towards determining the ancestral set of MADS-box genes in seed plants

23 Pages·2014·0.78 MB·English

AnnalsofBotany114:1407–1429,2014
doi:10.1093/aob/mcu066,availableonlineatwww.aob.oxfordjournals.org
PARTOFASPECIALISSUEONFLOWERDEVELOPMENT
MADS goes genomic in conifers: towards determining the ancestral set
of MADS-box genes in seed plants
LydiaGramzow,LisaWeilandtandGu¨nterTheißen*
DepartmentofGenetics,FriedrichSchillerUniversityJena,Philosophenweg12,07743Jena,Germany
*[email protected] D
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Received:9December2013 Returnedforrevision:3January2014 Accepted:10March2014 Publishedelectronically:22May2014 lo
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†BackgroundandAimsMADS-boxgenescompriseagenefamilycodingfortranscriptionfactors.Thisgene fro
familyexpandedgreatlyduringlandplantevolutionsuchthatthenumberofMADS-boxgenesrangesfromone m
ortwoingreenalgaetoaround100inangiosperms.GiventhecrucialfunctionsofMADS-boxgenesfornearly h
allaspectsofplantdevelopment,theexpansionofthisgenefamilyprobablycontributedtotheincreasingcomplexity ttp
s
ofplants.However,theexpansionofMADS-boxgenesduringoneimportantstepoflandplantevolution,namelythe ://a
originofseedplants,remainspoorlyunderstoodduetothepreviouslackofwhole-genomedataforgymnosperms. ca
†MethodsThenewlyavailablegenomesequencesofPiceaabies,PiceaglaucaandPinustaedawereusedtoiden- de
m
tifythecompletesetofMADS-boxgenesintheseconifers.Inaddition,MADS-boxgeneswereidentifiedinthe ic
growingnumberoftranscriptomesavailableforgymnosperms.Withthesedatasets,phylogenieswereconstructed .o
u
todeterminetheancestralsetofMADS-boxgenesofseedplantsandtoinfertheancestralfunctionsofthesegenes. p
.c
†KeyResultsTypeIMADS-boxgenesareunder-representedingymnospermsandonlyaminimumoftwoTypeI o
m
MADS-boxgeneshavebeenpresentinthemostrecentcommonancestor(MRCA)ofseedplants.Incontrast,alarge /a
numberofTypeIIMADS-boxgeneswerefoundingymnosperms.TheMRCAofextantseedplantsprobablypos- ob
sfoesusnedd,astulcehastth1a1tt–h1e4MTRypCeAIIoMfAexDtaSn-tbgoyxmgennoessp.eIrnmgsyhmandoastpleeramsts1tw4–o1d6upTlyicpaetiIoInMsoAfDTySp-beoIIxMgeAnDesS.-boxgeneswere /artic
le
†ConclusionsTheimpliedancestralsetofMADS-boxgenesforseedplantsshowssimplicityforTypeIMADS-box -a
b
genesandremarkablecomplexityforTypeIIMADS-boxgenesintermsofphylogenyandputativefunctions.The s
analysisoftranscriptomedatarevealsthatgymnospermMADS-boxgenesareexpressedinagreatvarietyoftissues, tra
c
indicatingdiverserolesofMADS-boxgenesforthedevelopmentofgymnosperms.Thisstudyisthefirstthatprovides t/1
acomprehensiveoverviewofMADS-boxgenesinconifersandthuswillprovideaframeworkforfutureworkon 14
MADS-boxgenesinseedplants. /7
/1
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Keywords:MADS-boxgene,flowerdevelopment,gymnosperm,conifer,seedplant,spruce,pine,Picea,Pinus, 7
/2
ancestralgeneset,mostrecentcommonancestor,MRCA. 7
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4
4
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INTRODUCTION Gramzow et al., 2010). MADS-box genes in land plants have y
g
been further subdivided into groups and clades based on their u
e
MADS-boxgenescomprisealargegenefamilycodingfortran- phylogeny and structural features (Gramzow and Theissen, st o
scriptionsfactors(Gramzowetal.,2010).Theyarecharacterized 2010). The Type I genes have been subdivided into the three n
1
bythepresenceofaMADS-boxthatencodestheDNA-binding groups, Ma, Mb and Mg based solely on phylogenetic criteria, 8
domain of the corresponding MADS-domain proteins. Two while,inthecaseofTypeIIgenes,MIKCC-andMIKC*-group No
v
typesofMADS-boxgenesaredistinguished,TypeIorSRF-like genes are distinguished by different lengths of their encoded e
m
andTypeIIorMEF2-like,whichhadprobablyalreadybeenestab- K-domains and also on phylogenetic criteria (Henschel et al., b
e
lished in the most recent common ancestor (MRCA) of extant 2002; Parenicova et al., 2003; Kwantes et al., 2012). Finally, r 2
0
eukaryotes(Alvarez-Buyllaetal.,2000;Gramzowetal.,2010). about a dozen ancient clades of MIKCC-group genes have been 1
8
Inlinewiththis,bothtypesofMADS-boxgeneshavebeeniden- recognized in angiosperms (Becker and Theissen, 2003), and a
tifiedinmosteukaryotesstudiedsofar,eventhoughtaxaexistin fewothercladesinmossesandferns(Mu¨nsteretal.,2002).
which theone orother type of MADS-box geneshas been lost Our knowledge of the functional importance of the different
(Gramzowetal.,2010).Inplants,theproteinsencodedbyType types of MADS-box genes differs greatly in plants. Onlyafew
II MADS-box genes have a conserved, characteristic domain Type I genes have been functionally characterized and it has
structure, with the MADS (M) domain followed by an beenshownthattheyaremainlyinvolvedinfemalegametophyte,
Intervening (I) domain, a Keratin-like (K) domain and a embryo and seed development (Yoo et al., 2006; Bemer et al.,
C-terminal domain. The genes encoding these types of proteins 2008;Kangetal.,2008;Steffenetal.,2008;Waliaetal.,2009).
havethereforebeentermedMIKC-typegenes(Maetal.,1991). Large fractions of their function maybe hiddenbyredundancy,
Inplants,thetotalnumberofMADS-boxgenesincreasedgreatly andmanyofthesegenesmayhaveafunctionthatisonlyweak,
toabout100infloweringplants(angiosperms),whileitremained atbest (Bemeretal.,2010).Incontrast,thecrucialfunctionsof
low in all othereukaryotic groups (Alvarez-Buylla et al., 2000; MIKC-typegeneshavelongbeenrecognizedbasedoninformative
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1408 Gramzowetal.—MADS-boxgenesinconifers
mutantphenotypesandarewellstudied(Schwarz-Sommeretal., given the function of these genes in developmental control of
1990;Yanofskyetal.,1990;Trobneretal.,1992;Mandeletal., extantorganisms,thatMADS-boxgenesareprobablyinvolved
1992;Pelazetal.,2001).Thesegenesareinvolvedincontrolling inthephenotypicevolutionofplants(Theissenetal.,2000).
nearlyallaspectsofsporophyteandmalegametophytedevelop- Duetothepreviouslackofwholegenomedataforfernsand
ment (for recent reviews see Gramzow and Theissen, 2010; allies and gymnosperms, the set of MADS-box genes in the
Smaczniaketal.,2012).Mostprominentaretheirrolesinflower MRCA of euphyllophytes and seed plants, respectively, could
andfruitdevelopmentofangiosperms. notbereliablyinferred.Fromstudiesaimingattheisolationof
Land plants evolved from unicellular green algae (Cronk, MADS-box genes from gymnosperm transcriptomes, it is
2001).Thetransitiontolandwasaccompaniedbytheevolution known that gymnosperms have several MIKCC-group genes
ofstructuresthatallowtheregulationofwaterloss,suchascuticles thatareorthologoustothoseknownfromangiosperms(Becker
andstomata(Petersonetal.,2010).Landplantscompriseliver- et al., 2000; Futamura et al., 2008; Carlsbecker et al., 2013). D
o
worts,mosses,hornworts(collectivelycalledbryophytes)andtra- Hence, the MRCA of seed plants also probably contained at w
n
cheophytes.Thetracheophytesevolvedrootsandvasculartissue least ten MIKCC-group genes. However, whether the MRCA lo
a
for the transport of water and nutrients, with lycophytes being ofextantseedplantscontainedevenmoreMIKCC-groupgenes de
d
the most basal group that has these structures. The next clade and what the ancestral number of Type I and MIKC*-group fro
thatbranchesofffromthetracheophytetreecomprisesfernsand genesisinseedplantsisnotknown. m
theirallies such as horsetails. These ‘euphyllophytes’represent Recently,thegenomesofPiceaabies,PiceaglaucaandPinus http
the most basal group of land plants having true leaves. taeda have been sequenced (Birol et al., 2013; Nystedt et al., s
Thereafter,seedsevolvedfacilitatingthe dispersal ofthe corre- 2013). Together with the increasing amount of transcriptome ://a
c
spondingplants.Seedplantscomprisetheancestralgymnosperms data(Wegrzynetal.,2008;Lorenzetal.,2012),itisnowpossible ad
e
andangiosperms.Accordingtomostmolecularanalyses,extant to get a much more detailed picture about the ancestral set of m
gymnosperms, comprising conifers, gnetophytes, cycads and MADS-box genes in seed plants and correlate MADS-box ic.o
Ginkgo, are monophyletic (Bowe and Coat, 2000; Chaw et al., gene evolution with the evolution of seeds and flowers. Here up
2000; Xiet al.,2013).However,the phylogenetic relationships weusethistreasuretroveofdatatogetamoredetailedpicture .co
m
between the different gymnosperm groups remain equivocal; of the dynamics of MADS-box gene evolution during the /a
nevertheless, the most recent comprehensive analyses suggest originofseedplantsandthediversificationofconifers. ob
tehxattancytcagdysmpnluosspGeirnmksgo(fWorumaetclaadl.e,th2a0t1is1;sisXteirtoetalalrl.e,m2a0in1i3n)g. /article
Angiospermsevolvedhavingflowersthatdevelopovulesenclosed -a
b
in carpels, and seeds protected and distributed byfruitsas new MATERIALS AND METHODS stra
structural features. Hence, the evolution of some land plant c
IdentificationofMADS-boxgenes t/1
lineages is characterized by the addition of new structures 1
4
leadingtomorecomplexbodyplans. ThewholegenomeassemblyofPiceaabies(Nystedtetal.,2013) /7
Wholegenomesequencesareavailableforseveralgreenalgae wasdownloadedfromUmeaUniversityinJuly2012(nowavailable /14
0
species, the moss Physcomitrella patens, the spikemoss (lyco- at http://congenie.org/). The whole genome assembly of Picea 7
/2
phyte)Selaginellamoellendorffiiandanumberof angiosperms glauca (Birol et al., 2013) was downloaded from SMarTForests 7
6
(The Arabidopsis Genome Initiative, 2000; Goff et al., 2002; (http://www.smartforests.ca/)inJanuary2013andthe0.8version 90
4
Derelle et al., 2006; Tuskan et al., 2006; Jaillon et al., 2007; of the assembly of Pinus taeda was downloaded from 4
b
Merchantetal.,2007;Rensingetal.,2008;Banksetal.,2011). PINEREFSEQ (http://pinegenome.org/pinerefseq/). Similarly, y
g
Hence,thecompletesetofMADS-boxgenesinthegenomesof transcriptome data were downloaded from Dendrome (http:// u
e
greenalgae,moss,spikemossandangiospermscanbeevaluated. dendrome.ucdavis.edu/;Wegrzynetal.,2008).Further-more,tran- st o
WhileonlyoneortwoMADS-boxgeneshavebeenidentifiedin scriptomedatadescribedbyLorenzetal.(2012)weredownloaded n
1
green algae, moss and spikemoss genomes encode around 20 fromtheNCBIShortReadArchive(Sayersetal.,2012). 8
N
MADS-box genes (Gramzow et al., 2012; Barker and Ashton, Thescaffoldsofthewholegenomeassembliesaswellasthe o
v
2013). This number further increases in angiosperms, which assembled transcriptomes were translated in all six possible em
have roughly about 100 MADS-box genes (Parenicova et al., reading frames to create amino acid sequences using a custo- be
2003;Lesebergetal.,2006;Aroraetal.,2007). mized perl script. These amino acid sequences were searched r 2
0
Withthesedata,itisalsopossibletoinfertheminimalances- forMADS domains usinghmmsearchof theHMMerpackage 1
8
tralsetsofMADS-boxgenesintheMRCAofplants,landplants, (Eddy, 1996) with a customized Hidden Markov Model for
vascularplantsandangiosperms.TheMRCAofplantsprobably plant MADS domains (Gramzowand Theißen, 2013). For the
encodedonlyfewMADS-boxgenes,butatleastoneTypeIand whole genome data, all results with a length of at least 30
oneTypeIIgene(GramzowandTheissen,2010).Incontrast,the amino acids were keptand usedfor phylogeny reconstruction.
MRCAofmossesandvascularplantsandtheMRCAofvascular Fortranscriptomedata,thecompletetranscriptsequenceswere
plantsbothprobablyencodedatleasttwoTypeI,oneMIKCC- obtainedforeachoftheHMMresultswithalengthofatleast
andoneMIKC*-groupgene.TheMRCAofextantangiosperms 30 amino acids. The identified transcript sequences were
probablyalreadypossessedatleastthreeTypeI,11MIKCC-and assembled separately for each gymnosperm species using
twoMIKC*-groupgenes,agreatincreaseascomparedwiththe Sequencher v 5.1 (Gene Codes Corp., Ann Arbor, MI, USA)
ancestorofvascularplants.Lookingatthesenumbersitappears with a minimum match percentage of 95 and a minimum
likelythattheincreaseinthenumberofMADS-boxgenesiscor- overlapof20.Fortheassembledtranscriptsequences,theopen
relatedwiththeincreasingcomplexityoflandplantsandhence, readingframes(ORFs)werethendeterminedusingBatchORF
Gramzowetal.—MADS-boxgenesinconifers 1409
Finder at greengene.uml.edu or ORF Finder at NCBI (Sayers BasedontheMLphylogenyforTypeIIproteins,theseproteins
etal.,2012).TheORFsweretranslatedintoproteinsequences. wereseparatedinto11clades.Totestthestabilityoftheseclades,
TodeterminewhichtranscriptsequencesofP.abies,P.glauca separate datasets were compiled containing all the MADS
andP.taedacorrespondtowhichMADS-domainsequencesiden- sequencesbelongingtoaspecificcladeaccordingtotheTypeII
tifiedfromthesegenomes,weconductedlocalBLASTsearches ML phylogeny plus MADS sequences from other clades of
(Altschul et al., 1990) using the transcript sequences as query A. thaliana, V. vinifera and O. sativa. Separate phylogenies for
sequences and the whole genome assemblies of the P. abies, each of the 12 clades were constructed as described above for
P. glauca and P. taeda genomes as database. If atranscript se- TypeIandTypeIIphylogenies.Finally,datasetsforeachclade
quencehadamatchwithmorethan97%sequenceidentityover werecompiledcontainingthe MADSproteinsbelongingtothe
alengthofapprox.180nucleotidestoagenomicregionthatwas correspondingcladeintheTypeIIaswellasintheseparatephy-
identifiedtohaveaMADS-domainbyhmmsearch,onlythetran- logeny.SEP3fromA.thalianawasusedasarepresentativeofthe D
o
scriptsequencewaskeptandtheMADS-domainsequenceidenti- outgroup for all clades except the clade containing AGL2-, w
n
fied from the genome was removed from the dataset. As the AGL6-, FLC- and SQUA-like genes, in which AGL12 of lo
a
number of transcript sequences for which no genomic region A. thaliana was used as an outgroup representative, and the de
d
was identified was quite high using these stringent criteria, we cladecontainingAG-likegenesinwhichAGL15ofA.thaliana fro
lateralsoconsideredshorterBLASTresults. was used. Alignments and phylogenies were constructed as m
The remaining MADS-domain-containing sequences identi- described above. For each clade, two phylogenies were recon- http
fied from gymnosperm genomes and transcriptomes were structedinthisway,onecontainingallMADSproteinsbelonging s
combinedandidenticalsequenceswerekeptonlyonceusingthe to this clade and anotheronly containing MADS proteins with ://a
c
function ‘Remove Redundancy’ with a threshold of 100 of the transcriptsupport.Totestthestabilityofthephylogeniesforthe ad
e
program Jalview (Waterhouse et al., 2009). The MADS- differentcladesofTypeIIproteins,wealsoreconstructedphylo- m
domain-containing sequences remaining after this step were genieswithMrBayes(RonquistandHuelsenbeck,2003)wherewe ic.o
named using a two- or three-letter code for the species from onlyincludedMADSproteinswithtranscriptsupport.Weusedthe up
which the sequences were identified followed by the keyword WAGmodelofaminoacidsubstitutions(WhelanandGoldman, .co
m
‘MADS’andincrementingnumbers. 2001), generated 6 million generations, sampled every 1000th /a
phylogenyandexcludedthefirst25%fromfurtheranalysis. ob
/a
rtic
Phylogenyreconstruction le-a
Geneexpressiondata b
s
froTmhegyremdnuocespdedrmatagseetnoomf eMsAaDndS-tdroanmsacirnipstoemqueesncweesreideanlitgifineedd Informationaboutgeneexpressionasrevealedbytranscrip- trac
with all MADS-domain proteins from Arabidopsis thaliana, tomedatawasobtainedfromtheNCBIexpressedsequencetag t/11
Oryzasativa,Populustrichocarpa,Vitisvinifera,Amborellatri- databaseandtheNCBIshortreadarchive(Sayersetal.,2012). 4/7
chopoda, Selaginella moellendorffii and Physcomitrella patens /14
0
andwithMADS-domainproteinsannotatedfromgymnosperms 7
/2
andfernsasfarasknownusinghmmalign(Eddy,1996)witha RESULTS 7
6
hiddenMarkovmodelforplantMADSdomains(Gramzowand 90
NumberofMADSsequencesingymnosperms 4
Theißen,2013)andthe –trimoptiontoremovenon-homologous 4
b
residues from the alignment. Furthermore, amino acids corre- OursearchofgenomeandtranscriptomedataforGnetumgnemon y
g
sponding to insert states were removed from the alignment. and 18 conifer species (Fig. 1) identified 1064 MADS-domain u
e
The alignment was used to reconstruct a phylogeny using the sequences (Table 1, Supplementary Data Table S1). In species st o
RAxML program (Stamatakis, 2006) with the -f option to for which whole-genome information isavailable, Picea abies, n
1
conduct a rapid bootstrap analysis with 1000 replicates and PiceaglaucaandPinustaeda,261,121and367MADSsequences 8
N
search for the best-scoring maximum-likelihood (ML) tree in werefound,respectively.However,thereisevidenceoftranscrip- o
v
oneprogramrunattheCIPRESSciencegateway(Milleretal., tiononlyfor49,23and60oftheseMADSsequences,respective- em
2010).BasedonthisMLphylogeny,MADS-domainsequences ly. We found a total of 43 sequences in transcriptome data of be
from gymnosperms were classified into Type I and Type II P.abies,P.glaucaandP.taedaforwhichwedidnotfindacorre- r 2
0
MADS domains depending on their grouping relative to the spondingsequenceinthegenome.Thisnumberdecreasesto19 1
8
Type I and Type II MADS-domain sequences of the other transcriptome sequences that were not found in the genomes
specieswhichhadbeenclassifiedpreviously(Parenicovaetal., when lessstringentcriteriawere used. The factthat we did not
2003;Lesebergetal.,2006;Aroraetal.,2007;Diaz-Riquelme find some transcript sequences in the genomes may be due to
etal.,2009;Gramzowetal.,2012;BarkerandAshton,2013). the incomplete knowledge of gymnosperm genomes. For
WethenalignedTypeIandTypeIIMADS-domainsequences P. abies and P. glauca, only about 61% of the whole genome
separately using sequences from the same species as described has been sequenced (Birol et al., 2013; Michael and Jackson,
above and the program Probalign (Roshan and Livesay, 2006). 2013;Nystedtetal.,2013).Whilemostofthemissingsequence
ForTypeIMADS-domainproteins,thealignmentwascropped is supposed to represent repetitive elements, some genes may
tocontainonlytheMADSdomain,andforTypeIIproteins,the havealsoescapedsequencingsofar.Webelieve,however,with
alignment was cropped to contain only the MADS and K our combination of genome and transcriptome data, that we
domains.Phylogeniesforthesetwodatasetswerereconstructed have made a major leap towards a complete overview of the
usingRAxMLasdescribedabove. MADS-boxgenesingymnospermgenomes.
1410 Gramzowetal.—MADS-boxgenesinconifers
BasedonourRAxMLphylogeny(SupplementaryDataFig. wholegenomeinformationisavailable,P.abies,P.glaucaand
S1), we classified the identified MADS sequences into Type I P.taeda,thenumberofTypeIIsequencesis249,118and350,
andTypeII.Bootstrapvaluesarequitelow.However,thegroup- respectively.
ingoftheknownMADS-boxgenesiscorrectandthustheclas-
sificationofthegymnospermsequencesintoTypeIandTypeII
TypeIgenes
should also be accurate in most cases. Only 35 gymnosperm
MADS sequences were classified as Type I. Even for the InourphylogeniesofTypeIgenestherearetwomajorbranches
species for which whole-genome information is available, the that may represent large clades (Fig. 2, Supplementary Data
percentage of Type I sequences is always less than 5% of all Fig.S2).OneputativecladecontainsgenesclassifiedasMagenes
MADS sequences. In contrast, 1029 gymnosperm MADS in A. thaliana, P. trichocarpa and O. sativa (Parenicova et al.,
sequences were classified as Type II. In the species for which 2003;Leseberg etal.,2006; Arora etal.,2007) and MADS-box D
o
w
genes from V. vinifera, gymnosperms, S. moellendorffii and n
P. patens. The other clade comprises genes classified as Mb loa
Sequoia andMggenesinA.thaliana,P.trichocarpaandO.sativaand de
Cypressaceae d
Cryptomeria MADS-boxgenesfromV.vinifera,gymnosperms,S.moellen- fro
Taxus Taxaceae dorffii and P. patens. This suggests that there are two ancient m
CSWceoipallhedamolpoiaittayxsus CSAcreaipauhdcaoalproiitataycxaeacaceeeaaee cbolouaordtresestsruaolpftsTvcayolpnueceusIrsMwupiAtphDoprStr-ienbvgoioxtuhgseesnttwuedsoiiengsr(loGaunrpdasmpazlraoenwtqsue.tiAtaelg.la,oi2wn0,1bt2hu)et. https://ac
Podocarpus Podocarpaceae a
Asmentionedabove,thenumberofTypeIgenesidentifiedin d
Pinus gymnospermsisverylow.The35TypeIgenesofgymnosperms em
Picea ic
Pinaceae are distributed approximately evenly between the two clades .o
Pseudotsuga u
with14genesbelongingtoputativecladeI(Ma)and21genes p
Cedrus .c
belongingtoputativecladeII(Mb/Mg).Evidenceoftranscrip- o
Gnetum Gnetaceae m
Angiosperms tionexistsonlyforfewofthesegenes.Ouranalysisoftranscrip- /ao
tome data revealed that two genes of clade I are expressed in b
Ferns /a
Lycophytes mixedshoottissues.ForcladeII,fourgeneswerefoundintran- rtic
Mosses scriptomedataderivedfrombud,maleconeandembryotissues. le-a
Liverworts bs
tra
eFtIaGl..1(1.99R7e)laatniodnGshuigpesrloifectoanl.if(e2r00g1en).eGranceotunmsi,daenregdiohsepreer,mms,ofdeirfinesd,layfcteorphCyhtaews, CladesofTypeIIgenes ct/1
mossesandliverwortsareshownasoutgrouprepresentatives;Gnetumisshown BasedonourphylogenyofTypeIIsequences(Supplementary 14
onadashedlineduetotheunresolvedposition.Thedifferentcoloursofthe DataFig.S3),wedefined11branches(putativeclades,hereafter /7/1
branchesareusedinthefollowingfigurestoindicatethehosttaxaofthecorre- termed ‘clades’ for simplicity) for separate analyses (MIKC*, 40
spondinggenes. 7
AGL2/AGL6/FLC/SQUA,DEF/GLO/OsMADS32/GGM13, /2
7
6
9
0
4
TABLE 1. NumberofMADSsequencesidentifiedfromgymnospermgenomeandtranscriptomedata 4 b
y
g
Order Family Abbreviation Total TypeI TypeII ue
s
t o
Gnetales Gnetaceae Gnetumgnemon GgMADS 41 0 41 n
Coniferales Pinaceae Cedrusatlantica CaMADS 13 0 13 18
Piceaabies PaMADS 253(41)+8 12 249 N
Piceaglauca PgMADS 107(9)+14 3 118 ov
e
Piceasitchensis PsMADS 17 1 16 m
Pinusbanksiana PbMADS 2 0 2 be
Pinuscontorta PcMADS 14 0 14 r 2
Pinuslambertiana PlMADS 41 0 41 01
Pinuspalustris PpaMADS 21 0 21 8
Pinuspinaster PpiMADS 10 0 10
Pinustaeda PtaMADS 346(39)+21 17 350
Pseudotsugamenziesii PmeMADS 40 0 40
Podocarpaceae Podocarpusmacrophyllus PmaMADS 16 1 15
Araucariaceae Wollemianobilis WnMADS 11 0 11
Sciadopityaceae Sciadopitysverticillata SvMADS 22 1 21
Taxaceae Taxusbaccata TbMADS 3 0 3
Cephalotaxaceae Cephalotaxusharringtonia ChMADS 35 0 35
Cryptomericajaponica CjMADS 10 0 10
Cupressaceae Sequoiasempervirens SsMADS 19 0 19
Forspeciesforwhichwhole-genomeinformationisavailablethenumbersaregivenasfollows:numberofMADSsequencesidentifiedfromgenomedata
(numberofMADSsequencesidentifiedfromgenomedataandsupportedbytranscriptomedata)+numberofMADSsequencesidentifiedfromtranscriptome
dataforwhichthegenomiclocuscouldnotbeidentified.
Gramzowetal.—MADS-boxgenesinconifers 1411
60 Angiosperm
Ma-like genes
Angiosperm
2
Ma-like genes
21
Angiosperm
0 Ma-like genes
2
0
62 Angiosperm D
Ma-like genes o
w
1 8 Angiosperm nlo
Ma-like genes a
5 d
Angiosperm e
19 Ma-like genes 100 PPTIM4 (Moss) Ma d fro
PPTIM5 (Moss) m
OsMADS71 h
2 ttp
1 AMnag-iloiksep geermnes s://a
c
a
98SmMADS14 (Lycophyte) de
0 30SmMADS15 (Lycophyte) m
PPTIM2 (Moss) ic
12 PPTIM3 (Moss) .o
5 59 PtaMADS287_G PtaMADS296_G up
12 SvMADS11 .c
62 PaMADS154_G om
53113431PPatMaMADASDP1tSa41M70_A4GD_GS165_G /aob
11 PtaMADS153_G /a
1238PPttaaMMAADDSS8136_7G_G rtic
37PtaMADS173_G le
32 PmaMADS1598 PaMADS263_G -ab
PaMADS276_G s
87 AMnbg-liioksep geermnes tract/1
1
100 AMnbg-liioksep geermnes 4/7/1
4
0
22 7
/2
7
6
Angiosperm 9
1 Mg-like genes 04
PtaMADS274_G 4
b
19 y
g
37 4 ue
s
21 Angiosperm t o
Mb-like genes n
Mb/g 1
8
72 978P0PaPMggMMAADADSDS3S48931__GG Nove
15 41 PtaMADS316_G m
23 PPttaaMMAADDSS227356__GG be
8 PtaMADS277_G r 2
PtaMADS310_G 0
4 PtaMADS37 1
38 PgMADS58_G 8
61
PaMADS33
42 84 PaMADS235a_G
89 66 PaMADS235b_G
80PaMADS270_G
PaMADS306_G
PtaMADS207_G
50
31PtaMADS212_G
PsMADS10
PPTIM7 (Moss)
31 SmMADS11 (Lycophyte)
100 SmMADS12 (Lycophyte)
PaMADS303_G
PaMADS302_G
FIG.2. PhylogenyofTypeIMADS-boxgenes.ThetwomajorputativecladesofTypeIgenes,MaandMb/g(Gramzowetal.,2012),aremarkedbylabelledlineson
theright.ThecoloursofthebranchescorrespondtothecoloursofthegymnospermgenerainFig.1.AbbreviationsofgymnospermgenenamesaredescribedinTable1.
LycophytesequencesarefromSelaginellamoellendorffiiandmosssequencesarefromPhyscomitrellapatens.Cladesofangiospermgenesarecollapsedintotriangles
andtheirclassificationaccordingto(Parenicovaetal.,2003;Lesebergetal.,2006;Aroraetal.,2007)isshownontheright.Numbersatnodesdenotebootstrapvalues.
ThefullyresolvedphylogenyisavailableasSupplementaryDataFig.S2.
1412 Gramzowetal.—MADS-boxgenesinconifers
AGAMOUS,AGL12,AGL15,AGL17,GpMADS4,StMADS11, (SupplementaryDataFig.S5a).Ofthe gymnospermsequences
TM3andTM8).Allofthesecladescontaingymnospermsequences identified here, 19 belong to the clade of AGL2/AGL6-like
(Table2). genes and 67 belong to the clade of FLC/SQUA-like genes.
Whenwerestrictourphylogenytosequencesforwhichwehave
MIKC*-group genes. In our type II phylogeny,50 gymnosperm evidence of transcription,the AGL2- andthe AGL6-like genes
sequences grouped together with MIKC*-group genes from of angiosperms form sister clades as well as the FLC- and
mosses, ferns and angiosperms (Supplementary Data Fig. S3). SQUA-like genes of angiosperms, with the exception of FLC-
Only eight of these are supported by expression data. The likegenesfromrice,whichclusterwithinacladeofgymnosperm
bryophytesequencesbranchoffbeforethedifferentiationofthe sequences(Fig.4).Thissuggeststhattheduplicationsgivingrise
S- and P-clade, each containing sequences from ferns, gymnos- to AGL2- and AGL6-like genes and to FLC- and SQUA-like
permsandangiosperms(SupplementaryDataFig.S4).Inthephyl- genes occurred in angiosperms and that the MRCA of extant D
o
ogeny containing only sequences with expression data (Fig. 3), seedplantspossessedatleastoneAGL2/AGL6-likegeneandat w
n
PaMADS17 is the only gymnosperm member of the S-clade. leastoneFLC/SQUA-likegene.Asthebootstrapvaluesforour lo
a
Interestingly, also 39 genomic sequences from P. taedawithout RAxMLphylogenieswerequitelowwealsoreconstructedaphyl- de
eTveindgenycmenoofsptrearnmscsreipqtuieonncbeesl(osnegvetnowthiethSe-vcildaednecienoofutrrapnhsycrloipgteionny). osigsetenry-gruosuipngrelMatrioBnasyheisps(SofupApGleLm2e-natanrdyADGaLta6-Flikige.g5ecn)e.sTanhde d from
fsopremrmtsh.eTPh-eclaasdseoctoiagteiothnerofwtihthesgeyqmueonscpeesrmfrosmequfeernncsesantdoathnegiPo-- oinf FoLurC-MarnBdaySeQsUpAh-ylliokgeegneynewsitohf asntrgoinogseprersmusppwoarst c(opnofistremrieodr https
andS-cladesinourMrBayesphylogenyisconsistentwiththatin probability of 0.51 and 0.90, respectively). However, all ://a
c
ourRAxMLphylogenies(SupplementaryDataFig.S4).Theiden- gymnospermsequencesappearmorecloselyrelatedtoAGL2/ ad
tified MIKC*-group genes of the P-clade, PaMADS29, AGL6-likegenesthantoFLC/SQUA-likegenesinourMrBayes em
PaMADS31 and PaMADS26 from P. abies, show expression in phylogeny. ic.o
stem, wood, vegetative shoots and female cones. PaMADS26 Analysingtranscriptomedata,wefoundexpressionofgymno- up
wasalsoidentifiedfrommalecones.cDNAofGgMADS1from sperm genes belonging to the AGL2/AGL6 clade in shoots, .co
m
G.gnemonwasisolatedfromfemalecones.Incontrast,theexpres- needles and reproductive tissues. FLC/SQUA-like genes from /a
sionofthesingleS-cladegenePaMADS17wasdetectedonlyin gymnosperms were identified in transcriptome data from a ob
malecones. wcoindees)v.ariety of tissues (roots, shoots, stems, bark and female /article
AGL2/AGL6/FLC/SQUA-likegenes. Recently,itwasestablished -a
b
thatFLC-likegenesbelongtothesupercladeofAGL2-,AGL6- DEF/GLO/OsMADS32/GGM13-like genes. In our phylogenies, s
and SQUA-like genes (Ruelens et al., 2013). Hence, we here OsMADS32-like genes cluster with DEF-like, GLO-like and tra
c
analysedthesegenestogether.Inourphylogenies,twolargesub- GGM13-like genes (Supplementary Data Fig. S3). Conse- t/1
1
cladescanbedefined,oneconsistingofAGL2-andAGL6-like quently,weanalysedthesecladestogether.BasedonourTypeII 4
/7
genes and the other consisting of FLC- and SQUA-like genes phylogeny including MADS sequences identified from genome /1
4
0
7
/2
7
TABLE 2. NumberofgymnospermgenesindifferentcladesofTypeIIMADS-boxgenes 69
0
4
4
DEF/GLO/ b
AGL2/AGL6/ OsMADS32/ y g
Species MIKC* FLC/SQUA GGM13 AGAMOUS AGL12 AGL15 AGL17 GpMADS4 StMADS11 TM3 TM8 u
e
s
t o
S.sempervirens 0 0 0 0 0 0 0 0 8 4 4 n
C.japonica 0 1 6 1 0 0 0 0 0 0 1 18
T.baccata 0 0 0 0 0 0 0 0 1 1 1 N
C.harringtonia 0 3 0 0 0 0 0 0 6 10 13 ov
e
S.verticillata 0 1 0 0 0 0 0 0 0 6 14 m
W.nobilis 1 0 0 0 0 0 0 0 4 0 6 b
e
P.macrophyllus 0 1 1 0 0 0 0 0 6 1 6 r 2
P.taeda 1(41) 11(38) 2(64) 3(24) 4(15) 0(0) 0(4) 0(2) 7(20) 25(77) 6(58) 0
1
P.pinaster 0 1 0 0 0 0 0 0 2 4 3 8
P.palustris 0 1 0 0 0 0 0 0 0 15 5
P.lambertiana 0 6 1 0 1 0 0 2 6 19 6
P.contorta 0 1 0 0 0 0 0 1 2 8 2
P.banksiana 0 0 0 0 0 0 0 0 0 1 1
P.abies 4(5) 2(15) 8(20) 2(15) 0(5) 0(1) 0(13) 2(9) 14(53) 14(85) 4(27)
P.glauca 0(1) 3(10) 1(8) 2(5) 1(6) 0(0) 0(6) 0(4) 7(26) 8(32) 1(17)
P.sitchensis 0 1 0 1 0 0 0 0 5 6 3
P.menziesii 0 1 2 2 0 0 0 0 1 31 3
C.atlantica 0 2 0 0 0 0 0 0 3 8 0
G.gnemon 2 4 3 4 0 0 0 0 4 2 18
Sum 8(50) 39(86) 24(105) 15(52) 6(27) 0(1) 0(23) 5(18) 76(147) 163(310) 97(188)
Generally,thenumberofexpressedgenesisgiven.ForP.taeda,P.abies,P.glaucaandsum,thetotalnumberofgenesincludingthosesolelyidentifiedfrom
genomedatawithoutevidenceoftranscriptionisgiveninparentheses.
Gramzowetal.—MADS-boxgenesinconifers 1413
0·1 PpMADS3 (Moss)
PPM6 (Moss)
56 PPMA9 (Moss)
2373PPMA10 (Moss)
24 PPMA11 (Moss)
92PPM4 (Moss)
PPM3 (Moss)
33 PPMA12 (Moss)
29 PpMADS2 (Moss)
PPMA8 (Moss)
81 CRM15 (Fern)
14 CRM14 (Fern)
40 32 PaMADS17 VvMADS11 (Angiosperm) Do
43 S-clade w
36 Angiosperm nlo
a
S-clade genes d
e
d
10 CRM13 (Fern) fro
GgMADS36 m
96 99 9410808 GPgtaMMAADDSS135 PaMADS31 https
45 76 PaMADS29 97 APn-cglaiodsep egremnes ://acad
P-clade e
m
47 51 Angiosperm ic.o
u
P-clade genes p
52 26 .c
o
m
46 WnMADS2 /a
PaMADS26 o
42 47SmMADS4_1 (Lycophyte) SmMADPtSM1A0D_1S 9(L8y (cAonpghiyotsep)erm) b/artic
MpMADS1 (Liverwort) le
SEP3 -a
b
s
FIG.3. PhylogenyofMIKC*-groupgenes.Coloursofthebranches,genenames,trianglesandnumbersatnodesareasdescribedinFig.2.ThesubcladesSandP(Nam tra
pethaylt.e,s2e0q0u3e)nacreesmaraerfkreodmbSyellaabgeilnleedllalimneoseollnenthdeorrifgfihi,tm.TohsessleivqeurewnocretssaerqeuferonmcePishfyrsocmomMitarercllhaapnattieanpsoalnydmtohrepsheap,afreartneasenqguioesnpceersmarseeqfruoemncCeesraarteofprotemriVsritiicshvainrdifiei,ralyacnod- ct/11
fromPopulustrichocarpa.ThefullyresolvedphylogenyisavailableasSupplementaryDataFig.S4b. 4/7
/1
4
0
and transcriptome projects, 105 sequences belong to this clade Gymnosperm DEF/GLO/OsMADS32-like sequences are 7
/2
(SupplementaryData Fig.S3a).Inourseparatephylogeniesfor mainly derived from transcriptome data of male reproductive 7
6
this superclade, DEF- and GLO-like genes from angiosperms tissues. GGM13-like genes were mainly identified from 90
4
form sister clades that in turn are sister clades to OsMADS32- mixedtissues.OnlyforGGM13-likegenesofP.abiesarespecific 4
b
likegenesofangiosperms(SupplementaryDataFig.S6a).This tissues,namelyfemalereproductiveorgans,given. y
g
suggests that there have been two duplications near the base of u
e
extant angiosperms giving rise first to OsMADS32- and DEF/ AGAMOUS-andAGL12-likegenes.Inourphylogenies,52ofall st o
GLO-likegenesandthentoDEF-andGLO-likegenes.Aclade identified gymnosperm MADS sequences belong to the n
1
of60MADSsequencesfromgymnospermsissistertotheDEF/ AGAMOUS clade (Supplementary Data Fig. S7a). When we 8
N
GLO/OsMADS32-superclade. GGM13-like genes from angio- excludesequencesforwhichnoevidenceoftranscriptionexists, o
v
sperms form a clade in our phylogeny to which 45 MADS 15 sequences remain. In this reduced phylogeny, all AG-like em
sequencesfromgymnospermsarerelated. genesofgymnospermsaresistertotheAG-likegenesofangio- be
WhenweexcludegymnospermMADSsequencessolelypre- sperms,suggestingthatthe MRCA of extantseedplantshadat r 2
0
dicted on genomic sequences (Fig. 5), 24 MADS sequences least one but may not have had more than one AG-like gene 1
8
from eight gymnosperm species cluster with DEF/GLO/ (Fig.6).Furthermore,theAG-likegenesofgymnospermsform
OsMADS32-like genes from angiosperms and nine MADS threesubcladesinourRAxMLphylogenyandtwosubcladesin
sequences from five gymnosperm species cluster with ourMrBayesphylogeny(SupplementaryDataFig.7c),indicating
GGM13-likegenesfromangiosperms.Thephylogenyanddistri- that the MRCA of extant gymnosperms may have possessed at
butionofthesequencessuggeststhattherehasbeenatleastone leasttwotothreeAG-likegenes.Interestingly,AG-likesequences
DEF/GLO/OsMADS32-like gene and one GGM13-like gene in derivedfromtranscriptswereidentifiedfromavarietyoftissues,
the MRCA of extant seed plants. In our MrBayes phylogeny, including roots, shoots, stems, bark, leaves and reproductive
somegymnospermsequencesaresistertotheOsMADS32-and organs.
to the DEF-like genes from angiosperms, respectively, rather ThesistergroupofAG-likegenesaretheAGL12-likegenes
than being ancestral to a superclade of DEF-, GLO- and (Becker and Theissen, 2003). We identified 27 AGL12-like
OsMADS32-likegenesfromangiosperms(SupplementaryData sequencesfromgymnosperms,wherethereisevidenceoftran-
Fig.6c). scriptionforsixofthem(Fig.7,SupplementaryDataFig.S8).
1414 Gramzowetal.—MADS-boxgenesinconifers
Angiosperm
84
AGL2-like genes
7
41 Angiosperm
AGL6-like genes
7
33
PtaMADS352
55 PaMADS8_DAL14 AGL2/AGL6
PrMADS2_AGL6
21
30 PmeMADS10
PtaMADS2
66
12 PtaMADS351 D
20 84 GGM11_AGL6 o
GgMADS14 w
47 78 95ChMADS31 CjMADS8 nloa
79 93 CChhMMAADDSS2373 ded
82 PIMADS6 PmaMADS13 fro
PIMADS39 m
h
ttp
s
58 EFLuCdi-cliokte genes ://a
c
a
d
e
24 m
ic
.o
72 Angiosperm up
25 SQUA-like genes .co
m
92 Rice /a
18 FLC-like genes o
16 24 94 92GgMAGGDSggMM37AADDSS4300 b/artic
99 PsMADS3 le
66 PgMADS118 FLC/SQUA -a
72 PtaMADS6 b
SvMADS7 PtaMADS17 stra
11 99 GGM9 c
9 GpMADS3 t/1
PpaMADS17 1
3524 CaMADS5 4/7
7 PPggMMAADDSS910_DAL1 /14
43PtaMADS365 0
13 13 27PtaMPAIMDASD36S05 7/27
PtaMADS358 6
30 45 78 P98IMADS14 PIMADS7 904
PgMADS109 4
414PPrtaM7M0APADcDPSMSI3MA3_AD6A3DSGS1L216 by gue
10P16taPMtAaMDSA5DS364 st o
AGL12PpiMADS10 n 18
N
FIG.4. PhylogenyofAGL2/AGL6/FLC/SQUA-likegenes.Coloursofthebranches,genenames,trianglesandnumbersatnodesareasdescribedinFig.2.Thesub- ov
cladesAGL2/AGL6andFLC/SQUAaremarkedbylabelledlinesontheright.ThefullyresolvedphylogenyisavailableasSupplementaryDataFig.S5b. em
b
e
Expressionwasmostlyfoundinmixedtissues,twiceinrootand Nevertheless, the alignments also reveal three positions each r 20
1
onceinshootand/orneedles.Ourphylogenysuggeststhatone where the gymnosperm sequences are different from all of the 8
AGL12-likegenewaspresentintheMRCAofextantseedplants. angiosperm MADS sequences of the respective clades. Hence,
furtheranalysesareneededtoconfirmordenytheassociationof
AGL15-andAGL17-likegenes.InourTypeIIphylogeny,onese- the gymnosperm sequences to the clades of AGL15- and
quenceofP.abiesclusterswithAGL15-likegenesfromangio- AGL17-likegenes.
spermsand23sequencesfromP.taeda,P.abiesandP.glauca
clusterwithAGL17-likegenesofangiosperms(Supplementary GpMADS4-like genes. We found 18 sequences belonging to the
DataFigs S3,S9andS10).However,allofthesegymnosperm gymnosperm-specific clade of GpMADS4-like genes
sequences have been identified solely from genomic sequences (Supplementary Data Fig. S3). The known genes DAL10 of
and no evidence of transcription exists for these genes. P. abies and GpMADS4 of G. parvifolium constitute the
Alignments reveal strong similarity of at least some of these GpMADS4-subclade together with 13 other gymnosperm
gymnospermsequencestotheAGL15-andAGL17-likeMADS sequences (Supplementary Data Fig. S11). DAL21 of P. abies
sequences of angiosperms, respectively (Figs 8 and 9). forms another subclade together with three other gymnosperm
Gramzowetal.—MADS-boxgenesinconifers 1415
Angiosperm
91
DEF-like genes
42
83 Angiosperm
GLO-like genes
13
60 Angiosperm
OsMADS32-like genes D
o
w
15 n
DEF/GLO/ lo
GGM2 OsMADS32 ad
17 97CjMADS1 ed
100 CjMADS10 fro
m
42 CjMADS2 h
75 CjMADS5 ttp
CjMADS3 s
24 100 PIMADS23 ://ac
PaMADS22_DAL11 a
d
59 PtaMADS23 em
60 PaMADS25_DAL13 ic
96 93PgMADS112 .o
u
40 PrDGL p.c
PaMADS18 o
m
29 70 PmaMADS14 /a
o
30 CjMADS4 b
PaMADS137_DAL12 /artic
le
-a
49 Angiosperm bs
GGM13-like genes tra
c
20 t/11
8 4
90GGM13 /7
GgMADS9 /14
34 4P0aMPAtDaSM3A_DDSA3L564 GGM13 07/2
27 7
PmeMADS24 6
9
42 PaMADS9 04
66 4
PaMADS16_DAL23 b
87 PmeMADS19 y g
PaMADS40_DAL22 u
e
s
SEP3 t o
n
1
FsuIbGc.l5ad.ePshDyEloFg/GenLyOo/fODsEMFA/GDLSO32/OasnMdAGDGSM3123/GaGreMm1a3rk-leidkebgyelnaebse.lCleodloliunressoofnthtehebrraignhcht.eTs,hgeefnuellnyarmeseosl,vtreidanpghlyelsoagnednnyuismabvearislaabtnleoadsesSaurpepalsemdeesnctraibryedDiantaFiFgi.g2..ST6hbe. 8 N
o
v
e
m
b
sequences,termedtheDAL21-subcladehere.Thesecladesarealso number in the corresponding clade. These two subclades er 2
stableinourMrBayesphylogeny(SupplementaryDataFig.S11b). also appear in our MrBayes phylogeny (Supplementary Data 0
1
Forbothsubclades,therearethreegenessupportedbyexpression Fig. S12c). For the PaMADS19 subclade, sequences of all the 8
data (Fig. 10), where expression was found in bark, shoots, conifer species studied here were found with the exception of
stems,needles,budsandcones. thoseofS.verticillata.ThesubcladePaMADS20containsonly
sequences of P. macrophyllusandthe family Pinaceae. Inboth
StMADS11-like genes. In our phylogenies, 147 gymnosperm
subclades several species-specific duplications occurred, for
sequences cluster together with StMADS11-like genes from
example in S. sempervirens, W. nobilis, C. harringtonia,
angiosperms,ofwhich76gymnospermsequenceshaveexpres-
P.macrophyllusandspeciesofthefamilyPinaceae.Expression
sion data support (Supplementary Data Fig. S12a). There are
of gymnosperm StMADS11-like genes was found mainly in
twosubcladesofStMADS11-likegenesfromconifersinourphyl-
shoots,butalsoinothertissuessuchasroots,stems,wood,bark
ogeny(Fig.11),suggestingthattherewasaduplicationofanan-
andneedles.
cestralStMADS11-likegenenearthebaseofextantconifers.We
termed the two resulting subclades PaMADS19-like and TM3-like genes. The clade of TM3-like genes includes 310
PaMADS20-like genes after the P. abies gene with the lowest gymnosperm sequences from the species studied here, 163 of
1416 Gramzowetal.—MADS-boxgenesinconifers
4 Angiosperm
AG-like genes
GgMADS7
38
GBM5
9
CyAG
1
GBM5
17 PsMADS11
71
PtaMADS1 D
65 o
w
PtaMADS26 n
lo
PaMADS2 a
36 100 de
43 61 PgMADS108 d fro
PgMADS113 m
38 GGM3 h
87GGM3 ttp
9G0gMADS26 s://a
20 GgMADS16 cad
65PmeMADS28 em
92 PaMADS1_DAL2 ic.o
u
SAG1 DAL2 p
86 .co
PtaMADS3 m
88 /a
PmeMADS22 o
b
GgMADS33 /a
rtic
CjMADS7 le
-a
AGL15 b
s
FIG.6. PhylogenyofAG-likegenes.Coloursofthebranches,genenames,trianglesandnumbersatnodesareasdescribedinFig.2.ThreeputativesubcladesofAG-like trac
genesingymnosperms,GBM5,GGM3andDAL2,aremarkedbylabelledlinesontheright.ThefullyresolvedphylogenyisavailableasSupplementaryDataFig.S7b. t/1
1
4
/7
/1
PgMADS117 Hence,eventhoughthesheernumberofTM3-likegenesinconifers 40
7
32 60 PIMADS35 indicatesanumberofduplications,thetimingandextentofduplica- /2
7
PtaMADS10 tions cannot be determined based on our data. Transcripts of 6
39 29 PtaMADS24 TM3-like genes were isolated from various tissues, such as 90
4
58 58 PtaMADS18 shoots,stems,needles,buds,andfemaleandmalecones. 4 b
PtaMADS14 y
GGM10 TM8-likegenes.TheangiospermTM8-likegenesgroupwith188 gu
e
100 Angiosperm MADS-sequences from gymnosperms (Supplementary Data st o
AGL12-like genes Fig. S14a). For 97 sequences expression data are available. In n
1
SEP3 the reduced phylogeny including only genes with expression 8
N
data,twosubclades are evident,termedasGgMADS2-like and o
tFriIaGn.g7le.sPanhdylnougmenbyerosfaAtGnoLd1e2s-alirkeeagsedneessc.riCboeldoiunrsFoigf.t2h.eTbhraenfcuhlleysr,egseonlevendampheys-, GgMADS25-likegenes(Fig.13).ThecladeofGgMADS2con- vem
logenyisavailableasSupplementaryDataFig.S8b. tains numerous sequences of the family Pinaceae and 16 be
sequencesofG.gnemon.ThePinaceaesequencescanbefurther r 2
0
which have expression support, andfoursequences fromPinus subdivided into the subclades PaMADS15 and PaMADS24. 1
8
radiata(SupplementaryDataFig.S13).Inourreducedphylogeny Transcript-based sequences belonging to these two subclades
there are two main clades of gymnosperm sequences, one wereisolatedfrombuds,shoots,needlesandstemsandonlyone
comprising genes from the conifer species S. verticillata, sequenceofP.taedaPtaMADS27,belongingtothePaMADS15
S.sempervirens,T.baccataandC.harringtonia(Fig.12),andthe subclade, was found to be expressed in roots. The clade of
other containing sequences of G. gnemon, P. macrophyllus and GgMADS25-likegenescontainssequencesofnon-Pinaceaecon-
Pinaceae species. The clade of Pinaceae TM3-like genes can be ifers and two sequences from G. gnemon. The clades of
furthersubdividedintothreesubclades,termedDAL19-,DAL3- GgMADS25-likegenesalsoappearsinourMrBayesphylogeny
and PrMADS4-like genes here. There are a number of species- (SupplementaryDataFig.S14c).However,thegenesbelonging
specificexpansionsofTM3-likegenes,forexampleinPinaceae, totheGgMADS2-likecladeinourRAxMLphylogenyaresplit
S. verticillata, S. sempervirens and C. harringtonia. The intotwocladesthatarenotsistercladesinourMrBayesphylogeny.
MrBayesphylogenyshowsdifferentsubcladestothoseobserved Expressionofgenesfromthiscladewasalsofoundinavarietyof
in our RAxML phylogenies (Supplementary Data Fig. S13c). tissues,suchasshoots,needlesandmalestrobili.