( Hurst(LD.(2002.(The(Ka/Ks(ratio:(diagnosing(the(form(of(sequence(evolution.(Trends(Genet( 18:486.( Inagaki(Y,(Simpson(AGB,(Dacks(JB,(Roger(AJ.(2004.(Phylogenetic(artifacts(can(be(caused(by( leucine,(serine,(and(arginine(codon(usage(heterogeneity:(dinoflagellate(plastid(origins(as(a( case(study.(Systemat(Biology(53:582[593.(

! 23! Ishikawa(SA,(Inagaki(Y,(Hashimoto(T.(2012.(RY[Coding(and(non[homogeneous(models(can( ameliorate(the(maximum[likelihood(inferences(from(nucleotide(sequence(data(with(parallel( compositional(heterogeneity.(Evol(Bioinf(8:357[371.( Janouskovec(J,(Horák(A,(Oborník(M,(Lukes(J,(Keeling(PJ.(2010.(A(common(red(algal(origin(of( the(apicomplexan,(dinoflagellate,(and(heterokont(plastids.(Proc(Natl(Acad(Sci(USA( 107:10949[10954.( Janouskovec(J,(Sobotka(R,(Lai(DH,(Flegontov(P,(Koník(P,(Komenda(J,(Ali(S,(Prásil(O,(Pain(A,( Oborník(M,(et(al.(2013.(Split(photosystem(protein,(linear[mapping(topology,(and(growth(of( structural(complexity(in(the(plastid(genome(of(Chromera-velia.(Mol(Biol(Evol(30:2447[2462.( Janouškovec(J,(Tikhonenkov(D,(Mikhailov(K,(Simdyanov(T,(Aleoshin(V,(Mylnikov(A,(Keeling(P.( 2013.(Colponemids(represent(multiple(ancient(alveolate(lineages.(Curr(Biol,(23:(2546[2552.( Kamiya(N,(Shen(JR.(2003.(Crystal(structure(of(oxygen[evolving(photosystem(II(from( Thermosynechococcus-vulcanus(at(3.7[A(resolution.(Proc(Natl(Acad(Sci(USA(100:98[103.(

Kearse(M,(Moir(R,(Wilson(A,(Stones[Havas(S,(Cheung(M,(Sturrock(S,(Buxton(S,(Cooper(A,( Downloaded from Markowitz(S,(Duran(C,(et(al.(2012.(Geneious(Basic:(An(integrated(and(extendable(desktop( software(platform(for(the(organization(and(analysis(of(sequence(data.(Bioinf(28:1647[1649.( Keeling(PJ.(2010.(The(endosymbiotic(origin,(diversification(and(fate(of(plastids.(Phil(Trans(R( Soc(Biol(Sci(365:729[748.( Keeling(PJ,(Burki(F,(Wilcox(HM,(Allam(B,(Allen(EE,(Amaral[Zettler(LA,(Armbrust(EV,(Archibald( http://mbe.oxfordjournals.org/ JM,(Bharti(AK,(Bell(CJ,(et(al.(2014.(The(Marine(Microbial(Eukaryote(Transcriptome( Sequencing(Project((MMETSP):(illuminating(the(functional(diversity(of(eukaryotic(life(in(the( oceans(through(transcriptome(sequencing.(PLoS(Biol(12:1001889.( Kim(S,(Bachvaroff(TR,(Handy(SM,(Delwiche(CF.(2011.(Dynamics(of(actin(evolution(in( dinoflagellates.(Mol(Biol(Evol(28:1469[1480.( Kim(SW,(Jung(WH,(Ryu(JM,(Kim(JB,(Jang(HW,(Jo(YB,(Jung(JK,(Kim(JH.(2008.(Identification(of(an( alternative(translation(initiation(site(for(the(Pantoea-ananatis(lycopene(cyclase((crtY)(gene(in( at University of Dundee on November 11, 2016 E.-coli(and(its(evolutionary(conservation.(Protein(Expr(Purif(58:23[31.( Lartillot(N,(Lepage(T,(Blanquart(S.(2009.(PhyloBayes(3:(a(Bayesian(software(package(for( phylogenetic(reconstruction(and(molecular(dating.(Bioinf(25:2286[2288.( Leterme(SC,(Seuront(L,(Edwards(M.(Differential(contribution(of(diatoms(and(dinoflagellates( to(phytoplankton(biomass(in(the(NE(Atlantic(Ocean(and(the(North(Sea.(Mar(Ecol(Prog(Ser( 312:9.( Lin(S.(2011.(Genomic(understanding(of(dinoflagellates.(Res(Microbiol(162:551[569.( Miller(MA,(Schwartz(T,(Pickett(BE,(He(S,(Klem(EB,(Scheuermann(RH,(Passarotti(M,(Kaufman(S,( O'Leary(MA.(2015.(A(RESTful(API(for(access(to(phylogenetic(tools(via(the(CIPRES(science( gateway.(Evol(Bioinform(Online(11:43[48.( Moore(RB,(Obornik(M,(Janouskovec(J,(Chrudimsky(T,(Vancova(M,(Green(DH,(Wright(SW,( Davies(NW,(Bolch(CJS,(Heimann(K,(et(al.(2008.(A(photosynthetic(alveolate(closely(related(to( apicomplexan(parasites.(Nature(451:959[963.( Morse(D,(Salois(P,(Markovic(P,(Hastings(JW.(1995.(A(nuclear[encoded(form(II(RuBisCO(in( dinoflagellates.(Science(268:1622[1624.( Moszczynski(K,(Mackiewicz(P,(Bodyl(A.(2012.(Evidence(for(horizontal(gene(transfer(from( Bacteroidetes(bacteria(to(dinoflagellate(minicircles.(Mol(Biol(Evol(29:887[892.( Mungpakdee(S,(Shinzato(C,(Takeuchi(T,(Kawashima(T,(Koyanagi(R,(Hisata(K,(Tanaka(M,(Goto( H,(Fujie(M,(Lin(S,(et(al.(2014.(Massive(gene(transfer(and(extensive(RNA(editing(of(a(symbiotic( dinoflagellate(plastid(genome.(Genome(Biol(Evol(6:1408[1422.( Nakamura(Y.(1998.(Growth(and(grazing(of(a(large(heterotrophic(dinoflagellate,(Noctiluca( scintillans,(in(laboratory(cultures.(J(Plankton(Res(20:1711[1720.( Nassoury(N,(Morse(D.(2005.(Protein(targeting(to(the(chloroplasts(of(photosynthetic( eukaryotes:(getting(there(is(half(the(fun.(Biochimic(Biophys(Acta(1743:5[19.(

! 24! Nassoury(N,(Wang(Y,(Morse(D.(2005.(Brefeldin(a(inhibits(circadian(remodeling(of(chloroplast( structure(in(the(dinoflagellate(gonyaulax.(Traffic(6:548[561.( Nelson(MJ,(Dang(YK,(Filek(E,(Zhang(ZD,(Yu(VWC,(Ishida(K,(Green(BR.(2007.(Identification(and( transcription(of(transfer(RNA(genes(in(dinoflagellate(plastid(minicircles.(Gene(392:291[298.( Nelson(MJ,(Green(BR.(2005.(Double(hairpin(elements(and(tandem(repeats(in(the(non[coding( region(of(Adenoides(eludens(chloroplast(gene(minicircles.(Gene(358:102[110.( Nisbet(RER,(Koumandou(VL,(Barbrook(AC,(Howe(CJ.(2004.(Novel(plastid(gene(minicircles(in( the(dinoflagellate(Amphidinium-operculatum.(Gene(331:141[147.( Oudot[Le(Secq(MP,(Grimwood(J,(Shapiro(H,(Armbrust(EV,(Bowler(C,(Green(BR.(2007.( Chloroplast(genomes(of(the(diatoms(Phaeodactylum-tricornutum(and(Thalassiosira- pseudonana:(comparison(with(other(plastid(genomes(of(the(red(lineage.(Molecular(Genet( Genom(277:427[439.( Pochon(X,(Putnam(HM,(Gates(RD.(2014.(Multi[gene(analysis(of(Symbiodinium(dinoflagellates:( a(perspective(on(rarity,(symbiosis,(and(evolution.(PeerJ(2:394.( Downloaded from Probert(I,(Siano(R,(Poirier(C,(Decelle(J,(Biard(T,(Tuji(A,(Suzuki(N,(Not(F.(2014.( Brandtodinium(gen.(nov.(and(B.-nutricula(comb.(nov.((),(a(dinoflagellate( commonly(found(in(symbiosis(with(polycystine(radiolarians.(J(Phycol(50:388[399.( Puthiyaveetil(S,(Kavanagh(TA,(Cain(P,(Sullivan(JA,(Newell(CA,(Gray(JC,(Robinson(C,(van(der( Giezen(M,(Rogers(MB,(Allen(JF.(2008.(The(ancestral(symbiont(sensor(kinase(CSK(links( http://mbe.oxfordjournals.org/ photosynthesis(with(gene(expression(in(chloroplasts.(Proc(Natl(Acad(Sci(USA(105:10061[ 10066.( Reynolds(JM,(Bruns(BU,(Fitt(WK,(Schmidt(GW.(2008.(Enhanced(photoprotection(pathways(in( symbiotic(dinoflagellates(of(shallow[water(corals(and(other(cnidarians.(Proc(Natl(Acad(Sci( USA(105:13674[13678.( Robbens(S,(Derelle(E,(Ferraz(C,(Wuyts(J,(Moreau(H,(Van(de(Peer(Y.(2007.(The(complete( chloroplast(and(mitochondrial(DNA(sequence(of(Ostreococcus-tauri:(organelle(genomes(of( at University of Dundee on November 11, 2016 the(smallest(eukaryote(are(examples(of(compaction.(Mol(Biol(Evol(24:956[968.( Ronquist(F,(Teslenko(M,(van(der(Mark(P,(Ayres(DL,(Darling(A,(Höhna(S,(Larget(B,(Liu(L,( Suchard(MA,(Huelsenbeck(JP.(2012.(MrBayes(3.2:(efficient(Bayesian(phylogenetic(inference( and(model(choice(across(a(large(model(space.(Syst(Biol(61:539[542.( Sato(S,(Nakamura(Y,(Kaneko(T,(Asamizu(E,(Tabata(S.(1999.(Complete(structure(of(the( chloroplast(genome(of(Arabidopsis-thaliana.(DNA(Res(6:283[290.( Shalchian[Tabrizi(K,(Skanseng(M,(Ronquist(F,(Klaveness(D,(Bachvaroff(TR,(Delwiche(CF,( Botnen(A,(Tengs(T,(Jakobsen(KS.(2006.(Heterotachy(processes(in(rhodophyte[derived( secondhand(plastid(genes:(implications(for(addressing(the(origin(and(evolution(of( dinoflagellate(plastids.(Mol(Biol(Evol(23:1504[1515.( Siano(R,(Montresor(M,(Probert(I,(Not(F,(de(Vargas(C.(2010.(Pelagodinium(gen.(nov.(and(P.( béii(comb.(nov.,(a(dinoflagellate(symbiont(of(planktonic(foraminifera.(Protist(161:385[399.( Stamatakis(A.(2014.(RAxML(version(8:(a(tool(for(phylogenetic(analysis(and(post[analysis(of( large(phylogenies.(Bioinf(30:1312[1313.( Walker(JE.(2013.(The(ATP(synthase:(the(understood,(the(uncertain(and(the(unknown.( Biochemical(Society(Transactions(41:1[16.( Wang(D,(Zhang(Y,(Zhang(Z,(Zhu(J,(Yu(J.(2010.(KaKs_Calculator(2.0:(a(toolkit(incorporating( gamma[series(methods(and(sliding(window(strategies.(Genom(Proteom(Bioinf(8:77[80.( Wang(YL,(Morse(D.(2006.(Rampant(polyuridylylation(of(plastid(gene(transcripts(in(the( dinoflagellate(Lingulodinium.(Nucl(Acids(Rese(34:613[619.( Wisecaver(JH,(Hackett(JD.(2011.(Dinoflagellate(genome(evolution.(Ann(Rev(Microbiol(65:369[ 387.( Xia(X.(2013.(DAMBE5:(a(comprehensive(software(package(for(data(analysis(in(molecular( biology(and(evolution.(Mol(Biol(Evol(30:1720[1728.(

! 25! Xia(X,(Xie(Z,(Salemi(M,(Chen(L,(Wang(Y.(2003.(An(index(of(substitution(saturation(and(its( application.(Mol(Phylogenet(Evol(26:1[7.( Yang(Z.(2007.(PAML(4:(phylogenetic(analysis(by(maximum(likelihood.(Mol(Biol(Evol(24:1586[ 1591.( Yang(Z,(Bielawski(JP.(2000.(Statistical(methods(for(detecting(molecular(adaptation.(Trends( Ecol(Evol(15:496[503.( Zauner(S,(Greilinger(D,(Laatsch(T,(Kowallik(KV,(Maier(UG.(2004.(Substitutional(editing(of( transcripts(from(genes(of(cyanobacterial(origin(in(the(dinoflagellate(Ceratium(horridum.( FEBS(Lett(577:535[538.( Zhang(H,(Hou(Y,(Miranda(L,(Campbell(DA,(Sturm(NR,(Gaasterland(T,(Lin(S.(2007.(Spliced( leader(RNA(trans[splicing(in(dinoflagellates.(Proc(Natl(Acad(Sci(USA(104:4618[4623.( Zhang(Z,(Green(BR,(Cavalier[Smith(T.(1999.(Single(gene(circles(in(dinoflagellate(chloroplast( genomes.(Nature(400:155[159.(

Ševčíková(T,(Horák(A,(Klimeš(V,(Zbránková(V,(Demir[Hilton(E,(Sudek(S,(Jenkins(J,(Schmutz(J,( Downloaded from Přibyl(P,(Fousek(J,(et(al.(2015.(Updating(algal(evolutionary(relationships(through(plastid( genome(sequencing:(did(alveolate(plastids(emerge(through(endosymbiosis(of(an( ochrophyte?(Sci(Rep(5:10134.( ( * http://mbe.oxfordjournals.org/ * * * * * * * at University of Dundee on November 11, 2016 * * * * * * * * * * * * * * * * * * * * * * * * *

! 26! Allen(JF.(1993.(Control(of(gene(expression(by(redox(potential(and(the(requirement(for( chloroplast(and(mitochondrial(genomes.(J(Theor(Biol(165:609[631.( Bachvaroff(TR,(Concepcion(GT,(Rogers(CR,(Herman(EM,(Delwiche(CF.(2004.(Dinoflagellate( expressed(sequence(tag(data(indicate(massive(transfer(to(the(nuclear(genome(sequence.( Protist(155:65[78.( Bachvaroff(TR,(Gornik(SG,(Concepcion(GT,(Waller(RF,(Mendez(GS,(Lippmeier(JC,(Delwiche(CF.( 2014.(Dinoflagellate(phylogeny(revisited:(Using(ribosomal(proteins(to(resolve(deep( branching(dinoflagellate(clades.(Mol(Phyl(Evol(70:314[322.( Bachvaroff(TR,(Sanchez[Puerta(MV,(Delwiche(CF.(2006.(Rate(variation(as(a(function(of(gene( origin(in(plastid[derived(genes(of(peridinin[containing(dinoflagellates.(J(Mol(Evol(62:42[52.( Barbrook(AC,(Dorrell(RG,(Burrows(J,(Plenderleith(LJ,(Nisbet(RER,(Howe(CJ.(2012.( Polyuridylylation(and(processing(of(transcripts(from(multiple(gene(minicircles(in(chloroplasts( of(the(dinoflagellate(Amphidinium-carterae.(Plant(Mol(Biol(79:347[357.(

Barbrook(AC,(Howe(CJ.(2000.(Minicircular(plastid(DNA(in(the(dinoflagellate(Amphidinium( Downloaded from operculatum.(Molecular(and(General(Genetics(263:152[158.( Barbrook(AC,(Santucci(N,(Plenderleith(LJ,(Hiller(RG,(Howe(CJ.(2006.(Comparative(analysis(of( dinoflagellate(chloroplast(genomes(reveals(rRNA(and(tRNA(genes.(BMC(Genom(7:(297.( Barbrook(AC,(Symington(H,(Nisbet(RER,(Larkum(A,(Howe(CJ.(2001.(Organisation(and( expression(of(the(plastid(genome(of(the(dinoflagellate(Amphidinium-operculatum.(Mol( http://mbe.oxfordjournals.org/ Genet(Genom(266:632[638.( Barbrook(AC,(Voolstra(CR,(Howe(CJ.(2014.(The(chloroplast(genome(of(a(Symbiodinium(sp( clade(C3(isolate.(Protist(165:1[13.( Bendtsen(JD,(Nielsen(H,(von(Heijne(G,(Brunak(S.(2004.(Improved(prediction(of(signal( peptides:(SignalP(3.0.(J(Mol(Biol(340:783[795.( Bowler(C,(Allen(AE,(Badger(JH,(Grimwood(J,(Jabbari(K,(Kuo(A,(Maheswari(U,(Martens(C,( Maumus(F,(Otillar(RP,(et(al.(2008.(The(Phaeodactylum(genome(reveals(the(evolutionary( at University of Dundee on November 11, 2016 history(of(diatom(genomes.(Nature(456:239[244.( Butterfield(ER,(Howe(CJ,(Nisbet(RER.(2013.(An(analysis(of(dinoflagellate(metabolism(using( EST(data.(Protist(164:218[236.( Chen(X,(Kindle(KL,(Stern(DB.(1995.(The(initiation(codon(determines(the(efficiency(but(not(the( site(of(translation(initiation(in(Chlamydomonas(chloroplasts.(Plant(Cell(7:1295[1305.( Crick(FH.(1968.(The(origin(of(the(genetic(code.(J(Mol(Biol(38:367[379.( Cummins(CA,(McInerney(JO.(2011.(A(method(for(inferring(the(rate(of(evolution(of( homologous(characters(that(can(potentially(improve(phylogenetic(inference,(resolve(deep( divergence(and(correct(systematic(biases.(Syst(Biol(60:833[844.( Dang(Y,(Green(BR.(2009.(Substitutional(editing(of(Heterocapsa-triquetra-chloroplast( transcripts(and(a(folding(model(for(its(divergent(chloroplast(16S(rRNA.(Gene(442:73[80.( de(Vargas(C,(Audic(S,(Henry(N,(Decelle(J,(Mahé(F,(Logares(R,(Lara(E,(Berney(C,(Le(Bescot(N,( Probert(I,(et(al.(2015.(Ocean(plankton.(Eukaryotic(plankton(diversity(in(the(sunlit(ocean.( Science(348:1261605.( Decelle(J,(Siano(R,(Probert(I,(Poirier(C,(Not(F.(2014.(Multiple(microalgal(partners(in(symbiosis( with(the(acantharian(Acanthochiasma(sp.((Radiolaria).(Symbiosis(58:233[244.( Derelle(E,(Ferraz(C,(Rombauts(S,(Rouzé(P,(Worden(AZ,(Robbens(S,(Partensky(F,(Degroeve(S,( Echeynié(S,(Cooke(R,(et(al.(2006.(Genome(analysis(of(the(smallest(free[living(eukaryote( Ostreococcus-tauri(unveils(many(unique(features.(Proc(Natl(Acad(Sci(USA(103:11647[11652.( Dorrell(RG,(Drew(J,(Nisbet(RE,(Howe(CJ.(2014.(Evolution(of(chloroplast(transcript(processing( in(Plasmodium(and(its(chromerid(algal(relatives.(PLoS(Genet(10:1004008.( Dorrell(RG,(Hinksman(GA,(Howe(CJ.(2016.(Diversity(of(transcripts(and(transcript(processing( forms(in(plastids(of(the(dinoflagellate(alga(Karenia-mikimotoi.(Plant(Mol(Biol(90:233[247.(

! 27! Dorrell(RG,(Howe(CJ.(2012.(Functional(remodeling(of(RNA(processing(in(replacement( chloroplasts(by(pathways(retained(from(their(predecessors.(Proc(Natl(Acad(Sci(USA(109:( 18879[18884.( Dorrell(RG,(Howe(CJ.(2015.(Integration(of(plastids(with(their(hosts:(lessons(learnt(from( dinoflagellates(Proc(Natl(Acad(Sci(USA(112:(10247–10254.( Edgar(RC.(2004.(MUSCLE:(multiple(sequence(alignment(with(high(accuracy(and(high( throughput.(Nucl(Acids(Res(32:1792[1797.( Emanuelsson(O,(Brunak(S,(von(Heijne(G,(Nielsen(H.(2007.(Locating(proteins(in(the(cell(using( TargetP,(SignalP(and(related(tools.(Nat(Protocol(2:953[971.( Gavelis(GS,(White(RA,(Suttle(CA,(Keeling(PJ,(Leander(BS.(2015.(Single[cell(transcriptomics( using(spliced(leader(PCR:(evidence(for(multiple(losses(of(photosynthesis(in(polykrikoid( dinoflagellates.(BMC(Genom(16:528.( Gornik(SG,(Ford(KL,(Mulhern(TD,(Bacic(A,(McFadden(GI,(Waller(RF.(2012.(Loss(of(nucleosomal(

DNA(condensation(coincides(with(appearance(of(a(novel(nuclear(protein(in(dinoflagellates.( Downloaded from Curr(Biol(22:2303[2312.( Gruber(A,(Rocap(G,(Kroth(PG,(Armbrust(EV,(Mock(T.(2015.(Plastid(proteome(prediction(for( diatoms(and(other(algae(with(secondary(plastids(of(the(red(lineage.(Plant(J(81:519[528.( Hallegraeff(GM.(2010.(Ocean(climate(change,(phytoplankton(community(responses,(and( harmful(algal(blooms:(a(formidable(predictive(challenge.(J(Phycol(46:220[235.( http://mbe.oxfordjournals.org/ Haxo(FT,(Kycia(JH,(Somers(GF,(Bennett(A,(Siegelman(HW.(1976.(Peridinin(chlorophyll(a( proteins(of(dinoflagellate(Amphidinium-carterae-(Plymouth(450).(Plant(Physiol(57:297[303.( Hiller(RG.(2001.('Empty'(minicircles(and(petB/atpA(and(psbD/psbE((cytb(559)(alpha)(genes(in( tandem(in(Amphidinium-carterae(plastid(DNA.(FEBS(Lett(505:449[452.( Hinder(SL,(Hays(GC,(Edwards(M,(Roberts(EC,(Waine(AW,(Gravenor(MB.(2012.(Changes(in( marine(dinoflagellate(and(diatom(abundance(under(climate(change.(Nat(Climate(Change.(2:( 271[275.( at University of Dundee on November 11, 2016 Hoppenrath(M,(Leander(BS.(2010.(Dinoflagellate(phylogeny(as(inferred(from(heat(shock( protein(90(and(ribosomal(gene(sequences.(PLoS(One(5:(13220.( Howe(CJ,(Nisbet(RER,(Barbrook(AC.(2008.(The(remarkable(chloroplast(genome(of( dinoflagellates.(J(Exp(Bot(59:1035[1045.( Huesgen(PF,(Alami(M,(Lange(PF,(Foster(LJ,(Schröder(WP,(Overall(CM,(Green(BR.(2013.( Proteomic(amino[termini(profiling(reveals(targeting(information(for(protein(import(into( complex(plastids.(PLoS(One(8:74483.( Hurst(LD.(2002.(The(Ka/Ks(ratio:(diagnosing(the(form(of(sequence(evolution.(Trends(Genet( 18:486.( Inagaki(Y,(Simpson(AGB,(Dacks(JB,(Roger(AJ.(2004.(Phylogenetic(artifacts(can(be(caused(by( leucine,(serine,(and(arginine(codon(usage(heterogeneity:(dinoflagellate(plastid(origins(as(a( case(study.(Systemat(Biology(53:582[593.( Ishikawa(SA,(Inagaki(Y,(Hashimoto(T.(2012.(RY[Coding(and(non[homogeneous(models(can( ameliorate(the(maximum[likelihood(inferences(from(nucleotide(sequence(data(with(parallel( compositional(heterogeneity.(Evol(Bioinf(8:357[371.( Janouskovec(J,(Horák(A,(Oborník(M,(Lukes(J,(Keeling(PJ.(2010.(A(common(red(algal(origin(of( the(apicomplexan,(dinoflagellate,(and(heterokont(plastids.(Proc(Natl(Acad(Sci(USA( 107:10949[10954.( Janouskovec(J,(Sobotka(R,(Lai(DH,(Flegontov(P,(Koník(P,(Komenda(J,(Ali(S,(Prásil(O,(Pain(A,( Oborník(M,(et(al.(2013.(Split(photosystem(protein,(linear[mapping(topology,(and(growth(of( structural(complexity(in(the(plastid(genome(of(Chromera-velia.(Mol(Biol(Evol(30:2447[2462.( Janouškovec(J,(Tikhonenkov(D,(Mikhailov(K,(Simdyanov(T,(Aleoshin(V,(Mylnikov(A,(Keeling(P.( 2013.(Colponemids(represent(multiple(ancient(alveolate(lineages.(Curr(Biol,(23:(2546[2552.( Kamiya(N,(Shen(JR.(2003.(Crystal(structure(of(oxygen[evolving(photosystem(II(from( Thermosynechococcus-vulcanus(at(3.7[A(resolution.(Proc(Natl(Acad(Sci(USA(100:98[103.(

! 28! Kearse(M,(Moir(R,(Wilson(A,(Stones[Havas(S,(Cheung(M,(Sturrock(S,(Buxton(S,(Cooper(A,( Markowitz(S,(Duran(C,(et(al.(2012.(Geneious(Basic:(An(integrated(and(extendable(desktop( software(platform(for(the(organization(and(analysis(of(sequence(data.(Bioinf(28:1647[1649.( Keeling(PJ.(2010.(The(endosymbiotic(origin,(diversification(and(fate(of(plastids.(Phil(Trans(R( Soc(Biol(Sci(365:729[748.( Keeling(PJ,(Burki(F,(Wilcox(HM,(Allam(B,(Allen(EE,(Amaral[Zettler(LA,(Armbrust(EV,(Archibald( JM,(Bharti(AK,(Bell(CJ,(et(al.(2014.(The(Marine(Microbial(Eukaryote(Transcriptome( Sequencing(Project((MMETSP):(illuminating(the(functional(diversity(of(eukaryotic(life(in(the( oceans(through(transcriptome(sequencing.(PLoS(Biol(12:1001889.( Kim(S,(Bachvaroff(TR,(Handy(SM,(Delwiche(CF.(2011.(Dynamics(of(actin(evolution(in( dinoflagellates.(Mol(Biol(Evol(28:1469[1480.( Kim(SW,(Jung(WH,(Ryu(JM,(Kim(JB,(Jang(HW,(Jo(YB,(Jung(JK,(Kim(JH.(2008.(Identification(of(an( alternative(translation(initiation(site(for(the(Pantoea-ananatis(lycopene(cyclase((crtY)(gene(in(

E.-coli(and(its(evolutionary(conservation.(Protein(Expr(Purif(58:23[31.( Downloaded from Lartillot(N,(Lepage(T,(Blanquart(S.(2009.(PhyloBayes(3:(a(Bayesian(software(package(for( phylogenetic(reconstruction(and(molecular(dating.(Bioinf(25:2286[2288.( Leterme(SC,(Seuront(L,(Edwards(M.(Differential(contribution(of(diatoms(and(dinoflagellates( to(phytoplankton(biomass(in(the(NE(Atlantic(Ocean(and(the(North(Sea.(Mar(Ecol(Prog(Ser( 312:9.( http://mbe.oxfordjournals.org/ Lin(S.(2011.(Genomic(understanding(of(dinoflagellates.(Res(Microbiol(162:551[569.( Miller(MA,(Schwartz(T,(Pickett(BE,(He(S,(Klem(EB,(Scheuermann(RH,(Passarotti(M,(Kaufman(S,( O'Leary(MA.(2015.(A(RESTful(API(for(access(to(phylogenetic(tools(via(the(CIPRES(science( gateway.(Evol(Bioinform(Online(11:43[48.( Moore(RB,(Obornik(M,(Janouskovec(J,(Chrudimsky(T,(Vancova(M,(Green(DH,(Wright(SW,( Davies(NW,(Bolch(CJS,(Heimann(K,(et(al.(2008.(A(photosynthetic(alveolate(closely(related(to( apicomplexan(parasites.(Nature(451:959[963.( at University of Dundee on November 11, 2016 Morse(D,(Salois(P,(Markovic(P,(Hastings(JW.(1995.(A(nuclear[encoded(form(II(RuBisCO(in( dinoflagellates.(Science(268:1622[1624.( Moszczynski(K,(Mackiewicz(P,(Bodyl(A.(2012.(Evidence(for(horizontal(gene(transfer(from( Bacteroidetes(bacteria(to(dinoflagellate(minicircles.(Mol(Biol(Evol(29:887[892.( Mungpakdee(S,(Shinzato(C,(Takeuchi(T,(Kawashima(T,(Koyanagi(R,(Hisata(K,(Tanaka(M,(Goto( H,(Fujie(M,(Lin(S,(et(al.(2014.(Massive(gene(transfer(and(extensive(RNA(editing(of(a(symbiotic( dinoflagellate(plastid(genome.(Genome(Biol(Evol(6:1408[1422.( Nakamura(Y.(1998.(Growth(and(grazing(of(a(large(heterotrophic(dinoflagellate,(Noctiluca( scintillans,(in(laboratory(cultures.(J(Plankton(Res(20:1711[1720.( Nassoury(N,(Morse(D.(2005.(Protein(targeting(to(the(chloroplasts(of(photosynthetic( eukaryotes:(getting(there(is(half(the(fun.(Biochimic(Biophys(Acta(1743:5[19.( Nassoury(N,(Wang(Y,(Morse(D.(2005.(Brefeldin(a(inhibits(circadian(remodeling(of(chloroplast( structure(in(the(dinoflagellate(gonyaulax.(Traffic(6:548[561.( Nelson(MJ,(Dang(YK,(Filek(E,(Zhang(ZD,(Yu(VWC,(Ishida(K,(Green(BR.(2007.(Identification(and( transcription(of(transfer(RNA(genes(in(dinoflagellate(plastid(minicircles.(Gene(392:291[298.( Nelson(MJ,(Green(BR.(2005.(Double(hairpin(elements(and(tandem(repeats(in(the(non[coding( region(of(Adenoides(eludens(chloroplast(gene(minicircles.(Gene(358:102[110.( Nisbet(RER,(Koumandou(VL,(Barbrook(AC,(Howe(CJ.(2004.(Novel(plastid(gene(minicircles(in( the(dinoflagellate(Amphidinium-operculatum.(Gene(331:141[147.( Oudot[Le(Secq(MP,(Grimwood(J,(Shapiro(H,(Armbrust(EV,(Bowler(C,(Green(BR.(2007.( Chloroplast(genomes(of(the(diatoms(Phaeodactylum-tricornutum(and(Thalassiosira- pseudonana:(comparison(with(other(plastid(genomes(of(the(red(lineage.(Molecular(Genet( Genom(277:427[439.( Pochon(X,(Putnam(HM,(Gates(RD.(2014.(Multi[gene(analysis(of(Symbiodinium(dinoflagellates:( a(perspective(on(rarity,(symbiosis,(and(evolution.(PeerJ(2:394.(

! 29! Probert(I,(Siano(R,(Poirier(C,(Decelle(J,(Biard(T,(Tuji(A,(Suzuki(N,(Not(F.(2014.( Brandtodinium(gen.(nov.(and(B.-nutricula(comb.(nov.((Dinophyceae),(a(dinoflagellate( commonly(found(in(symbiosis(with(polycystine(radiolarians.(J(Phycol(50:388[399.( Puthiyaveetil(S,(Kavanagh(TA,(Cain(P,(Sullivan(JA,(Newell(CA,(Gray(JC,(Robinson(C,(van(der( Giezen(M,(Rogers(MB,(Allen(JF.(2008.(The(ancestral(symbiont(sensor(kinase(CSK(links( photosynthesis(with(gene(expression(in(chloroplasts.(Proc(Natl(Acad(Sci(USA(105:10061[ 10066.( Reynolds(JM,(Bruns(BU,(Fitt(WK,(Schmidt(GW.(2008.(Enhanced(photoprotection(pathways(in( symbiotic(dinoflagellates(of(shallow[water(corals(and(other(cnidarians.(Proc(Natl(Acad(Sci( USA(105:13674[13678.( Robbens(S,(Derelle(E,(Ferraz(C,(Wuyts(J,(Moreau(H,(Van(de(Peer(Y.(2007.(The(complete( chloroplast(and(mitochondrial(DNA(sequence(of(Ostreococcus-tauri:(organelle(genomes(of( the(smallest(eukaryote(are(examples(of(compaction.(Mol(Biol(Evol(24:956[968.(

Ronquist(F,(Teslenko(M,(van(der(Mark(P,(Ayres(DL,(Darling(A,(Höhna(S,(Larget(B,(Liu(L,( Downloaded from Suchard(MA,(Huelsenbeck(JP.(2012.(MrBayes(3.2:(efficient(Bayesian(phylogenetic(inference( and(model(choice(across(a(large(model(space.(Syst(Biol(61:539[542.( Sato(S,(Nakamura(Y,(Kaneko(T,(Asamizu(E,(Tabata(S.(1999.(Complete(structure(of(the( chloroplast(genome(of(Arabidopsis-thaliana.(DNA(Res(6:283[290.( Shalchian[Tabrizi(K,(Skanseng(M,(Ronquist(F,(Klaveness(D,(Bachvaroff(TR,(Delwiche(CF,( http://mbe.oxfordjournals.org/ Botnen(A,(Tengs(T,(Jakobsen(KS.(2006.(Heterotachy(processes(in(rhodophyte[derived( secondhand(plastid(genes:(implications(for(addressing(the(origin(and(evolution(of( dinoflagellate(plastids.(Mol(Biol(Evol(23:1504[1515.( Siano(R,(Montresor(M,(Probert(I,(Not(F,(de(Vargas(C.(2010.(Pelagodinium(gen.(nov.(and(P.( béii(comb.(nov.,(a(dinoflagellate(symbiont(of(planktonic(foraminifera.(Protist(161:385[399.( Stamatakis(A.(2014.(RAxML(version(8:(a(tool(for(phylogenetic(analysis(and(post[analysis(of( large(phylogenies.(Bioinf(30:1312[1313.( at University of Dundee on November 11, 2016 Walker(JE.(2013.(The(ATP(synthase:(the(understood,(the(uncertain(and(the(unknown.( Biochemical(Society(Transactions(41:1[16.( Wang(D,(Zhang(Y,(Zhang(Z,(Zhu(J,(Yu(J.(2010.(KaKs_Calculator(2.0:(a(toolkit(incorporating( gamma[series(methods(and(sliding(window(strategies.(Genom(Proteom(Bioinf(8:77[80.( Wang(YL,(Morse(D.(2006.(Rampant(polyuridylylation(of(plastid(gene(transcripts(in(the( dinoflagellate(Lingulodinium.(Nucl(Acids(Rese(34:613[619.( Wisecaver(JH,(Hackett(JD.(2011.(Dinoflagellate(genome(evolution.(Ann(Rev(Microbiol(65:369[ 387.( Xia(X.(2013.(DAMBE5:(a(comprehensive(software(package(for(data(analysis(in(molecular( biology(and(evolution.(Mol(Biol(Evol(30:1720[1728.( Xia(X,(Xie(Z,(Salemi(M,(Chen(L,(Wang(Y.(2003.(An(index(of(substitution(saturation(and(its( application.(Mol(Phylogenet(Evol(26:1[7.( Yang(Z.(2007.(PAML(4:(phylogenetic(analysis(by(maximum(likelihood.(Mol(Biol(Evol(24:1586[ 1591.( Yang(Z,(Bielawski(JP.(2000.(Statistical(methods(for(detecting(molecular(adaptation.(Trends( Ecol(Evol(15:496[503.( Zauner(S,(Greilinger(D,(Laatsch(T,(Kowallik(KV,(Maier(UG.(2004.(Substitutional(editing(of( transcripts(from(genes(of(cyanobacterial(origin(in(the(dinoflagellate(Ceratium(horridum.( FEBS(Lett(577:535[538.( Zhang(H,(Hou(Y,(Miranda(L,(Campbell(DA,(Sturm(NR,(Gaasterland(T,(Lin(S.(2007.(Spliced( leader(RNA(trans[splicing(in(dinoflagellates.(Proc(Natl(Acad(Sci(USA(104:4618[4623.( Zhang(Z,(Green(BR,(Cavalier[Smith(T.(1999.(Single(gene(circles(in(dinoflagellate(chloroplast( genomes.(Nature(400:155[159.( Ševčíková(T,(Horák(A,(Klimeš(V,(Zbránková(V,(Demir[Hilton(E,(Sudek(S,(Jenkins(J,(Schmutz(J,( Přibyl(P,(Fousek(J,(et(al.(2015.(Updating(algal(evolutionary(relationships(through(plastid(

! 30! genome(sequencing:(did(alveolate(plastids(emerge(through(endosymbiosis(of(an( ochrophyte?(Sci(Rep(5:10134.( ( ( ( ( ( ( ( ( ( ( (

( Downloaded from ( ( ( ( ( http://mbe.oxfordjournals.org/ ( ( ( ( ( ( ( at University of Dundee on November 11, 2016 ( ( (

! 31! 90

80

70 MMETSP 60

50

40 NCBI ESTs

30 Downloaded from

20 Previous Number species with sequencesNumber species with 10 sequence accessions http://mbe.oxfordjournals.org/ 0

( Fig.*1.*New*peridinin*plastid*coding*sequences*identified*from*transcriptome*libraries.*This( graph(shows(the(number(of(peridinin(dinoflagellates(for(which(sequences(of(each(of(the(

twelve(protein[coding(genes(present(in(peridinin(plastids(have(been(identified,(both( at University of Dundee on November 11, 2016 previously,(and(from(the(data(presented(in(this(study.(Sequences(are(only(counted(if(they( are(>(200bp(length((for(atpA,-atpB,-psaA,-psaB,-psbA,-psbB,-psbC-and(psbD),(>(100(bp((for( petB,-petD,-psbE)(or(>(50bp((for(psbI).(( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

! 32! Cyanidioschyzon merolae Porphyridium purpureum Rhodomonas salina

0.63/ Guillardia theta 60 Pavlova lutheri 1/91 1/86 Emiliania huxleyi Aureococcus anophageferrens 1/52 Ectocarpus siliculosus 0.96/55 1/58 Phaeodactylum tricornutum Amphidinium massartii 1/95 Symbiodinium clade A* Amphidinium carterae Togula jolla 1/96 1/65 Ceratium fusus Lingulodinium polyedra Gambierdiscus australes 1/ Alexandrium margalefi 76 0.97/52 Alexandrium tamarense 1/82 0.55/51 Alexandrium monilatum Gonyaulacales

Alexandrium catenella Downloaded from 0.74/ 40 Alexandrium andersonii Pyrodinium bahamense 1/ Gonyaulax spinifera 1/97 81 Protoceratium reticulatum 1/87 Symbiodinium CCMP421 1/72 Symbiodinium clade C15 Symbiodinium clade C1 http://mbe.oxfordjournals.org/ Symbiodinium clade Mp Suessiales Symbiodinium clade D1a 1/83 1/94 Symbiodinium clade A 0.99/99 Symbiodinium CCMP2430 1/60 Polarella glacialis Azadinium spinosum # Heterocapsa rotundata $ $ 1/66 Heterocapsa arctica Brandtodinium nutriculum 1/76 Peridinium aciculiferum Peridiniales 1/76 Scrippsiella hangoei SHTV5 1/99 Scrippsiella hangoei SHHI4 at University of Dundee on November 11, 2016 Scrippsiella trochoidea 1/62 (1/2) Pelagodinium beii ‡ 1/64 Proro- Prorocentrum minimum CCMP1329 centrales Prorocentrum minimum CCMP2233 Cyanophora paradoxa

1/90 Ostreococcus tauri Chlorella variabilis Camellia sinensis Marchantia polymorpha 0.05 * * Fig.*2.*Multigene*protein*phylogeny*of*peridinin*dinoflagellates.*This(figure(shows(the( Bayesian(obtained(for(the(48(species(x(3410(aa(alignment(of(the(twelve(proteins(that(are( plastid[encoded(in(peridinin(dinoflagellates.(Filled(circles(at(each(node(indicate(support(with( a(posterior(probability(of(1.0((for(Bayesian(inference)(and(100%(bootstrap(values((for( RAxML);(elsewhere,(support(values(for(each(node(are(given(in(the(format((MrBayes/( RAxML).(The(branch(leading(to(Pelagodinium-beii-(marked(with(double(crossed(lines)(has( been(reduced(to(half(its(true(length((true(length(0.463,(displayed(length(0.232)(to( accommodate(the(branch(within(the(figure.(Two(phylogenetically(distinct(populations(of( sequences(identified(from(the(Clade(A(Symbiodinium-library(are(shown,(one(grouping(with( other(Symbiodinium-sequences,(and(the(other((asterisked)(within(Amphidinium.( Dinoflagellate(orders((Gymnodiniales,(Gonyaulacales,(Peridiniales,(Suessiales,(and( Prorocentrales)(are(labelled(on(the(diagram.(Species(that(do(not(resolve(with(other( members(of(the(same(order(are(individually(labelled,(as(follows:(#((Azadinium-spinosum),( previously(assigned(to(Gonyaulacales(but(identified(here(as(a(sister(group(to(the(Suessiales;( $((Heterocapsa-sp.),(assigned(to(Peridiniales(but(identified(here(as(a(sister(group(to( Gonyaulacales(and(Suessiales;(‡((Pelagodinium-beii),(assigned(to(Suessiales(but(identified( here(as(a(sister(group(to(Prorocentrales.( (

! 33! ( (

Total Sub tauri 421

2430 salina

margalefi paradoxa

variabilis polymorpha . 2233 . 1329

tricornutum . anophageferrens Togula jolla Togula Proro Proro . Pavlovalutheri Ceratiumfusus Guillardiatheta Emilianiahuxleyi Polarellaglacialis Camelliasinensis SymbiodiniumC1 Pelagodiniumbeii SymbiodiniumMp Chlorella Symbiodinium Ostreococcus ScrippsiellaSHHI4 SymbiodiniumD1a SymbiodiniumC15 Phaeo ScrippsiellaSHTV5 Symbiodinium Gonyaulaxspinifera Rhodomonas Heterocapsaarctica Azadiniumspinosum Amphidiniumcarterae Vitrella brassicaformis Vitrella Ectocarpussiliculosus Alexandriumcatenella Cyanophora Alexandrium Amphidiniummassartii Scrippsiellatrochoidea Heterocapsarotundata Lingulodiniumpolyedra Peridiniumaciculiferum Alexandriummonilatum Marchantia Alexandriumandersonii Pyrodiniumbahamense Alexandriumtamarense Aureo Gambierdiscusaustrales Porphyridiumpurpureum Brandtodiniumnutriculum Protoceratiumreticulatum Ka/Ks Cyanidioschyzonmerolae

Cyanophora paradoxa 892$ 1054$ 893$ 1026$ 982$ 944$ 929$ 966$ 953$ 1077$ 877$ 920$ 920$ 1315$ 1673$ 1577$ 1380$ 1818$ 1633$ 1624$ 1568$ 1606$ 1680$ 1741$ 1701$ 1379$ 1401$ 1539$ 1462$ 1573$ 1554$ 1335$ 1511$ 1562$ 1562$ 1538$ 1435$ 1403$ 1405$ 1398$ 1423$ 1431$ 1432$ 1435$ 1461$ 1437$

Chlorella variabilis 0.0290274$ 955$ 850$ 996$ 1079$ 1036$ 1008$ 1021$ 956$ 1069$ 934$ 992$ 992$ 1312$ 1653$ 1613$ 1460$ 1826$ 1715$ 1708$ 1617$ 1663$ 1697$ 1748$ 1702$ 1408$ 1437$ 1587$ 1533$ 1643$ 1629$ 1383$ 1564$ 1614$ 1614$ 1581$ 1483$ 1450$ 1465$ 1456$ 1486$ 1490$ 1486$ 1493$ 1485$ 1480$

Ostreococcus tauri 0.0192244$ 0.0137476$ 1113$ 1171$ 1265$ 1205$ 1148$ 1116$ 1026$ 1137$ 1123$ 1074$ 1074$ 1352$ 1602$ 1583$ 1535$ 1826$ 1719$ 1713$ 1657$ 1711$ 1705$ 1733$ 1740$ 1483$ 1464$ 1674$ 1627$ 1666$ 1669$ 1450$ 1612$ 1670$ 1670$ 1676$ 1623$ 1580$ 1583$ 1581$ 1615$ 1588$ 1608$ 1602$ 1618$ 1606$

Marchantia polymorpha 0.0122238$ 0.00854982$ 0.0129098$ 652$ 990$ 923$ 1021$ 1074$ 1081$ 1115$ 961$ 1060$ 1060$ 1324$ 1769$ 1693$ 1406$ 1793$ 1660$ 1654$ 1574$ 1621$ 1700$ 1742$ 1687$ 1475$ 1520$ 1565$ 1492$ 1597$ 1602$ 1376$ 1543$ 1600$ 1604$ 1544$ 1445$ 1429$ 1433$ 1420$ 1445$ 1451$ 1459$ 1464$ 1455$ 1464$

Camellia sinensis 0.0157472$ 0.0119583$ 0.0173718$ 0.00946879$ 1110$ 1046$ 1153$ 1174$ 1143$ 1189$ 1085$ 1137$ 1137$ 1355$ 1758$ 1735$ 1512$ 1829$ 1694$ 1685$ 1617$ 1688$ 1717$ 1753$ 1710$ 1537$ 1550$ 1633$ 1586$ 1653$ 1645$ 1419$ 1604$ 1665$ 1663$ 1614$ 1533$ 1527$ 1521$ 1515$ 1551$ 1542$ 1552$ 1553$ 1566$ 1555$

Cyanidioschyzon merolae 0.0161656$ 0.0182014$ 0.0244598$ 0.0159487$ 0.0193712$ 977$ 995$ 1061$ 1070$ 1108$ 947$ 1021$ 1021$ 1268$ 1720$ 1676$ 1451$ 1777$ 1557$ 1555$ 1544$ 1675$ 1638$ 1673$ 1638$ 1491$ 1522$ 1587$ 1514$ 1585$ 1573$ 1345$ 1523$ 1573$ 1572$ 1548$ 1461$ 1460$ 1460$ 1446$ 1482$ 1463$ 1463$ 1487$ 1486$ 1467$

Porphyridium purpureum 0.0138877$ 0.0149573$ 0.0216508$ 0.0117902$ 0.01608$ 0.0106869$ 883$ 941$ 951$ 998$ 903$ 911$ 911$ 1231$ 1689$ 1661$ 1427$ 1743$ 1541$ 1537$ 1557$ 1632$ 1660$ 1678$ 1659$ 1472$ 1509$ 1544$ 1476$ 1549$ 1530$ 1313$ 1499$ 1560$ 1561$ 1543$ 1453$ 1417$ 1412$ 1419$ 1437$ 1451$ 1441$ 1463$ 1449$ 1459$ Ka/Ks

Guillardia theta 0.0167529$ 0.0185806$ 0.0248137$ 0.0170271$ 0.0215152$ 0.0161879$ 0.0109683$ 755$ 941$ 993$ 842$ 920$ 920$ 1239$ 1698$ 1618$ 1399$ 1789$ 1620$ 1615$ 1565$ 1682$ 1670$ 1742$ 1704$ 1428$ 1447$ 1530$ 1459$ 1576$ 1550$ 1323$ 1500$ 1560$ 1564$ 1551$ 1454$ 1410$ 1399$ 1404$ 1432$ 1450$ 1445$ 1438$ 1450$ 1438$

Rhodomonas salina 0.0165795$ 0.0188915$ 0.0256002$ 0.0179824$ 0.0224973$ 0.0176808$ 0.012153$ 0.00290949$ 891$ 991$ 943$ 921$ 921$ 1257$ 1624$ 1594$ 1390$ 1779$ 1620$ 1612$ 1557$ 1664$ 1671$ 1750$ 1720$ 1389$ 1410$ 1619$ 1563$ 1613$ 1581$ 1360$ 1535$ 1599$ 1600$ 1566$ 1543$ 1496$ 1501$ 1492$ 1515$ 1525$ 1514$ 1523$ 1539$ 1522$ 0.3

Emiliania huxleyi 0.0224378$ 0.0346831$ 0.0513253$ 0.017644$ 0.0217613$ 0.0170986$ 0.011728$ 0.0150324$ 0.0308185$ 953$ 819$ 840$ 840$ 1248$ 1581$ 1555$ 1404$ 1775$ 1620$ 1621$ 1561$ 1639$ 1636$ 1705$ 1666$ 1371$ 1367$ 1588$ 1508$ 1588$ 1565$ 1335$ 1522$ 1579$ 1578$ 1555$ 1488$ 1443$ 1437$ 1440$ 1473$ 1463$ 1457$ 1475$ 1477$ 1464$

Pavlova lutheri 0.0192608$ 0.0192236$ 0.0245059$ 0.0183294$ 0.0228886$ 0.0174124$ 0.0132487$ 0.0154234$ 0.0167783$ 0.012906$ 986$ 950$ 950$ 1237$ 1628$ 1560$ 1456$ 1783$ 1671$ 1664$ 1607$ 1684$ 1670$ 1722$ 1681$ 1446$ 1463$ 1589$ 1538$ 1614$ 1598$ 1378$ 1549$ 1606$ 1605$ 1570$ 1528$ 1482$ 1472$ 1477$ 1497$ 1503$ 1499$ 1509$ 1519$ 1511$ 0.2

Phaeodactylum tricornutum 0.0288961$ 0.0170735$ 0.0225988$ 0.0107338$ 0.0151883$ 0.0145704$ 0.0103795$ 0.0251584$ 0.0137634$ 0.0294395$ 0.010201$ 762$ 762$ 1244$ 1650$ 1611$ 1353$ 1792$ 1632$ 1628$ 1561$ 1625$ 1633$ 1705$ 1658$ 1344$ 1349$ 1541$ 1458$ 1574$ 1564$ 1342$ 1531$ 1590$ 1587$ 1525$ 1424$ 1402$ 1391$ 1400$ 1434$ 1432$ 1426$ 1438$ 1434$ 1420$ Aureo. anophageferrens 0.0325004$ 0.0208312$ 0.0255391$ 0.0174307$ 0.0215789$ 0.0165064$ 0.011127$ 0.0173973$ 0.0265024$ 0.0379823$ 0.0136953$ 0.025523$ 831$ 1229$ 1620$ 1578$ 1381$ 1785$ 1604$ 1600$ 1621$ 1649$ 1656$ 1730$ 1681$ 1390$ 1409$ 1571$ 1505$ 1590$ 1589$ 1356$ 1539$ 1583$ 1587$ 1535$ 1461$ 1419$ 1416$ 1416$ 1453$ 1457$ 1462$ 1460$ 1484$ 1454$ 0.1 Ectocarpus siliculosus 0.0164938$ 0.0168233$ 0.0218982$ 0.0147307$ 0.0194135$ 0.0151546$ 0.0103796$ 0.0192105$ 0.0142622$ 0.0136857$ 0.0143386$ 0.0135657$ 0.0116997$ 1253$ 1699$ 1643$ 1413$ 1760$ 1589$ 1585$ 1570$ 1610$ 1628$ 1683$ 1631$ 1390$ 1415$ 1500$ 1432$ 1538$ 1533$ 1312$ 1486$ 1540$ 1544$ 1466$ 1402$ 1352$ 1354$ 1350$ 1377$ 1384$ 1380$ 1397$ 1409$ 1398$

Vitrella brassicaformis 0.0278391$ 0.0288034$ 0.0355734$ 0.0279353$ 0.0351696$ 0.0272222$ 0.0241653$ 0.0264285$ 0.0273737$ 0.0255925$ 0.0266182$ 0.0223024$ 0.0227727$ 0.0222699$ 1705$ 1706$ 1595$ 1847$ 1719$ 1711$ 1696$ 1742$ 1714$ 1746$ 1740$ 1570$ 1580$ 1760$ 1688$ 1679$ 1703$ 1483$ 1646$ 1724$ 1726$ 1725$ 1642$ 1631$ 1614$ 1623$ 1668$ 1649$ 1649$ 1657$ 1664$ 1646$

Amphidinium carterae 0.0717624$ 0.072804$ 0.0814014$ 0.0721771$ 0.080973$ 0.0824176$ 0.0755749$ 0.0746307$ 0.0751148$ 0.0722678$ 0.0749244$ 0.0742739$ 0.0783983$ 0.0712525$ 0.0847839$ 842$ 1526$ 1838$ 1732$ 1727$ 1671$ 1675$ 1671$ 1730$ 1733$ 1374$ 1367$ 1713$ 1696$ 1673$ 1655$ 1438$ 1607$ 1679$ 1675$ 1674$ 1678$ 1641$ 1636$ 1646$ 1688$ 1659$ 1672$ 1679$ 1678$ 1675$ 0.067

Amphidinium massartii 0.0685731$ 0.07001$ 0.0794234$ 0.0682859$ 0.0799688$ 0.0759613$ 0.0718978$ 0.0711623$ 0.0712308$ 0.0698486$ 0.07814$ 0.0709613$ 0.0746423$ 0.0673684$ 0.0808913$ 0.011078$ 1505$ 1785$ 1727$ 1722$ 1630$ 1642$ 1661$ 1716$ 1706$ 1356$ 1334$ 1656$ 1631$ 1639$ 1639$ 1412$ 1564$ 1615$ 1612$ 1638$ 1599$ 1584$ 1573$ 1589$ 1628$ 1593$ 1617$ 1622$ 1630$ 1621$

Togula jolla 0.050625$ 0.0529054$ 0.0637326$ 0.0496384$ 0.0606553$ 0.0564768$ 0.0525115$ 0.0538318$ 0.0639474$ 0.0544402$ 0.0570809$ 0.0504446$ 0.0540102$ 0.0503545$ 0.0630747$ 0.0554559$ 0.0542693$ 1660$ 1371$ 1376$ 1353$ 1543$ 1522$ 1566$ 1528$ 1110$ 1124$ 1320$ 1236$ 1328$ 1328$ 1121$ 1290$ 1321$ 1318$ 1325$ 1244$ 1130$ 1138$ 1148$ 1192$ 1176$ 1184$ 1184$ 1207$ 1174$ 0.033

Pelagodinium beii 0.0934023$ 0.0939762$ 0.106744$ 0.0833326$ 0.100929$ 0.0980152$ 0.0816855$ 0.0940623$ 0.0956916$ 0.0973225$ 0.0901938$ 0.090978$ 0.0923074$ 0.0886728$ 0.108753$ 0.0977754$ 0.0881718$ 0.0736495$ 1671$ 1668$ 1659$ 1778$ 1691$ 1738$ 1747$ 1667$ 1677$ 1732$ 1676$ 1649$ 1639$ 1400$ 1570$ 1615$ 1618$ 1692$ 1720$ 1647$ 1655$ 1655$ 1664$ 1661$ 1668$ 1666$ 1671$ 1679$

Prorocentrum CCMP2233 0.0707482$ 0.0744805$ 0.0894128$ 0.0698045$ 0.0825844$ 0.0727908$ 0.0730825$ 0.0719356$ 0.0765765$ 0.0749701$ 0.0795952$ 0.0575999$ 0.0744094$ 0.068962$ 0.0803972$ 0.0797486$ 0.0770262$ 0.0450831$ 0.0708432$ 11$ 1415$ 1626$ 1516$ 1555$ 1540$ 1470$ 1492$ 1473$ 1409$ 1468$ 1453$ 1245$ 1415$ 1453$ 1450$ 1452$ 1396$ 1330$ 1336$ 1345$ 1388$ 1373$ 1384$ 1385$ 1360$ 1390$ 0

Prorocentrum CCMP1329 0.0714853$ 0.0749876$ 0.0898438$ 0.0704321$ 0.083135$ 0.0727642$ 0.0730416$ 0.0722684$ 0.076926$ 0.0748749$ 0.0799775$ 0.0575174$ 0.0744065$ 0.069032$ 0.0805295$ 0.0799597$ 0.0772929$ 0.0450835$ 0.0711793$ 0.001$ 1422$ 1623$ 1513$ 1551$ 1541$ 1475$ 1488$ 1471$ 1412$ 1472$ 1458$ 1250$ 1420$ 1461$ 1458$ 1453$ 1401$ 1334$ 1339$ 1350$ 1391$ 1375$ 1386$ 1388$ 1362$ 1394$

Scrippsiella trochoidea 0.0635914$ 0.05473$ 0.0778879$ 0.051797$ 0.06677$ 0.0668277$ 0.0633029$ 0.0666613$ 0.06815$ 0.0663632$ 0.0634502$ 0.0643342$ 0.052031$ 0.0529794$ 0.0761924$ 0.0713339$ 0.0618145$ 0.0401096$ 0.0724674$ 0.0443074$ 0.0445067$ 1506$ 1430$ 1461$ 1454$ 1365$ 1363$ 1406$ 1368$ 1428$ 1412$ 1197$ 1341$ 1384$ 1378$ 1357$ 1310$ 1264$ 1272$ 1286$ 1311$ 1326$ 1324$ 1331$ 1336$ 1331$

Brandtodinium nutriculum 0.0577878$ 0.060843$ 0.0701042$ 0.0556737$ 0.072193$ 0.0654872$ 0.0597846$ 0.0655416$ 0.066083$ 0.0622825$ 0.0668332$ 0.0598472$ 0.0599729$ 0.0575667$ 0.0807287$ 0.0684679$ 0.0596429$ 0.0476329$ 0.0889139$ 0.0588817$ 0.0590214$ 0.0481384$ 1596$ 1644$ 1608$ 1484$ 1521$ 1538$ 1507$ 1649$ 1636$ 1399$ 1566$ 1600$ 1599$ 1504$ 1470$ 1441$ 1434$ 1442$ 1482$ 1464$ 1472$ 1474$ 1481$ 1480$

Peridinium aciculiferum 0.0822778$ 0.0669614$ 0.0752776$ 0.0656765$ 0.078503$ 0.0722995$ 0.0682998$ 0.0677297$ 0.0681315$ 0.0669084$ 0.072738$ 0.0606151$ 0.0608312$ 0.0615188$ 0.0855171$ 0.0749904$ 0.0778882$ 0.0545463$ 0.0843524$ 0.0543131$ 0.0541948$ 0.0468177$ 0.0613864$ 408$ 410$ 1486$ 1479$ 1557$ 1515$ 1548$ 1562$ 1348$ 1503$ 1554$ 1549$ 1513$ 1492$ 1459$ 1442$ 1461$ 1509$ 1473$ 1478$ 1479$ 1493$ 1515$

Scrippsiella SHHI4 0.0673616$ 0.0690164$ 0.07853$ 0.0667857$ 0.0807024$ 0.073938$ 0.0699273$ 0.0695789$ 0.0718438$ 0.0707454$ 0.0758629$ 0.0635715$ 0.0648101$ 0.06263$ 0.0872146$ 0.0774335$ 0.0723441$ 0.0462963$ 0.0862805$ 0.0559694$ 0.0558608$ 0.0485969$ 0.0641365$ 0.0167286$ 348$ 1546$ 1562$ 1570$ 1536$ 1578$ 1586$ 1367$ 1531$ 1585$ 1581$ 1545$ 1519$ 1484$ 1472$ 1484$ 1537$ 1496$ 1504$ 1516$ 1525$ 1542$

Scrippsiella SHTV5 0.0656911$ 0.0662565$ 0.0768267$ 0.0634467$ 0.0786119$ 0.0722303$ 0.0678755$ 0.0683688$ 0.0699451$ 0.0685943$ 0.0730555$ 0.0609673$ 0.0632945$ 0.0607853$ 0.0860663$ 0.0756772$ 0.0702376$ 0.0448364$ 0.0860113$ 0.0551239$ 0.0550129$ 0.0466751$ 0.0610866$ 0.0107966$ 0.0125502$ 1519$ 1538$ 1543$ 1495$ 1549$ 1553$ 1334$ 1494$ 1558$ 1550$ 1512$ 1485$ 1441$ 1426$ 1441$ 1490$ 1449$ 1461$ 1459$ 1473$ 1495$

Heterocapsa arctica 0.0480394$ 0.0489421$ 0.0596834$ 0.046292$ 0.055706$ 0.0557845$ 0.0498927$ 0.0512751$ 0.0527041$ 0.0518583$ 0.0547235$ 0.0477512$ 0.0490347$ 0.0466054$ 0.0565682$ 0.047198$ 0.0477733$ 0.0254899$ 0.0702053$ 0.0404869$ 0.0406806$ 0.0336316$ 0.0411363$ 0.0489017$ 0.0416104$ 0.0395136$ 305$ 1165$ 1127$ 1296$ 1257$ 1066$ 1211$ 1262$ 1256$ 1222$ 1170$ 962$ 964$ 993$ 1064$ 1017$ 1027$ 1040$ 1087$ 1040$

Heterocapsa rotundata 0.048569$ 0.0505439$ 0.0596325$ 0.0477286$ 0.0566341$ 0.0569025$ 0.0514423$ 0.052728$ 0.0543518$ 0.0515851$ 0.0553743$ 0.0484558$ 0.0510952$ 0.0474698$ 0.0558686$ 0.0465389$ 0.0466063$ 0.0253331$ 0.0712909$ 0.0405328$ 0.0404116$ 0.0330794$ 0.0417224$ 0.0488093$ 0.0414919$ 0.0393549$ 0.0374194$ 1187$ 1154$ 1309$ 1267$ 1071$ 1221$ 1279$ 1274$ 1255$ 1170$ 989$ 992$ 1011$ 1085$ 1044$ 1056$ 1067$ 1100$ 1067$

Azadinium spinosum 0.0492679$ 0.0545269$ 0.0656468$ 0.0476735$ 0.0634231$ 0.0573744$ 0.0533164$ 0.0512614$ 0.0544937$ 0.057786$ 0.05864$ 0.0482332$ 0.0510824$ 0.0479861$ 0.0785274$ 0.0642735$ 0.0629542$ 0.0311103$ 0.0749765$ 0.0366357$ 0.0367159$ 0.0333064$ 0.0509548$ 0.0543386$ 0.0486883$ 0.04685$ 0.0165127$ 0.0165407$ 679$ 1138$ 1095$ 894$ 1040$ 1075$ 1074$ 1063$ 950$ 477$ 509$ 499$ 576$ 611$ 601$ 587$ 638$ 668$ Sub

Polarella glacialis 0.0506922$ 0.0547719$ 0.0670147$ 0.0476141$ 0.0601659$ 0.0560908$ 0.0526656$ 0.0533208$ 0.057662$ 0.0577723$ 0.0611595$ 0.0427065$ 0.056837$ 0.0492101$ 0.0683839$ 0.0609734$ 0.0595048$ 0.0270276$ 0.0655209$ 0.0430122$ 0.0430605$ 0.0298473$ 0.0462013$ 0.0527177$ 0.0453144$ 0.0433925$ 0.0186447$ 0.0189695$ 0.210913$ 984$ 925$ 745$ 873$ 903$ 903$ 1045$ 910$ 427$ 458$ 452$ 523$ 541$ 533$ 522$ 591$ 600$ Symbiodinium Mp 0.0649637$ 0.0588362$ 0.0676121$ 0.0527209$ 0.0696361$ 0.0615708$ 0.0591279$ 0.0569144$ 0.0597557$ 0.0591758$ 0.0626973$ 0.0525066$ 0.0537662$ 0.0542686$ 0.0804322$ 0.0693377$ 0.0704825$ 0.036269$ 0.0703106$ 0.0528753$ 0.0529467$ 0.0398503$ 0.0593964$ 0.0509564$ 0.0523961$ 0.0516792$ 0.0306608$ 0.0309767$ 0.052245$ 0.0482712$ 304$ 243$ 329$ 415$ 423$ 1303$ 1219$ 1010$ 1032$ 1034$ 1067$ 1070$ 1070$ 1083$ 1114$ 1097$ 2000 Symbiodinium CCMP2430 0.0630282$ 0.0698543$ 0.0816638$ 0.0595153$ 0.0751936$ 0.0695964$ 0.0659321$ 0.0659928$ 0.071054$ 0.0702213$ 0.0734341$ 0.0509862$ 0.0692753$ 0.0611592$ 0.0814702$ 0.0712741$ 0.0691453$ 0.0358068$ 0.0699636$ 0.052888$ 0.0529783$ 0.038684$ 0.0583967$ 0.062025$ 0.0539153$ 0.0526502$ 0.0293491$ 0.0299269$ 0.081584$ 0.0752659$ 0.0466023$ 64$ 180$ 358$ 362$ 1292$ 1218$ 959$ 981$ 980$ 1027$ 1031$ 1031$ 1044$ 1058$ 1057$ Symbiodinium D1a 0.0566285$ 0.0621373$ 0.075578$ 0.0532969$ 0.0675884$ 0.0617382$ 0.0585071$ 0.0580784$ 0.0628531$ 0.062234$ 0.0648983$ 0.0555539$ 0.0612332$ 0.0544183$ 0.0703513$ 0.0641548$ 0.0623586$ 0.0313173$ 0.0618273$ 0.0459156$ 0.0461214$ 0.040105$ 0.0514103$ 0.0560917$ 0.0477911$ 0.0463172$ 0.0254722$ 0.0258601$ 0.0765607$ 0.0734518$ 0.0660293$ 0.141817$ 118$ 149$ 154$ 1078$ 1020$ 772$ 799$ 787$ 826$ 825$ 830$ 845$ 864$ 863$ 1600 Symbiodinium CCMP421 0.0636949$ 0.069566$ 0.0830441$ 0.0596009$ 0.0752101$ 0.0694651$ 0.0662483$ 0.0660983$ 0.0702538$ 0.0703059$ 0.0712564$ 0.0504028$ 0.06917$ 0.0608001$ 0.0814965$ 0.0713961$ 0.0687087$ 0.0362165$ 0.068822$ 0.052182$ 0.0523449$ 0.0404234$ 0.0595322$ 0.0618352$ 0.0541876$ 0.0522884$ 0.0299064$ 0.0304295$ 0.0890351$ 0.0818248$ 0.0645046$ 0.145607$ 0.184445$ 190$ 195$ 1227$ 1162$ 911$ 932$ 933$ 974$ 985$ 991$ 1000$ 1010$ 1011$

Symbiodinium C1 0.0626166$ 0.0551796$ 0.0815349$ 0.0499037$ 0.0673212$ 0.0685667$ 0.0649216$ 0.0645459$ 0.0688395$ 0.0698557$ 0.0615396$ 0.0627736$ 0.0515365$ 0.0518313$ 0.0805305$ 0.0702502$ 0.0668199$ 0.0354101$ 0.0671184$ 0.0500187$ 0.0501051$ 0.0451328$ 0.0566861$ 0.0601162$ 0.0522338$ 0.0507047$ 0.0294016$ 0.0298051$ 0.083477$ 0.0766692$ 0.0747016$ 0.0710596$ 0.0663275$ 0.083084$ 13$ 1268$ 1182$ 942$ 963$ 956$ 1008$ 998$ 1007$ 1015$ 1039$ 1039$

Symbiodinium C15 0.0629$ 0.0552027$ 0.0820439$ 0.0498866$ 0.0674135$ 0.0690136$ 0.0652552$ 0.0648818$ 0.0691728$ 0.0702251$ 0.0618636$ 0.0631198$ 0.0516244$ 0.0519282$ 0.0809581$ 0.0700204$ 0.0670704$ 0.0353728$ 0.0676818$ 0.0499477$ 0.0500299$ 0.04489$ 0.0568021$ 0.0595667$ 0.0520383$ 0.0505528$ 0.0294968$ 0.0298845$ 0.0822151$ 0.0780824$ 0.0743389$ 0.0728838$ 0.0701709$ 0.08322$ 0.0718949$ 1266$ 1182$ 939$ 960$ 953$ 1008$ 995$ 1004$ 1012$ 1036$ 1035$ 1200

Ceratium fusus 0.0480952$ 0.0527011$ 0.0618887$ 0.0486285$ 0.0641628$ 0.0554038$ 0.0531028$ 0.0526121$ 0.0550155$ 0.0542734$ 0.0612902$ 0.0489696$ 0.051268$ 0.0497543$ 0.0770084$ 0.0643634$ 0.0672919$ 0.037004$ 0.0735111$ 0.0448247$ 0.0448521$ 0.0359769$ 0.0517009$ 0.0578707$ 0.0530998$ 0.0505718$ 0.0243864$ 0.0241983$ 0.0757008$ 0.0858455$ 0.0353505$ 0.0417314$ 0.0482174$ 0.0406494$ 0.039472$ 0.0395005$ 1091$ 842$ 862$ 862$ 928$ 910$ 914$ 920$ 941$ 955$ Lingulodinium polyedra 0.0388127$ 0.0417128$ 0.050522$ 0.0385947$ 0.0528864$ 0.0460414$ 0.0426525$ 0.0441584$ 0.0455561$ 0.0420753$ 0.0478115$ 0.0402925$ 0.0422502$ 0.039849$ 0.062212$ 0.054585$ 0.0486686$ 0.022935$ 0.0683241$ 0.033309$ 0.0333754$ 0.028752$ 0.0451349$ 0.0432599$ 0.0445707$ 0.0426091$ 0.0148656$ 0.0155977$ 0.0495675$ 0.051107$ 0.0252444$ 0.0251716$ 0.0226853$ 0.025303$ 0.0250337$ 0.0251848$ 0.0247398$ 698$ 712$ 730$ 806$ 789$ 778$ 770$ 829$ 848$ 800 Protoceratium reticulatum 0.0381911$ 0.0405652$ 0.0612233$ 0.0366689$ 0.0503357$ 0.0448082$ 0.0411806$ 0.0485184$ 0.0516094$ 0.0518957$ 0.0487045$ 0.0384644$ 0.0485352$ 0.0382$ 0.0615304$ 0.0509397$ 0.0452116$ 0.0169311$ 0.0626864$ 0.028685$ 0.0287693$ 0.0235424$ 0.0412153$ 0.0397327$ 0.0405845$ 0.0385455$ 0.00761119$ 0.00830527$ 0.173538$ 0.195486$ 0.0429664$ 0.0507672$ 0.0635765$ 0.0553993$ 0.0545647$ 0.0545922$ 0.0656964$ 0.0462207$ 100$ 91$ 241$ 232$ 218$ 203$ 298$ 299$ Gonyaulax spinifera 0.037922$ 0.0404178$ 0.0506917$ 0.0365897$ 0.0496955$ 0.0447813$ 0.0406482$ 0.0413337$ 0.0422953$ 0.0421543$ 0.0474076$ 0.0375366$ 0.0389637$ 0.0377382$ 0.059711$ 0.0505237$ 0.0446973$ 0.0199665$ 0.0624428$ 0.0290869$ 0.029169$ 0.0240511$ 0.0407987$ 0.0394272$ 0.0401579$ 0.0382548$ 0.0074429$ 0.00824145$ 0.16349$ 0.15472$ 0.035016$ 0.042914$ 0.0397301$ 0.0491226$ 0.0631632$ 0.062759$ 0.0602157$ 0.0436854$ 0.0995469$ 139$ 282$ 270$ 252$ 240$ 338$ 347$ Pyrodinium bahamense 400 0.046686$ 0.0411097$ 0.0615622$ 0.0364271$ 0.0497247$ 0.0513702$ 0.0470382$ 0.0479748$ 0.0515086$ 0.0516956$ 0.0481075$ 0.045588$ 0.0399824$ 0.0383021$ 0.060625$ 0.0546699$ 0.0537125$ 0.021232$ 0.0657442$ 0.0299801$ 0.0300692$ 0.0289001$ 0.0424244$ 0.0467127$ 0.0419678$ 0.0399691$ 0.0114647$ 0.0121171$ 0.183743$ 0.198863$ 0.0443978$ 0.0519353$ 0.0654716$ 0.0590822$ 0.0735549$ 0.0727645$ 0.0699576$ 0.0507598$ 0.28375$ 0.145422$ 229$ 219$ 201$ 187$ 278$ 284$ Gambierdiscus australes 0.0485837$ 0.0430374$ 0.0629941$ 0.0392808$ 0.0532022$ 0.053362$ 0.0495257$ 0.0497528$ 0.054257$ 0.0546852$ 0.0503957$ 0.0478156$ 0.0514605$ 0.040724$ 0.0652763$ 0.0589199$ 0.0506593$ 0.0236346$ 0.0650329$ 0.0315261$ 0.0315385$ 0.0318907$ 0.0446847$ 0.0447638$ 0.0460371$ 0.0437684$ 0.0143601$ 0.0154247$ 0.211131$ 0.214309$ 0.048655$ 0.0588288$ 0.0721306$ 0.0620933$ 0.0815361$ 0.0806001$ 0.0694375$ 0.051651$ 0.277757$ 0.186834$ 0.275142$ 347$ 322$ 324$ 406$ 402$ Alexandrium andersonii 0 0.0491742$ 0.0436264$ 0.0616744$ 0.0387431$ 0.0524161$ 0.0548553$ 0.0489639$ 0.0506934$ 0.0546909$ 0.0546505$ 0.0502026$ 0.0474901$ 0.0414438$ 0.039817$ 0.0635029$ 0.0568266$ 0.0481367$ 0.0227535$ 0.0662335$ 0.032853$ 0.0328854$ 0.0309848$ 0.0452133$ 0.0433507$ 0.0439577$ 0.0422854$ 0.0133532$ 0.0143811$ 0.1519$ 0.165125$ 0.0408416$ 0.0492134$ 0.0653889$ 0.0537105$ 0.0720683$ 0.0713227$ 0.066659$ 0.045226$ 0.142425$ 0.111163$ 0.132487$ 0.180648$ 142$ 221$ 319$ 298$ Alexandrium catenella 0.0402612$ 0.0437736$ 0.0529448$ 0.0390539$ 0.0530483$ 0.0474642$ 0.0435894$ 0.0444257$ 0.0457892$ 0.0452894$ 0.0503617$ 0.0398151$ 0.0430602$ 0.0401608$ 0.0654512$ 0.0562712$ 0.0510119$ 0.020236$ 0.0674449$ 0.034668$ 0.0347276$ 0.0279603$ 0.0466001$ 0.0443541$ 0.0450079$ 0.0430651$ 0.010996$ 0.0117037$ 0.183346$ 0.191101$ 0.0443607$ 0.0534936$ 0.0693609$ 0.0574369$ 0.0570105$ 0.0570139$ 0.0690605$ 0.0505386$ 0.202967$ 0.149624$ 0.178869$ 0.2103$ 0.161035$ 178$ 298$ 294$ Alexandrium monilatum 0.0391864$ 0.0430661$ 0.0633638$ 0.0389213$ 0.0532431$ 0.0477335$ 0.0440328$ 0.0512397$ 0.0550566$ 0.0550956$ 0.0509089$ 0.0406093$ 0.0522704$ 0.0404775$ 0.0646888$ 0.0557943$ 0.0578521$ 0.0242689$ 0.0688203$ 0.0352501$ 0.0353116$ 0.0277299$ 0.04577$ 0.0498033$ 0.044891$ 0.0424141$ 0.0114796$ 0.0122906$ 0.194516$ 0.205413$ 0.0412774$ 0.0695824$ 0.0678711$ 0.0743431$ 0.0748635$ 0.0740483$ 0.0833652$ 0.0508047$ 0.224158$ 0.156785$ 0.193511$ 0.231715$ 0.148739$ 0.230463$ 286$ 269$ Alexandrium tamarense 0.0408883$ 0.0437316$ 0.0545459$ 0.0391727$ 0.0536081$ 0.0478414$ 0.0431844$ 0.0445083$ 0.045882$ 0.0454962$ 0.0516028$ 0.0410134$ 0.0433437$ 0.0408278$ 0.0663702$ 0.0554384$ 0.0571728$ 0.0261479$ 0.0670196$ 0.0330844$ 0.0332007$ 0.0285643$ 0.0456396$ 0.0500316$ 0.04426$ 0.0420701$ 0.0138884$ 0.014682$ 0.163639$ 0.182726$ 0.0445094$ 0.0772361$ 0.0771135$ 0.0777399$ 0.0796129$ 0.07691$ 0.0767339$ 0.0466029$ 0.150128$ 0.114971$ 0.134446$ 0.194898$ 0.124444$ 0.161775$ 0.176197$ 335$ Alexandrium margalefi 0.0508515$ 0.044319$ 0.0657105$ 0.0396411$ 0.0539704$ 0.0556289$ 0.0513929$ 0.0539995$ 0.0568062$ 0.0563156$ 0.0514496$ 0.0494364$ 0.0432967$ 0.0402592$ 0.0652621$ 0.06077$ 0.0509858$ 0.0251491$ 0.0674335$ 0.0346662$ 0.034704$ 0.0337338$ 0.0454399$ 0.0466815$ 0.0470293$ 0.0451102$ 0.0158753$ 0.0167169$ 0.143248$ 0.152636$ 0.0380105$ 0.0477433$ 0.0683762$ 0.0526069$ 0.0753762$ 0.075026$ 0.0586642$ 0.0414447$ 0.143984$ 0.113243$ 0.140363$ 0.188075$ 0.123487$ 0.147925$ 0.145489$ 0.185052$ ( ( Fig.*3.*Pairwise*substitution*ratios*in*peridinin*dinoflagellates.*This(heatmap(shows( pairwise(Ka/Ks(ratios((bottom(left(hand(sector)(and(total(number(of(pairwise(substitutions( (top(right(hand(sector)(between(every(dinoflagellate(and(non[dinoflagellate(sequence(in(the( multigene(nucleotide(alignment.(Sequences(are(shown(per(the(multigene(tree(topology( established(in(figure(2;(non[dinoflagellates(are(shown(on(grey(branches,(basal(dinoflagellates( (Gymnodiniales,(Peridiniales,(Prorocentrales(and(Pelagodinium)(with(dashed(branches;(and( Gonyaulacales(and(Suessiales(on(solid(black(branches.(The(scale(factors(associated(with(each( value(are(shown(on(the(right(hand(side(of(the(figure.(( (

! 34! Alternative initiation codons 20 87

Deletions 5 18 25 Ancestral

Clade-specific Insertions 12 22 77 Species-specific

Loss of atpA conserved residues 15 92 93

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Amphidinium carterae A 4 3 14 Amphidinium massartii Togula jolla Pelagodinium beii

1 4 Prorocentrum CCMP2233 12 5 15 B 2 1 8 Prorocentrum CCMP1329 1 5 Scrippsiella trochoidea 2 C 1 Brandtodinium nutriculum 2 1 13 1 1 Peridinium aciculiferum D 2 2 9 Scrippsiella SHHI4 1 1 Scrippsiella SHTV5 Heterocapsa arctica E 4 3 9 Key 2 Heterocapsa rotundata Azadinium spinosum 1 2 Polarella glacialis Alternative initiation codon Symbiodinium Mp 1 2 A atpA - TTG 1 1 Symbiodinium CCMP2430 G Symbiodinium D1a F 1 12 B psbB -ATC 1 1 2 Symbiodinium CCMP421 2 Symbiodinium C1 C petD – KTG; psaB- RTW H Symbiodinium C15 D petB- GTG; psaB- ATW; psbC- ATC; psbD- ATY;psbE- ATT 2 1 Ceratium fusus Lingulodinium polyedra E psaA- ATA; psbB- ATT Protoceratium reticulatum 1 2 F psaA- ATC Gonyaulax spinifera I Pyrodinium bahamense G atpA- TTG; atpB- GTG; psaB- ATC Gambierdiscus australes Alexandrium andersonii H psaB- GTA In-frame insertions 1 Alexandrium catenella I psbA- TTG Alexandrium monilatum In-frame deletions J Alexandrium tamarense J atpB- BTG; psaA- GTR Loss of otherwise conserved atpA K 1 Alexandrium margalefi K psaB- ATW residues ( ( Fig.*4.*Phylogenetic*reconstruction*of*clade?specific*evolutionary*events*in*peridinin* dinoflagellates.*This(figure(shows(discrete(changes(to(peridinin(plastid(sequences(that(have( occurred(throughout(dinoflagellate(evolution.(Panel(A(shows(the(number(of(instances(of( four(evolutionary(events((adoption(of(alternative(translation(initiation(codons,(in[frame( insertions(and(deletions,(and(the(loss(of(otherwise(conserved(residues(from(atpA)(that( originated(in(a(common(ancestor(of(all(dinoflagellates,(are(confined(to(specific(species,(or( that(originated(in(the(common(ancestors(of(specific(dinoflagellate(clades.(Panel(B(shows(the( phylogenetic(origins(of(each(of(the(clade[specific(features,(as(determined(by(comparison(to( the(multigene(tree(topology(obtained(in(Fig.(2.(Alternative(initiation(codons(are(shown(with( square(labels,(in[frame(insertions(and(deletions(with(triangular(labels,(and(discrete(changes( to(otherwise(conserved(atpA-residues(with(circular(labels.*

! 35! A) 7 6 5 4 3 /Ks%ra'o%

Ka 2 1 0

Posi,ve! B) All! Posi,ve!selec,on! selec,on! !! residues! ancestral!branch! within!dinos! Total! 4121! 331! 517! Transmembrane!domains! 1008! 45**$ 108**$ Stromal!residues! 691! 70**% 156**% Luminal!residues! 1488! 110! 223! Intersubunit!interfaces! 472! 29*$ 2**$ CofactorFbinding!residues! 393! 32! 0**$

( * Fig.*5.*Site?specific*evolution*in*dinoflagellates.*Panel(A(shows(the(Ka/Ks(residues(calculated( for(each(of(the(twelve(peridinin(plastid(genes.(Black(bars(show(the(Ka/Ks(ratios(inferred(for( the(dinoflagellate(common(ancestor,(divided(by(the(Ka/Ks(ratios(calculated(for(every(non[ dinoflagellate(reference(sequence(within(the(alignments.(Grey(bars(show(the(Ka/Ks(ratios( calculated(within(the(dinoflagellate(sequences,(again(divided(by(the(Ka/Ks(ratios(calculated( for(the(non[dinoflagellate(sequences(within(the(alignment.(Panel(B(tabulates(the(residues( with(adjusted(Ka/Ks(ratio(>(1,(and(Ka/Ks(ratio(significantly(greater(than(corresponding(non[ dinoflagellate(reference(Ka/Ks(ratio)(in(the(common(ancestor(of(all(studied(dinoflagellates(or( within(the(dinoflagellates((as(above,(except(adjusted(Ka/Ks(ratio(>(0.5)(and(their(predicted( functional(properties.(Values(that(are(significantly(higher(than(would(be(expected(through( random(distribution(of(these(sites(are(shown(in(bold(text,(and(values(that(are(significantly( lower(are(shown(in(italics.(Values(with(one(asterisk(are(significant(to(P(<(0.05,(and(values( with(two(asterisks(are(significant(to(P(<(1(x(10[04.( * ( * (

! 36!