Evolutionary history of the Afro-Madagascan Ixora species

20 Pages·2013·0.84 MB·English

AnnalsofBotany112:1723–1742,2013
doi:10.1093/aob/mct222,availableonlineatwww.aob.oxfordjournals.org
Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae): species
diversification and distribution of key morphological traits inferred from dated
molecular phylogenetic trees
J.Tosh1,2,*,S.Dessein3,S.Buerki4,I.Groeninckx1,A.Mouly5,6,B.Bremer6,E.F.Smets1,7,8andP.DeBlock3
1LaboratoryofPlantSystematics,KULeuven,KasteelparkArenberg31,POBox2437,BE-3001Leuven,Belgium,2AshdownHouse
School,ForestRow,EastSussexRH185JY,UK,3NationalBotanicGardenofBelgium,DomeinvanBouchout,BE-1860Meise,
Belgium,4JodrellLaboratory,RoyalBotanicGardens,Kew,Richmond,SurreyTW93AE,UK,5LaboratoireChrono-environnement,
UMRCNRS6249,Universite´deFranche-Comte´,16RoutedeGray,F-25030Besanc¸oncedex,France,6BergiusFoundation,Royal
SwedishAcademyofSciencesandBotanyDepartment,StockholmUniversity,SE-10691,Stockholm,Sweden,7NationalHerbarium
ofTheNetherlands,LeidenUniversityBranch,POBox9514,NL-2300RALeiden,TheNetherlandsand8NetherlandsCentrefor
D
BiodiversityNaturalis,POBox9517,NL-2300RALeiden,TheNetherlands o
w
*Forcorrespondence.E-mail:[email protected] n
lo
a
d
e
Received:9April2013 Returnedforrevision:20May2013 Accepted:6August2013 Publishedelectronically:18October2013 d fro
m
†BackgroundandAimsPreviousworkonthepantropicalgenusIxorahasrevealedanAfro-Madagascanclade,butas http
yetnostudyhasfocusedindetailontheevolutionaryhistoryandmorphologicaltrendsinthisgroup.Heretheevo- ://a
lutionaryhistoryofAfro-MadagascanIxoraspp.(acladeofapprox.80taxa)isinvestigatedandthephylogenetictrees o
b
comparedwithseveralkeymorphologicaltraitsintaxaoccurringinMadagascar. .o
x
†MethodsPhylogeneticrelationshipsofAfro-MadagascanIxoraareassessedusingsequencedatafromfourplastid fo
regions(petD,rps16,rpoB-trnCandtrnL-trnF)andnuclearribosomalexternaltranscribedspacer(ETS)andinternal rdjo
transcribed spacer (ITS) regions. The phylogenetic distribution of key morphological characters is assessed. urn
Bayesian inference (implemented in BEAST) is used to estimate the temporal origin of Ixora based on fossil als
evidence. .o
rg
†KeyResultsTwoseparatelineagesofMadagascantaxaarerecovered,oneofwhichisnestedinagroupofEast a/
Africantaxa.DivergenceinIxoraisestimatedtohavecommencedduringthemidMiocene,withextensiveclado- t S
genesisoccurringintheAfro-MadagascancladeduringthePlioceneonwards. toc
k
†ConclusionsBothlineagesofMadagascanIxoraexhibitmorphologicalinnovationsthatarerarethroughouttherest h
o
ofthegenus,includingatrendtowardspauciflorousinflorescencesandatrendtowardsextremecorollatubelength, lm
s
suggestingthatthesameecologicalandselectivepressuresareactingupontaxafrombothMadagascanlineages. U
Novelecologicalopportunitiesresultingfromclimate-inducedhabitatfragmentationandcorollatubelengthdiver- niv
sificationarelikelytohavefacilitatedspeciesradiationonMadagascar. ers
ite
Keywords:Rubiaceae,Ixora,Afro-Madagascan,molecularphylogenetics,moleculardating,biogeography,ETS, t o
n
ITS,petD,rps16,rpoB-trnC,trnL-trnF. M
a
rc
h
3
, 2
0
1
INTRODUCTION 1998). In continental Africa, Ixora mainly occurs in the 4
Guineo-Congolian Regional Centre of Endemism (RCE) (fol-
The pantropical genus Ixora is one of the largest genera in lowingWhite,1983),butalsointheAfromontanearchipelago-
Rubiaceae,withapprox.530speciesofshrubsandsmalltrees likeRCE,andextendsintotheZambezianRCE,theSwahilian
that typically grow in humid rain forest (Davis et al., 2009). RCE and the Swahilian/Maputaland regional transition zone
The centre of species diversity for the genus is in South-East (RTZ)(DeBlock,1998).InMadagascar,Rubiaceaearemostnu-
Asia,inparticularBorneo(Lorenceetal.,2007).Althoughno merousandspeciesrichintheevergreenhumidforests(Davis
modern monograph of Ixora exists, there have been a number and Bridson, 2003). Ixora is no exception to this, occurring
ofrevisionsfocusingonspecificgeographicalregions(e.g.De mostfrequentlyinthehumidevergreenforest(littoral,lowland
Block, 1998, revision of continental African Ixora spp.; De and montane) on the eastern coast of Madagascar, although
Block,2013,revisionofMadagascanIxoraspp.).Phylogenetic Ixora spp. also occur in the semi-deciduous forest of
studiesofIxorahaveprimarilyfocusedonthetribalplacement Madagascar(DeBlock,2003,2013).
and circumscription of the genus (Andreasen and Bremer, Ixora is one of the most easily recognizable genera in
1996,2000;Mouly,2007;Moulyetal.,2009a).Mostrecently, Rubiaceae,inpartduetotheoftenstrikinginflorescencesandtet-
Moulyetal.(2009b)identifiedsomewell-supported,geograph- ramerous flowers (Fig. 1). Diagnostic features for the genus
icallydefinedlineages,includingan‘Afro-Madagascan’clade. (adaptedfromDeBlock,2007)includearticulatedpetioles,nar-
Thereareapprox.80Afro-MadagascanIxoraspp.distributed rowly tubular tetramerous flowers, bilobed stigmas, bilocular
equallybetweencontinentalAfricaandMadagascar(DeBlock, ovaries and fruits (or, rarely, with more than two locules),
#TheAuthor2013.PublishedbyOxfordUniversityPressonbehalfoftheAnnalsofBotanyCompany.Allrightsreserved.
ForPermissions,pleaseemail:[email protected]
1724 Toshetal.—EvolutionaryhistoryoftheAfro-MadagascanIxoraspecies(Rubiaceae)
A B C
D E F
D
o
w
n
lo
a
d
e
d
G H fro
m
h
ttp
://a
o
b
.o
x
fo
rd
jo
u
rn
a
ls
.o
rg
I J K a/
t S
to
c
k
h
o
lm
s
U
n
iv
e
rs
ite
t o
n
M
a
rc
L M h
3
, 2
0
1
4
FIG.1. ExampleofmorphologicalvariationinIxora.(A)InflorescenceofIxoraregalis.(B)InflorescenceofIxoraelliotii.(C)InflorescenceofIxoraemirnensis.(D)
PendulousinflorescenceofIxoramangabensis.(E)InflorescenceofIxoradensithyrsa.(F)InflorescenceofIxorasiphonantha.(G)FrontviewofaflowerofIxora
guillotii.(H)FloweringbranchofIxorarakotonasoloi.(I)MaturefruitsofIxoraguillotii.(J)FruitsofIxoraquadrilocularis.(K)FloweringnodeofIxorahomolleae.
(L)ArticulatepetiolesofIxorafinlaysoniana.(M)ArticulatepetiolesofIxorahomolleae.
uniovulateloculesandseedswithalargeadaxialhilarcavity.In flowers (De Block, 1998, 2003). This is particularly the case
contrast, identification at the species level is difficult, with for the African representatives of the genus, which De Block
speciesdistinguishedonthebasisofminorandoftencontinuous (1998) described as ‘extremely homogeneous’ in theircharac-
characters,typicallyinvolvingfeaturesoftheinflorescenceand ters. On Madagascar, there are several morphological traits
Toshetal.—EvolutionaryhistoryoftheAfro-MadagascanIxoraspecies(Rubiaceae) 1725
occurringinIxorathatareabsentinthecontinentalAfricantaxa 25mL, and contained 1mL of each primer (100ng mL21),
andrareinthegenusasawhole.Theseinclude:(1)reductionof 0.35mLofBiotaqDNApolymerase,2.5mLof10×NH reac-
4
thenumberofflowersperinflorescencetowardssolitaryflowers; tionbuffer,1.5mLof50mMMgCl2,2.5mLof10mMdNTPs,
(2)increasefromtwo-tofour-locularovaries;and(3)increase 1mLofbovineserumalbumin(BSA;0.4%)and2mLoftotal
towards large flowers (corolla tubes .15cm long) and fruits genomic DNA. Amplification of petD, rps16 and trnL-trnF
(DeBlock,2007,2008,2013). used the following temperature profile: 948C for 3min; 32
Inthepresentstudy,wefurtherinvestigatethephylogenetic cycles of 948C for 1min, 508C for 1min, 728C for 1.5min;
relationships of Madagascan and continental African Ixora finalextensionof728Cfor7min.Theamplificationprofilefor
spp. using molecular sequence data from four plastid regions rpoB-trnCwas:948Cfor3min;32cyclesof948Cfor1min,
(petD,rps16,rpoB-trnCandtrnL-trnF)andnuclearribosomal 538C for 1min, 728C for 2min; final extension of 728C for
external transcribed spacer (ETS) and internal transcribed 7min.
spacer (ITS) regions. The purpose of this study is to improve PCR mixesfornuclear regions were the same as forplastid
taxon sampling of both African and Madagascan species in regions, except that 1mL of dimethylsulfoxide (DMSO) was
orderto:(1)testexistinghypothesesconcerningtheevolutionary addedper25mL.TheITSamplificationprofilewas:948Cfor
affinitieswithinandbetweenAfricanandMadagascanspecies; 3min;32cyclesof948Cfor1min,528Cfor1min,728Cfor
(2)assessthedistributionofkeymorphologicalinnovationsof 1min;finalextensionof728Cfor7min.TheETSamplification
D
the Madagascan species on our molecular phylogenetic trees; profilewas:978Cfor1min;40cyclesof978Cfor10s,558Cfor ow
n
and(3)investigatetheageofspeciesdiversificationanddispersal 30s,728Cfor30s;finalextensionof728Cfor7min.Allamp- lo
a
usingmoleculardatingtechniques. lification products were purified using Nucleospin purification d
e
d
columns and sent to Macrogen Inc. (Seoul, South Korea) for fro
sequencing. m
MATERIALS AND METHODS h
ttp
TaxonsamplingandDNApreparation Sequencealignmentandphylogeneticanalyses ://a
o
b
Extensive fieldwork was undertaken in eastern and northern Contiguous sequences were assembled and edited using the .ox
Madagascarinordertocollectherbarium,alcoholandDNAma- Stadensoftwarepackage(Stadenetal.,1998).Sequenceswere ford
terial of Madagascan Ixora spp. This material was used in the manually aligned in MacClade v. 4.04 (Maddison and jou
molecularandthemorphologicalstudy.Weincluded67Ixora Maddison,2002)withoutdifficultyduetolowlevelsofsequence rna
accessions,representingapprox.50speciesthatoccurthrough- variation.Allvariablenucleotidepositionswereverifiedagainst ls.o
oouutrtthaexognlobsaalmdpislitnrigbuitsiofnocoufstehdegoennuMsa(dTaagbalesc1a)n.IannpdarAtifcruiclaarn, tdhaetao;ripgointeanltieallelcytroinpfhoerrmogartiavmes.inGdaeplss wweerreetrceoadteeddaussminigssitnhge at Srg/
species. Where possible, we included multiple accessions for ‘simple indel coding’ method of Simmons and Ochoterena toc
each Madagascan species to test species monophyly. (2000).Tominimizethetimeandcomputationaleffortrequired kh
o
Thirty-eight Madagascan accessions were included, represent- forphylogeneticanalyses,weexcludedduplicateaccessionsofa lm
s
ing24species.Weincluded16accessions(14species)fromcon- species if the sequences from each accession were identical U
n
tinental Africa, and from west and east tropical Africa. The acrossallsixdatasets. iv
e
rMemasacianriennge 1(t2woIxsopreacieacs)c,esNsieoontrsopaircealA(sfoiaunr a(cthcreesesiosnpsecfireosm), hoCmoonggernueeintyceteostfitmhepldeamtaensetetsdwinasPAasUsePs*sevd.u4.s0inbg10th(eSwpaorftfiotirodn, rsitet o
three species) and Pacific Island (three species) taxa. 2003).Allconstantanduninformativecharacterswereexcluded. n M
Vangueria madagascariensis, representing the closely related Onethousandpermutationcycleswererun,eachconsistingofa arc
tribe Vanguerieae (e.g. Kainulainen et al., 2013) was selected heuristic maximum parsimony (MP) search of ten random se- h 3
asanoutgroup. quenceadditionreplicateswithTBR(treebisectionandrecon- , 2
0
TotalgenomicDNAwasisolatedfromeithersilicagelcollec- struction) branch swapping, holding ten trees at each step and 14
tions, fresh leaf material from the living collections of the savingnomorethanfivetreesperreplicate.
NationalBotanicGardenofBelgium(NBGB)orherbariumma- MaximumparsimonyanalyseswereperformedwithPAUP*
terial(BR,MO,P)usingastandardcetyltrimethylammonium v. 4.0b10. We conducted equal weighted parsimony heuristic
bromide(CTAB)protocol(DoyleandDoyle,1987).Asreported tree searches on: (a) individual data sets; (b) a combined
elsewhere (e.g. Rajaseger et al., 1997), Ixora leaves may be plastiddataset;and(c)acombinedplastid–nuclearDNAdata
highlycoriaceousandcancontainhighlevelsofphenoliccom- set.Eachanalysisconsistedof1000randomsequenceaddition
poundsthatmayaffectthequalityofisolatedDNA.Therefore, replicates,holdingtentreesateachstep,withTBRbranchswap-
we purified isolated DNA using Nucleospin purification pingandMulTreesineffect,DELTRANoptimizationandsaving
columns (Macherey-Nagel), following the manufacturer’s nomorethantentreesperreplicate.Supportforcladeswaseval-
instructions. uated with 1000 full-heuristic bootstrap pseudo-replicates
(Felsenstein,1985),usingthesamesettingsasoutlinedabove.
Bayesian analyses were implemented in MrBayes 3.1
Amplificationandsequencing
(HuelsenbeckandRonquist,2001).ThemodelofDNAsubstitu-
Primers for amplification of plastid and nuclear ribosomal tion for each region was determined using Modeltest v. 3.06
DNA(nrDNA)regionsarelistedinTable2.PCRandcyclese- (PosadaandCrandall,1998)undertheAkaikeinformationcri-
quencing was performed using a Perkin Elmer GeneAMPw terion(AIC;SupplementaryDataTableS1).Fourindependent
9700 thermocycler. Plastid PCR mixes were made up to Bayesian analyseswithfourchainswere runforeachdataset,
1726 Toshetal.—EvolutionaryhistoryoftheAfro-MadagascanIxoraspecies(Rubiaceae)
TABLE 1. Taxonaccessiondata(onlyfirstcollectorlistedforvoucher)
Taxon Voucher/Herbarium/Countryoforigin ETS ITS petD rps16 rpoB-trnC trnL-trnF
Vangueriamadagascariensis VanCaekenberghe82(BR),Mozambique — — HG315109 HG315176 HG315244 HG315312
J.F.Gmel.
IxoraaluminicolaSteyerm. Pre´vost4160(P),FrenchGuiana HG315378 HG315441 — HG315177 HG315245 —
I.ambrensisDeBlock Gautier5006(BR),Madagascar HG315379 HG315442 HG315110 HG315178 HG315246 HG315313
I.amplidentataDeBlock* DeBlock976(BR),Madagascar HG315380 HG315443 HG315111 HG315179 HG315247 —
I.ankazobensisDeBlock* Tosh30(BR),Madagascar HG315381 HG315444 HG315112 HG315180 HG315248 HG315314
I.ankazobensisDeBlock* Tosh245(BR),Madagascar HG315382 HG315445 HG315113 HG315181 HG315249 HG315315
I.ankazobensisDeBlock* DeBlock943(BR),Madagascar HG315383 HG315446 HG315114 HG315182 HG315250 HG315316
I.ankazobensisDeBlock* DeBlock857(BR),Madagascar HG315384 HG315447 HG315115 HG315183 HG315251 HG315317
I.ankazobensisDeBlock* Tosh256(BR),Madagascar HG315385 HG315448 HG315116 HG315184 HG315252 HG315318
I.batesiiWernham Dessein1455(BR),Cameroon — HG315449 HG315117 HG315185 HG315253 HG315319
I.borboniaeMouly& VanCaekenberghe42(BR),Mauritius HG315386 HG315450 HG315118 HG315186 HG315254 HG315320
B.Bremer
I.brachypodaDC. Bradley1022(MO),Gabon HG315387 HG315451 HG315119 HG315187 HG315255 HG315321
I.brachypodaDC. Walters1437(MO),Gabon — HG315452 HG315120 HG315188 HG315256 HG315322 D
I.brevifoliaBenth. Delpretes.n.(BR),Brazil HG315388 HG315453 HG315121 HG315189 HG315257 HG315323 ow
I.capuronianaDeBlock* Tosh400(BR),Madagascar HG315389 HG315454 HG315122 HG315190 HG315258 HG315324 nlo
I.caulifloraMontrouz. Mouly267(P),NewCaledonia HG315390 HG315455 HG315123 HG315191 HG315259 HG315325 ad
I.chinensisLam. VanCaekenberghe316(BR),China — — HG315124 HG315192 HG315260 HG315326 ed
I.collina(Montrouz.) Mouly236(P),NewCaledonia HG315391 HG315456 HG315125 HG315193 HG315261 HG315327 fro
Beauvis. m
IIB..lccorrceakmssiixpoersaBDoriavkineexDe GMroouelnyin6c5k9x(8P0),(CBoRm),oMroaIdsalagnadsscar HHGG331155339923 HHGG331155445578 HHGG331155112267 HHGG331155119945 HHGG331155226623 HHGG331155332289 http://ao
I.cremixoraDrake DeBlock987(BR),Madagascar HG315394 HG315459 HG315128 HG315196 HG315264 HG315330 b.o
I.densithyrsaDeBlock DeBlock1773(BR),Madagascar HG315395 HG315460 HG315129 HG315197 HG315265 HG315331 xfo
I.elliotiiDrakeexDeBlock DeBlock1977(BR),Madagascar HG315396 HG315461 HG315130 HG315198 HG315266 HG315332 rd
I.emirnensisBaker DeBlock1786(BR),Madagascar HG315397 HG315462 HG315131 HG315199 HG315267 HG315333 jo
u
I.emirnensisBaker DeBlock1788(BR),Madagascar HG315398 HG315463 HG315132 HG315200 HG315268 HG315334 rn
I.ferrea(Jacq.)Benth. Merello1716(MO),Commonwealthof HG315399 HG315464 HG315133 HG315201 HG315269 HG315335 als
Dominica(LesserAntilles) .org
I.ferrea(Jacq.)Benth. Taylor11693(MO),PuertoRico HG315400 HG315465 HG315134 HG315202 HG315270 HG315336 a/
I.foliosaHiern Onana566(P),Cameroon HG315401 HG315466 HG315135 HG315203 HG315271 HG315337 t S
I.foliicalyxGue´de`s Tosh352(BR),Madagascar HG315402 HG315467 HG315136 HG315204 HG315272 HG315338 to
c
I.foliicalyxGue´de`s DeBlock696(BR),Madagascar HG315403 HG315468 HG315137 HG315205 HG315273 HG315339 k
h
I.franciiSchltr. Mouly241(P),NewCaledonia HG315404 HG315469 HG315138 HG315206 HG315274 HG315340 olm
I.guillotiiHoch. DeBlock2091(BR),Madagascar HG315405 HG315470 HG315139 HG315207 HG315275 HG315341 s
I.guillotiiHoch. Tosh408B(BR),Madagascar HG315406 HG315471 HG315140 HG315208 HG315276 HG315342 U
n
I.guineensisBenth. Gereau5601(MO),Ghana HG315407 HG315472 HG315141 HG315209 HG315277 HG315343 iv
e
I.hartianaDeBlock Bamps4320(BR),Angola HG315408 HG315473 HG315142 HG315210 HG315278 HG315344 rs
I.hierniiScott-Elliot Adam23101(P),SierraLeone HG315409 HG315474 HG315143 HG315211 HG315279 HG315345 ite
I.hippoperiferaBremek. Dessein1669(BR),Cameroon HG315410 HG315475 HG315144 HG315212 HG315280 HG315346 t o
n
I.hippoperiferaBremek. VanValkenburg3083(WAG),Gabon HG315411 HG315476 HG315145 HG315213 HG315281 HG315347 M
IG.ohvoameortlsl†eaeDeBlock& Tosh107(BR),Madagascar HG315412 HG315477 HG315146 HG315214 HG315282 HG315348 arch
3
GI.ohvoameortlsl†eaeDeBlock& Tosh207(BR),Madagascar HG315413 HG315478 HG315147 HG315215 HG315283 HG315349 , 20
1
I.lagenifructaDeBlock* DeBlock2036(BR),Madagascar HG315414 HG315479 HG315148 HG315216 HG315284 HG315350 4
I.macilentaDeBlock Dessein1404(BR),Cameroon HG315415 HG315480 HG315149 HG315217 HG315285 HG315351
I.mangabensisDC. Tosh128(BR),Madagascar HG315416 HG315481 HG315150 HG315218 HG315286 HG315352
I.mangabensisDC. Tosh130(BR),Madagascar HG315417 HG315482 HG315151 HG315219 HG315287 HG315353
I.mangabensisDC. DeBlock2040(BR),Madagascar HG315418 HG315483 HG315152 HG315220 HG315288 HG315354
I.mangabensisDC. DeBlock2053(BR),Madagascar HG315419 HG315484 HG315153 HG315221 HG315289 HG315355
I.masoalensisDeBlock* Razafimandimbison654(BR),Madagascar HG315420 HG315485 HG315154 HG315222 HG315290 HG315356
I.microphyllaDrake DeBlock985(BR),Madagascar HG315421 — HG315155 HG315223 HG315291 HG315357
I.minutifloraHiern Dessein1440(BR),Cameroon HG315422 HG315486 HG315156 HG315224 HG315292 HG315358
I.mocquerysiiDC. Malcomber2805(MO),Madagascar HG315423 HG315487 HG315157 HG315225 HG315293 HG315359
I.moramangensisDeBlock* Tosh255(BR),Madagascar HG315424 — HG315158 HG315226 HG315294 HG315360
I.moramangensisDeBlock* DeBlock837(BR),Madagascar HG315425 — HG315159 HG315227 HG315295 HG315361
I.narcissodoraK.Schum. DeBlock418(BR),Kenya HG315426 HG315488 HG315160 HG315228 HG315296 HG315362
I.nematopodaK.Schum. Dessein1449(BR),Cameroon HG315427 HG315489 HG315161 HG315229 HG315297 HG315363
I.nitens(Poir.)Mouly& Friedmann2631(P),Mauritius HG315428 HG315490 HG315162 HG315230 HG315298 HG315364
B.Bremer
I.perrieriDeBlock* DeBlock841(BR),Madagascar HG315429 HG315491 HG315163 HG315231 HG315299 HG315365
I.perrieriDeBlock* Tosh232(BR),Madagascar HG315430 HG315492 HG315164 HG315232 HG315300 HG315366
I.platythyrsaBaker DeBlock773(BR),Madagascar HG315431 HG315493 HG315165 HG315233 HG315301 HG315367
Continued
Toshetal.—EvolutionaryhistoryoftheAfro-MadagascanIxoraspecies(Rubiaceae) 1727
TABLE 1. Continued
Taxon Voucher/Herbarium/Countryoforigin ETS ITS petD rps16 rpoB-trnC trnL-trnF
I.praetermissaDeBlock Dessein1519(BR),Cameroon HG315432 HG315494 HG315166 HG315234 HG315302 HG315368
I.quadrilocularisDeBlock* Tosh85(BR),Madagascar HG315433 HG315495 HG315167 HG315235 HG315303 HG315369
I.rakotonasoloiDeBlock Tosh316(BR),Madagascar HG315434 HG315496 HG315168 HG315236 HG315304 HG315370
I.regalisDeBlock* DeBlock835(BR),Madagascar HG315435 HG315497 HG315169 HG315237 HG315305 HG315371
I.regalisDeBlock* DeBlock2083(BR),Madagascar HG315436 — HG315170 HG315238 HG315306 HG315372
I.scheffleriK.Schum. Luke9162(P),Tanzania HG315437 HG315498 HG315171 HG315239 HG315307 HG315373
I.siphonanthaOliv. Tosh389(BR),Madagascar HG315438 HG315499 HG315172 HG315240 HG315308 HG315374
I.sp.‘Brunei’ Malcomber2980(MO),Brunei — HG315500 HG315173 HG315241 HG315309 HG315375
I.sp.‘Malaysia’ Billiet7327(BR),Malaysia HG315439 HG315501 HG315174 HG315242 HG315310 HG315376
I.tanzaniensisBridson Luke9304(P),Tanzania HG315440 HG315502 HG315175 HG315243 HG315311 HG315377
*sp.nov.ined.
†nom.nov.ined.
D
o
w
n
TABLE 2. Amplificationprimersforplastidandnuclearregions lo
a
d
e
d
Region Primer Primersequence(5’–3’) Reference fro
m
prpeotDB-trnC PPrpeeottBDB1-7F33685 CTATAAGCTACTCTCAYRGGCCGAYTTTTCYTTTTGTAAAAATTCAATGCGTAGTGGTGGAC LSho¨ahwneeatnadl.B(2o0r0sc5h)(2005) http://a
o
trnC-R CKACAAAAYCCYTCRAATTG b.o
rps16 rps16-F AAACGATGTGGTARAAAGCAAC Shawetal.(2005) x
fo
rps16-R AACATCWATTGCAASGATTCGATA rd
trnL–trnF trnL-c CGAAATCGGTAGACGCTACG Taberletetal.(1991) jo
u
trnF-f ATTTGAACTGGTGACACGAG rn
ETS 18S-ETS GCAGGATCAACCAGGTGACA Negro´n-OrtizandWatson(2002) als
ETS-ERIT CTTGTATGGGTTGGTTGGA BaldwinandMarkos(1998) .o
rg
ITS ITS1 TCCGTAGGTGAACCTGCGG Whiteetal.(1990) a/
ITS4 TCCTCCGCTTATTGATATGC t S
to
c
k
h
o
lm
s
startingfromrandomtrees,for5milliongenerations,sampling assessed the distribution of these morphological characters by U
n
trees every 1000 generations. TRACER v. 1.4 (Rambaut and mapping unambiguous character state changes onto our com- iv
e
Drummond,2007)wasusedtoassessifthesearchhadreached bined Bayesian MJ consensus tree using MacClade v. 4.04 rs
ite
stationarity and to check each parameter had an effective (MaddisonandMaddison,2002). t o
sample size (ESS) .100. The initial 1250 (25%) trees were n M
discardedasaconservativeburn-in.Post-burn-intreesfromthe a
four independent analyses were pooled and summarized by a Divergencetimeestimation rch 3
50% majority rule consensus tree using PAUP* v4.0b10 to ABayesianapproachwasappliedtoinferthetemporalframe- , 2
0
obtainposteriorprobabilities(PPs). workoftheevolutionofIxora.Duetothelimitedfossilrecord 14
that could be unequivocally assigned to the most recent
common ancestor of Ixora, an expanded family-level data set
Morphologyandoptimizationofmorphologicalcharacters
was constructed and divergence time estimation was inferred
Thematerialforthetaxonomicalandmorphologicalstudyof based on fossils (see the following section for more detail).
IxorainMadagascarconsistsofpreservedsamplesandherbar- This large-scale analysis allowed estimation of the temporal
iummaterialofthefollowinginstitutions:BM,BR,G,K,MO, originofIxoratogetherwitha95%confidenceinterval,which
P,S,TAN,TEF,UPS,W,WAGandZ(abbreviationsofinstitu- wassubsequentlyusedasapriortoperformasecondanalysisfo-
tionsfollowHolmgrenetal.,1990).Intotal,.1000herbarium cusing only on this genus. The family-level data set included
collections, each with several duplicates, were studied. representatives of all the major lineages in Rubiaceae (see
Morphological terminology generally follows Robbrecht Appendix 1) and was based on the group II plastid introns
(1988).FortheMadagascanspecies,morphologicalcharacters petD, rps16 and the trnL-trnF spacer (Groeninckx, 2009). In
were scored on herbarium material available for this species. the large-scale analysis, a sub-set of taxa representative of the
For the continental African species, morphological characters main clades in Ixora was included. These taxa were selected
were taken from the revision of African Ixora (De Block, basedonpreliminaryphylogeneticanalyses.Weoptedforthis
1998).Wechosemorphologicaltraitsofinterest(i.e.uniflorous approachratherthanpoolingallthedatasetsandtaxatogether
inflorescences,four-locularovaries,extremecorollatubelength) mainlytoavoidencounteringproblemsrelatedtomissingdata.
andcharactersofpotentialtaxonomicsignificance(Table3).We In addition to the group II plastid introns, the Ixora data set
1
7
2
8
TABLE 3. Selectedmorphologicalcharacters
Inflorescence Calyx Flower
Taxon Sessile/pedunculate Lax/compact Tubelength(mm) Lobelength(mm) Number Corollatubelength(cm) Ovary T
o
s
Africantaxa I.batesii Sessile Lax 0.4–0.5 0.4–0.6 30–70 0.5–1.3 Bilocular h
e
I.brachypoda Pedunculate Lax 0.4–0.8 0.3–1.0 50–200 3.3–11 Bilocular t
a
I.foliosa Pedunculate Compact 0.3–0.5 0.4–0.7 45–90 0.9–2.3 Bilocular l.
I.guineensis Sessile Lax 0.3–0.8 0.2–0.6 30–90 0.5–2.3 Bilocular —
I.hartiana Pedunculate Lax 0.2–0.5 0.3–0.6 9–30 1.2–2.4 Bilocular E
I.hiernii Sub-sessile Lax 0.2–0.7 0.2–0.5 30–90 1.4–3.3 Bilocular vo
I.hippoperifera Pedunculateorsessile Compact 0.2–0.4 0.2–0.4 30–120 1.6–2.7 Bilocular lu
I.macilenta Sessile Lax 0.2–0.5 0.2–0.6 9–20 0.9–2.2 Bilocular tio
I.minutiflora Sessile Compact 0.2–0.4 0.1–0.4 9–30 1.0–2.0 Bilocular n
a
I.narcissodora Sessile Lax 0.8–1.5 0.2–0.6 50–100 3.0–7.5 Bilocular r
y
I.nematopoda Pedunculate Lax 0.3–0.6 0.8–1.2 20–50 0,4–0,9 Bilocular h
I.praetermissa Sessile Lax 0.2–0.5 0.3–0.8 30–70 1.1–2.5 Bilocular is
I.scheffleri Pedunculate Compact 0.4–0.6 0.6–1.0 30–90 0.8–2.2 Bilocular tor
I.tanzaniensis Sessile Compact 0.3–0.7 0.2–0.4 20–50 2.2–3.2 Bilocular y
o
Madagascantaxa(Clade2) II..admenpsliitdheynrtsaata PPeedduunnccuullaattee LCaoxmpact 00..25––01..40 02..55––41..025 509––16200 11.58––23.33 BBiillooccuullaarr fthe
I.foliicalyx Sub-sessile Compact 2.0–4.0 1.5–6.0 45–90 3.5–8.5 Bilocular A
I.guillotii Pedunculate Lax 0.5–1.0 0.75–2.5 50–150 5.0–8.0 Bilocular fro
I.homolleae (Sub-)sessile Uniflorous 5.0–10 5.0–12 1 2.6–4.7 Four-locular -M
I.lagenifructa Pedunculate Lax 5.0–8.0 1.5–2.0 3(–15) 3.5–6.5 Four-locular a
I.microphylla (Sub-)sessile Lax 0.5–2.0 0.5–1.5 (1–)3–9 2.2–4.0 Bilocular da
I.mocquerysii Pedunculate Compact 0.5–1.0 2.0–6.0 3–45 5.5–11(–16) Bilocular g
a
I.platythyrsa Pedunculate Lax 0.2–0.4 0.9–1.4 50–150 1.4–2.5 Bilocular s
c
I.quadrilocularis Pedunculate Lax 4.0–7.0 (3–)5–15 3(–9) 4.0–8.0 Four-locular a
n
I.regalis Pedunculate Lax 0.5–1(–1.5) 1–5(–9) 50–120 1.3–5.5 Bilocular I
I.siphonantha Pedunculate Lax 0.5–1 3.0–11 12–80 (10–)15–22 Bilocular xo
r
Madagascantaxa(Clade3) I.ambrensis Pedunculate Lax 0.4–0.5 0.2–0.35 25–50 3.2–3.9 Bilocular a
s
I.ankazobensis Sessile(+pedunculate) Lax 0.5–0.75 0.3–0.75 7–18 4.0–6.3 Bilocular p
e
I.capuroniana Sessile Laxorcompact 0.3–0.6 0.1–0.8 40–90 1.5–3.5 Bilocular c
I.crassipes Sub-sessile Lax 0.75–1.25 0 15–50 17–22.5 Bilocular ies
I.cremixora Sessile Lax 0.2–0.75 ,0.3 50–120 4.5–7.8 Bilocular (R
I.elliotii Sessile Lax 0.2–0.4 0.3–0.5 30–90 1.8–3.1 Bilocular u
I.emirnensis Pedunculate Lax 0.2–0.3 0.3–0.4 9–50 1.0–1.5 Bilocular bia
I.mangabensis Pedunculate Lax 0.2–0.5 0.5–1.0 9–30 2.0–3.2 Bilocular c
e
I.masoalensis Sessile Lax 0.5–2.0 0.5–1.5 8–25 2.2–4.0 Bilocular a
I.moramangensis Pedunculate Lax 0.25–0.5 0.25–0.8 9–30 2.1–3.5 Bilocular e)
I.perrieri Pedunculate Lax 0.3–0.5 0.2–0.4 30–90 4.5–7.5 Bilocular
I.rakotonasoloi Sessile Uniflorous 0.3–0.5 0.3–0.5 1 2.2–2.8 Bilocular
AdaptedfromDeBlock(1998,2013).
4102 ,3 hcraM no tetisrevinU smlohkcotS ta /gro.slanruojdrofxo.boa//:ptth morf dedaolnwoD
Toshetal.—EvolutionaryhistoryoftheAfro-MadagascanIxoraspecies(Rubiaceae) 1729
included one additional plastid (rpoB-trnC) and two nuclear basedonfossilrecordsofpollenfromtheOligocene(Graham,
(ETSandITS)regions.Theseregionsprovedusefultoresolve 2009);(d)thestemoftheGaliumandRubiacladewasassigned
phylogeneticrelationshipsinIxorafurther. anoffsetof5.3Ma,followingthefirstoccurrenceoffossilpollen
With the exception of the numberof runs and length of the duringthelateMioceneforthisgroup(Graham,2009);(e)the
Monte Carlo Markov chain (MCMC), the settings of the stem of the Chiococca clade was calibrated with an offset of
Bayesiananalyseswereidenticalforthetwodatasets.Aparti- 5.3MabasedonleafmaterialfromthelateMiocene(Graham,
tioned Bayesian MCMC analysis was conducted in BEAST 2009);(f )thestemoftheEmmenopteryscladewassetwithan
v.1.7witharelaxedlog-normalmolecularclockandaYulespe- offset of 48Ma, following the first record of Rubiaceae fruits
ciationmodel.Thepartitionswereunlinkedforthemodelofevo- in the mid Eocene (Graham, 2009); and (g) an offset of
lution,butlinkedfortheestimationofthemolecularclockand 14.5Ma was assigned to the Gardenia clade, based on pollen
thetreetopology.Theotherparametersweresetasdefault(see datafromthemidMiocene(Graham,2009).Basedontheposter-
Drummond and Rambaut, 2007). The best-fit models foreach iordistributionofthedatinguncertaintyonthestemofIxora,the
DNAregionwerekeptidenticaltothoseintheMrBayesanalyses most recent common ancestor of the ingroup in the second
(seeabove).Inthecaseofthefamily-leveldataset,fourrunsof BEASTanalysiswassetwithanormaldistribution,ameanof
10 million generations were performed, sampling a tree every 18Maandans.d.of2.5.
1000 generations. To improve the tree topology research, the
D
MrBayesconsensustreewasprovidedasthestartingtreeinthe ow
n
BEASTfamily-levelanalysis.InthecaseoftheIxoradataset RESULTS lo
a
(which was rooted using Vangueria infausta), two runs of 5 d
The368novelsequencesgeneratedinthisstudywerecombined ed
million generations were performed, sampling a tree every with31sequencespreviouslygeneratedbyMouly(2007),result- fro
1000generations,andtheanalyseswereseededwitharandom m
tree. For each parameter, convergence of runs was confirmed ianpgprionxa.t5o0talIcxoomrabisnpepd.dLaetaveslestooff3g9e9nseetqicuevnacreias,tiroenprebseetwnteinegn http
bytheexaminationoftheirrespectiveposteriordistributionsin species were generally low across all six regions investigated ://a
TRACERv.1.4(RambautandDrummond,2004).Inaddition, ob
we considered the MCMC sampling sufficient when the ESS (Table4).Thetotalnumberofpotentiallyparsimonyinformative .ox
ow2lTen0rane0sgte4htA.)he.sns2Anae0ont0mtdoaufta9osxt5ririnemv%ge.us1Tmca.oR5fnt.cAe4filraCd(dbDeEeunrRruccnemr-veii.mdnni1tob(e.fni4rolvdirt(ayaRlenaaotdcrmnehRebnraaowumudnitetb,hsaaanuwmbdta,uesD2rdn0bira-0uuin7nmil)tbpmbrueaaosrnsiinnoecddhdg, fIhtpceonhiergrtastDhre1(aer13c(ms32ttIes..px43rrosoo%%rfrpaa))pon..sergTOptreicphoude.enrnfWlrttooaoafwxemgpoeeoo2nsbvtt1essapainernemritraivrcapbpeellidslnily1nitnt6agpyogaa,iernnitsnhdcvitemlatrurarEodnisaneTLpbdye-SitclmiriipnntfiuyrfFocolwtvrtismoepaed5lsqae8toutiaobevicnnsehcetcceahrehvsveseaevIirdTaotarhnSicines--. afordjournals.org/
of1millionwasapplied). ation in nine of these species. In contrast, varying amounts of t Sto
intraspecific sequence divergence were observed in the other ck
h
Fossilcalibration five species; multiple accessions from these five species were olm
Toestimateabsoluteagesforlineagedivergencesinthecase includedinallsubsequentphylogeneticanalyses. s U
n
ofthefamily-leveldataset,wehaveusedsevenfossilcalibration iv
e
points(seebelow)inRubiaceaetosetageconstraintsforseveral Phylogeneticanalyses rsite
nodes.AsinBuerkietal.(2011),foreachcalibrationpoint,the t o
n
oldest fossil record was selected and the upper (younger) Plastiddatasets.Phylogeneticanalysesofindividualplastiddata M
bound of the geological interval (Gradstein et al., 2004) in sets(petD,rpoB-trnC,rps16,trnL-trnF)generatedlargelyunre- arc
which the fossil was foundwasused torepresent theage con- solvedandpoorlysupportedphylogenetictrees(datanotshown). h 3
straint.Inthefamily-levelBayesiananalysis,allthecalibration Thepartitionhomogeneitytestdidnotrevealanysignificantin- , 2
0
points were modelled as follows: log-normal distribution, congruencebetweenplastiddatasets(P¼0.69)andtherewas 14
mean¼0.5,s.d.¼1,offset¼agefossil(seebelow). nosupportedincongruence(BayesianPP.0.95),sothesefour
Beforedetailingthefossilsusedhere,wewouldliketoreport regionswerecombinedforallsubsequentanalyses.
thecurrentknowledgeonfossilrecordsthathavebeententatively Thecharacteristicsoftheindividualandcombinedplastiddata
assignedtoIxora.PalaeobotanicalremainsofIxoraarepoorly setsarelistedinTable4.InboththeMPandBayesiananalyses
known, and Graham (2009) reported the presence of Ixora (Fig.2),theAsianandPacificOceantaxaaresistertotherest
pollen dating from the Miocene on the Marshall Islands of the genus [bootstrap (BS) 70, PP 1.00]. The Mascarene
(Micronesia).Inhisstudy,Graham(2009)stressedthatthisevi- Ixora clade is in turn sister to the Neotropical and
dencecouldbechallengedandthatfurtherstudieshavetobecon- Afro-MadagascanIxoraspp.(BS77,PP1.00).Thesisterrela-
ducted to assign these pollen unequivocally to existing taxa. tionship between the three Neotropical Central–South
Therefore,wehavenotconsideredthisrecordinourdivergence AmericantaxaandtheAfro-Madagascantaxaisweaklytomod-
timeestimations.Thefollowingcalibrationpointswereselected: eratelysupportedintheMP(BS56)andBayesian(PP0.96)ana-
(a) the stem group of Rubiaceae was set with an offset of 54 lyses (Fig. 2). In the Afro-Madagascan group, we identified a
millionyears(Ma),followingthefirstoccurrenceofRubiaceae clade containing African and Madagascan taxa (BS 72, PP
fossils during the Eocene (Malcomber, 2002); (b) the stem of 1.00; Clade 1) and an exclusively Madagascan clade (BS 59,
theFarameacladewassetwithanoffsetof40Ma,basedondis- PP0.99;Clade2).Thereisalackofresolutionandweaknode
tinctivediporatepollenfromthelateEocene(Graham,2009);(c) supportinClades1and2duetoapaucityofpotentiallyparsi-
theCoprosmacladewasconstrainedwithanoffsetof23.8Ma mony informative characters (Table 4). In Clade 1 there is
1730 Toshetal.—EvolutionaryhistoryoftheAfro-MadagascanIxoraspecies(Rubiaceae)
TABLE 4. Characteristicsofindividualandcombineddatasetsandtreestatistics
petD trnL-trnF rps16 rpoB-trnC Plastid ETS ITS nrDNA Total
Numberoftaxa 56 57 57 55 57 52 52 49 57
Totallength(bp) 1023 828 722 1016 3589 409 631 1040 4629
Non-parsimonyinformative 49 41 46 75 211 51 71 122 333
characters
Potentiallyparsimonyinformative 24(2.3%) 21(2.5%) 21(2.9%) 33(3.2%) 99(2.7%) 55(13.4%) 58(9.2%) 108(10.4%) 212(4.6%)
characters(%oftotal)
Indels 3 2 1 3 9 0 0 0 9
Treelength 81 68 76 140 376 159 196 357 758
Consistencyindex 0.914 0.956 0.921 0.850 0.872 0.780 0.791 0.759 0.801
Retentionindex 0.955 0.969 0.917 0.811 0.889 0.898 0.819 0.839 0.849
Numberoftreessaved 100 68 511 4887 8722 1151 9580 3464 5362
D
strong support for a clade of Madagascan and tropical East 1 is supported in both the Bayesian and MP analyses (BS 73, ow
Africantaxa(BS85,PP1.00;Clade3). PP1.00)followingtheexclusionofI.nematopoda(notshown). nlo
a
In Clade 1, the group of I. foliosa and I. hippoperifera is d
e
snerqDuNeAncedsatfaorseVtas.ngWueeriwaemreaduangaabslceartioenosbist,aidnueEtToSamanpdlifiIcTaS- wiceaalkWlyesstu–pCpoenrtterdal(ABfSri5c7a,nPtPax0a.9a2re),wbeultltswupopoothrteerdg:rIo.guupisnoefetnrosips-, d from
tAiosn oduifrficpurlitmiesaryandinatmerbesigtsuocuosnsceeqrunentcheereAadfrso,-rMesapdeacgtaivseclayn. II..hmairntuiatinfao,liIa.,mI.achiileernntiaiaannddII..pbraaetetesirim(iBssSa9(B2S, P8P5,1P.0P01).0a0n)d. http://a
elementofIxora,phylogeneticanalysesfromseparateandcom- Supportforthesisterrelationshipsbetween thesethreeWest– ob
bined nuclear DNA analyses were rooted using Asian and/or Central African clades, and between the widespread .ox
Pacific Ocean Ixora spp. Relationships in the Afro- I. brachypoda, is weak or lacking (Fig. 3). Branch lengths ford
Madagascan Clade were not affected by the root choice. The between these clades are short, which may be a contributing jo
u
characteristicsofthenuclearDNAdatasetsarelistedinTable4. factor in the phylogenetic uncertainty between these West– rna
The topologyof the ETS phylogenetic tree (Supplementary CentralAfricanspecies(SupplementaryDataFig.S3). ls.o
DthaetraeFarieg.soSm1)eidsisfifmerielnarcetos.tChlaatdoefst1heancdom2abriennedotpflualsltyidretcroeev,ebreudt PPN1e.s0t0e)d,icnoCmlpardiese1diosfthEeassttroAnfgrliycasnupapnodrtMedadCalgadasec3an(BsSpe1c0ie0s, at Srg/
in the ETS topology, with the deepest nodes in the (Fig. 3). The branch length subtending Clade 3 is relatively toc
k
Afro-Madagascan group being unresolved. Furthermore, there long, with eight synapomorphies (Supplementary Data Fig. h
o
isweaktomoderatesupport(BS78,PP0.93)forthesisterrela- S3).InClade3,theEastAfricanI.narcissodoraandI.tanzanien- lm
s
tionshipoftheGuineo-CongolianI.nematopodawithrespectto sis are sister to the Madagascan taxa (BS 66, PP 0.94). U
n
Neotropical Ixora and the remaining Afro-Madagascan Ixora PhylogeneticrelationshipsbetweentheseMadagascantaxaare iv
e
s(duipspcourstseeddibnytMheoEuTlySettoapl.o,l2o0g0y9(bB).SH9o4w,ePvPer1,.C0l0a)d.eT3heisIsTtrSontogply- pHooowrleyverr,etshoelrveedare(aFfigew. s3p;ecSieusprpelleamtioennstahriypstDhaattaareFwige.llsSu3p)-. rsitet o
ologyispoorlyresolvedandweaklysupported,inparticularin ported,suchasI.elliotiiandI.rakotonasoloi (BS99,PP1.00) n M
the MP strict consensus (Supplementary Data Fig. S2). Few andI.crassipesandI.cremixora(BS98,PP1.00).Theacces- arc
clades are well supported in both MP and Bayesian analyses, sionsofI.mangabensisdidnotgrouptogetherinouranalyses, h 3
andClades1–3werenotrecovered. due to the presence of two distinct plastid haplotypes in the , 2
0
fouraccessionsofthisspecies. 14
Totalcombineddataset.Despitesometopologicalinconsisten- The monophyly of Clade 2, comprised exclusively of
ciesbetweenthecombinedplastidandnuclearDNAdatasets, Madagascan taxa, is supported in the combined plastid–
results from the simultaneous partition homogeneity test did nuclearDNAdataset(BS70,PP1.00).Therearetwomainsub-
not reveal significant incongruence between data sets (P¼ cladesrecoveredintheMPstrictconsensusandBayesianmajor-
0.10).Thecombinedplastid–nuclearDNAdatasetcomprised ityruleconsensustrees,althoughsupportforthesesub-cladesis
4629characters.Intotal,212ofthe545variablecharactersare lacking (Fig. 3). Although the partition homogeneity test
potentiallyparsimonyinformative(Table4). revealed no significant incongruence between individual data
The Mascarene clade is sister to the Neotropical and sets, the phylogenetic placement of certain taxa (e.g.
Afro-Madagascanspecies(BS84,PP1.00).Themonophylyof I. densithyrsa and I. regalis 2) differed between plastid and
theAfro-Madagascangroupissupported(BS65,PP1.00),and nucleardatasets.Intheplastidphylogenetictree,I.regalis2is
in this group there is support for Clades 1–3 (Fig. 3). In the nested in the group containing I. regalis 1, I. guillotii and
Bayesian analysis, I. nematopoda and I. scheffleri are sister to I. amplidentata (Fig. 2). In contrast, I. regalis 2 is unresolved
therestofClade1(PP1.00).Althoughthisrelationshipisrecov- in the ETS phylogenetic tree (Supplementary Data Fig. S1)
eredintheMPstrictconsensus,itisnotsupportedbythebootstrap andsistertoI.homolleaeandI.quadrilocularisintheITSphylo-
analysis. The phylogenetic position of I. nematopoda differs genetic tree (Supplementary Data Fig. S2). Similarly,
between the ETS and the plastid and ITS data sets (Fig. 2; I. densithyrsa andI. mocquerysii demonstratedifferingphylo-
SupplementaryDataFigsS1andS2).ThemonophylyofClade genetic affinities in the plastid and nuclear data sets (Fig. 2;
Toshetal.—EvolutionaryhistoryoftheAfro-MadagascanIxoraspecies(Rubiaceae) 1731
V. madagascariensis
0·73/* I. aluminicola
1·00/56 I. ferrea Neotropical lxora
I. brevifolia
I. amplidentata
0·62/* I. guillotti
1·00/78 I. regalis 1
I. regalis 2
I. lagenifructa
1·00/75 II.. dheonmsoitlhleyarsea 1 e 2 Madagascan lxora
0·91/* d
0·99/59 0·98/60 II.. hqoumadorlilleoaceu la2ris Cla
I. mocquerysii
1·00/77 I. foliicalyx
1·00/71 I. microphylla
I. platythyrsa
I. siphonantha
I. ankazobensis 1 D
1·00/85 I. perrieri ow
I. ankazobensis 2 nlo
I. ankazobensis 3 ad
1·00/86 I. capuroniana ed
1·00/64 I. masoalensis fro
I. emirnensis m
0·96/56 1.00/85 1·00/62 III... mccrraaesnmsgiixapoberesan 1sis 1 ade 3 MEaasdta Agfarisccaann l xaonrda http://ao
I. cremixora 2 Cl b.o
1·00/96 I. elliotii xfo
I. rakotonasoloi rd
1·00/70 0·99/52 II.. mmaonragmabaenngseinss 2is e 1 journa
1·00/99 III... antamanrbzcarisensnioesdnisosrisa Clad als.org/
I. batesii t S
1·00/66 I. guineensis toc
k
1·00/67 I. hiernii h
o
I. minutiflora lm
1·00/100 I. brachypoda 1 s U
I. brachypoda 2 n
West and Central iv
1·00/100 1·00/72 0·86/54 II.. fhoipliopsoaperifera African lxora ersite
0·91/* I. hartiana t o
1·00/57 I. macilenta n M
I. praetermissa a
0·70/* I. nematopoda rch
I. scheffleri 3
1·00/100 I. nitens Mascarene lxora , 20
1
I. borboniae 4
I. cauliflora
1·00/100
I. collina Pacific lxora
0·99/60 I. francii
0·59/50 I. chinensis
1·00/97 I. sp. Malaysia Asian lxora
I. sp. Brunei
FIG.2. CombinedplastidBayesianmajorityruleconsensustree.Bayesianposteriorprobabilities.0.5andbootstrapvalues.50%areindicatedabovebranches(PP/
BS).Asterisks(*)denotenodesthathavebootstrapsupport,50intheMPanalysis.Clade1,‘Afro-Madagascanclade’;Clade2,‘Madagascanclade’;Clade3,‘East
African–Madagascanclade’.
Supplementary Data Figs S1 and S2). Exclusion of I. regalis I. regalis 1, I. guillotii, I. amplidentata, I. lagenifructa,
2andI.densithyrsa(datanotshown)fromtheBayesiananalyses I.siphonanthaandI.mocquerysii(PP0.95;notshown).
results in increased support forthe twosub-cladesof Clade 2,
with both sub-clades supported by a PP of 1.00. In the
Distributionofkeymorphologicalcharacters
first subclade, I. foliicalyx is sister to I. homolleae and
I.quadrilocularis(PP1.00;notshown).Inthesecondsub-clade, The phylogenetic distribution of key morphological charac-
I. microphylla and I. platythyrsa are sister to the group of tersisillustratedinFig.4.Pedunculateandsessileinflorescences
1732 Toshetal.—EvolutionaryhistoryoftheAfro-MadagascanIxoraspecies(Rubiaceae)
V. madagascariensis
1·00/84 I. aluminicola
0·64/* I. brevifolia Neotropical lxora
I. ferrea
0·92/* I. amplidentata
0·88/* I. lagenifructa
0·56/* I. guillotii
0·66/* I. regalis 1
0·67/* I. densithyrsa 2
0·69/* I. mocquerysii e Madagascan lxora
I. siphonantha d
1·00/85 I. microphylla a
1·00/70 I. platythyrsa Cl
I. foliicalyx
0·72/* 1·00/95 I. homolleae 1
1·00/84 1·00/99 I. homolleae 2
0·54/* I. quadrilocularis
I. regalis 2
1·00/83 I. ankazobensis 1
0·94/* I. ankazobensis 2
1·00/54 I. ankazobensis 3
I. perrieri
1·00/98 I. capuroniana
0·99/* I. masoalensis Do
I. emirnensis 3 w
0·94/66 1·00/98 II.. mcraasnsaigpaebsensis 1 de MEaasdta Agfarisccaann l xaonrda nloa
1·00/65 11··0000//9896 III... ccerrlleeiommtiiiixxoorraa 12 Cla ded fro
1·00/100 I. rakotonasoloi m
I. mangabensis 2 1 h
1·00/78 I. moramangensis e ttp
0·55/* 0·60/* 1·00/851·10·00/01/0808 IIIII..... anthmamaaanrrbczctiiarialseennsnniaoestdanisosrisa Clad ://aob.oxfo
I. praetermissa rd
0·55/* I. batesii jou
0·81/53 1·00/92 0·54/* I. hiernii rn
1·00/100 1·00/89 1·00/100 III... mgburianincuehtieyflpnoosraidsa 1 WAferiscta ann lxdo Craentral als.org
1·00/* 0·92/57 II.. bforlaiocshaypoda 2 at S/
I. hippoperifera to
0·77/* I. nematopoda c
k
I. scheffleri h
o
1·00/100 I. nitens Mascarene lxora lm
I. borboniae s
1·00/100 I. cauliflora U
0·67/60 I. collina Pacific lxora niv
0·92/61 I. francii e
1·00/970·89/59 II.. cshp.i nMeanlsaiyssia Asian lxora rsite
I. sp. Brunei t o
n
FIG. 3. Combinedplastid–nuclearBayesianmajorityruleconsensus.Bayesianposteriorprobabilities.0.5andbootstrapvalues.50%areindicatedabove Ma
branches.Asterisks(*)denotenodesthathavebootstrapsupport,50intheMPanalysis.Clade1,‘Afro-Madagascanclade’;Clade2,‘Madagascanclade’;Clade rc
h
3,‘EastAfrican–Madagascanclade’. 3
, 2
0
1
4
occur in African taxa and both clades of Madagascan the Miocene–Pliocene boundary. Based on the three-gene
taxa (Fig. 4A). All taxa from Clade 2 and the African Rubiaceae-wide data set (Supplementary Data Fig. S4, Table
I. nematopoda and the Madagascan I. masoalensis (Clade3) S2),thecrownageforIxoreae(node152)isestimatedat16.67
possess calyx lobes .1mm long (Fig. 4B). Calyx tubes millionyearsold(9.67–27.55,95%highestposteriordensity;
.1mmlongarefoundinallbuttwospeciesfromClade2,in hereafter HPD). The subsequent age estimates (discussed
addition to the African I. narcissodora and the Madagascan below)arebasedontheresultsofthesecondarydatinganalysis
I.masoalensisandI.crassipes(Fig.4C).Specieswithuniflorous (Fig.5;SupplementaryDataTableS3).Theonsetofdivergence
inflorescences or corolla tubes .15cm long occur in both betweentheAsian-PacificOceanIxora(node59)andtherestof
Madagascan clades, but species with four-locular ovaries are the genus is estimated at 15.37Ma (7.39–22.89, 95% HPD).
onlyfoundinClade2(Fig.4D–F). The estimated age of divergence between the Neotropical and
Afro-Madagascan taxa (node 66) is 9.51Ma (4.47–14.94, 95
% HPD), with the crown age for the Afro-Madagascan group
Divergencetimeestimation (node 67) estimated at 7.95 million years old (3.71–12.52, 95
Theestimateddivergencetimesfornodesofinterestaresum- % HPD). The crown age of Clade 1 (node 68) is estimated at
marizedinFig.5andSupplementaryDataFig.S4,TablesS2and 7.22 million years old (3.36–11.46, 95% HPD). The crown
S3.Theonsetofdiversificationofthegenusstartedduringthe ageofClade2(node98)isestimatedat6.24millionyearsold
mid Miocene, with the emergence of most of the lineages at (2.88–10.03, 95% HPD), with two separate periods of