Nuclear and plastid haplotypes suggest rapid diploid and polyploid speciation in the N Hemisphere Achillea millefolium complex (Asteraceae).

0.74 MB·English

by  Guo, Yan-Ping

Guoetal.BMCEvolutionaryBiology2012,12:2
http://www.biomedcentral.com/1471-2148/12/2
RESEARCH ARTICLE Open Access
Nuclear and plastid haplotypes suggest rapid
diploid and polyploid speciation in the N
Achillea millefolium
Hemisphere complex
(Asteraceae)
Yan-Ping Guo1*, Shuai-Zhen Wang1, Claus Vogl2 and Friedrich Ehrendorfer3
Abstract
Background: Species complexes or aggregates consist of a set of closely related species often of different ploidy
levels, whose relationships are difficult to reconstruct. The N Hemisphere Achillea millefolium aggregate exhibits
complex morphological and genetic variation and a broad ecological amplitude. To understand its evolutionary
history, we study sequence variation at two nuclear genes and three plastid loci across the natural distribution of
this species complex and compare the patterns of such variations to the species tree inferred earlier from AFLP
data.
Results: Among the diploid species of A. millefolium agg., gene trees of the two nuclear loci, ncpGS and SBP, and
the combined plastid fragments are incongruent with each other and with the AFLP tree likely due to incomplete
lineage sorting or secondary introgression. In spite of the large distributional range, no isolation by distance is
found. Furthermore, there is evidence for intragenic recombination in the ncpGS gene. An analysis using a
probabilistic model for population demographic history indicates large ancestral effective population sizes and
short intervals between speciation events. Such a scenario explains the incongruence of the gene trees and
species tree we observe. The relationships are particularly complex in the polyploid members of A. millefolium agg.
Conclusions: The present study indicates that the diploid members of A. millefolium agg. share a large part of their
molecular genetic variation. The findings of little lineage sorting and lack of isolation by distance is likely due to
short intervals between speciation events and close proximity of ancestral populations. While previous AFLP data
provide species trees congruent with earlier morphological classification and phylogeographic considerations, the
present sequence data are not suited to recover the relationships of diploid species in A. millefolium agg. For the
polyploid taxa many hybrid links and introgression from the diploids are suggested.
Background Hemisphere common yarrow taxa form such a complex,
Speciescomplexesoraggregatesconsistofasetofclosely i.e., the Achillea millefolium aggregate. Centered in SE
relatedspeciesoftenofdifferentploidylevels,whoserela- Europe and SW to C Asia, its diploid species are limited
tionships are difficult to reconstruct. Such species com- toEurasia,whereasthepolyploidshavespreadthroughout
plexes are common in angiosperms [1-5]. Rapid genetic, the N Hemisphere [10,11]. In N America, the 4x and 6x
phenotypic and ecological differentiation on one hand, cytotypes form a complex of ecological races adapted to
andhybridization/polyploidyontheother,playimportant manydifferent nicheswith marked genotypicdiversifica-
rolesin theirevolutionary bursts[6-9]. Thetemperate N tion [6,12,13]. By cultivation in experimental gardens,
Clausenetal.[6]documentedlocaladaptationofA.mille-
folium populations to environments along an altitudinal
*Correspondence:[email protected]
1MinistryofEducationKeyLaboratoryforBiodiversityScienceandEcological transectinCaliforniafromsealeveltoalpineregions.This
Engineering,andCollegeofLifeSciences,BeijingNormalUniversity,Beijing hasbecomeaclassicexampleofrapidadaptivedivergence
100875,China
ofplantpopulations[12-18].
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Guoetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
Guoetal.BMCEvolutionaryBiology2012,12:2 Page2of18
http://www.biomedcentral.com/1471-2148/12/2
Our earlier AFLP data have characterized the Achillea individuals from each population were analyzed. Broadly
millefoliumcomplexasaclade[10].Theinferredspecies the same individuals were sequenced for the two nuclear
relationshipsofthediploidmembersconformedtoabin- loci and three cpDNA fragments; minor exceptions were
ary bifurcating tree and generally agreed withtraditional due to repeated failures in sequencing a certain locus
species delimitations and taxonomic arrangements. The from a certain individual (see Table 1). Three diploid
polyploid members appeared to be polytomic and poly- species, taxonomically outside the A. millefolium aggre-
phyletic although geographic patterns can be recognized gate but included in previous AFLP analyses [10], were
[10,11]. Available data show that frequent exchange of also sampled for this study. They are the W-Eurasian
genetic materials have been involved in the origins of A. nobilis-2x, the C-Mediterranean A. ligustica-2x and
many polyploidy taxa. Meanwhile, it seems also to have the East Asian A. acuminata-2x (Table 1). In addition,
occurred during the divergence of the diploid species sequences of two cpDNA regions, trnH-psbA and trnC-
[10,11,19]. So far,we are stilluncertain about the demo- ycf6, of 43 North American populations available from
graphichistoryofA.millefoliumagg.andtheprogenitors the NCBI data base (GenBank accessions EU128982-
ofmanypolyploidtaxa. EU129456) [12] are incorporated into our plastid haplo-
Due totheirdominantnature, AFLP markersare diffi- type network analysis.
culttouseforinferringgeneticparametersofpopulations, To check ploidy levels of the populations studied, two
particularly ancestral population sizes, split times, and methods, either chromosome counting or DNA ploidy
migrationrates.Inprinciple,thiscanbeaccomplishedbet- leveldetermination,wereappliedusingyoungflowerbuds
ter with DNA sequences either from organelles or from or fresh or silica gel-dried leaves, respectively. Young
thenucleargenome[20,21].Yet,we stillmeetchallenges flower buds were collected in the field and fixed in Car-
inpractice:PlastidDNAvariationisoftentoolowtoinfer noy’sfluid(ethanol:aceticacid=3:1).Tocountthechro-
relevant gene trees with confidence. In addition, despite mosome number, fixed flower buds were stained and
thelowereffectivepopulationsizeofplastids,plastidgene squashed in 4% acetocarmine and observed under the
trees may still not reflect the species tree due to incom- microscope.DNAploidylevelswereinvestigatedwithpro-
plete lineage sorting. With nuclear genes, frequent birth pidium iodide flow cytometry [28,29] from the prepared
anddeathofgenecopies,lesslineagesortingduetohigher leaves. Information for ploidy levels obtained with the
effective populationsize,secondary introgressionamong above two methods are marked in Table 1 by c and f,
split species as well as intragenic recombination tend to respectively,whilethoseinferredfrompreviousstudies[12
hamper interpretation of the patterns of polymorphism andEhrendorferetal.,unpubl.Data]aremarkedbyi.
[22-25]. Voucherspecimensaredepositedintheherbariaofthe
Here, we survey DNA sequence variation sampled at Institute of Botany (WU) and the Department of Phar-
twonuclearlociandthreeplastidfragmentsfrompopula- macognosy (HBPh), both at the University of Vienna,
tions across diploid, tetraploid, and hexaploid species Austria,andoftheCollegeofLifeSciences,BeijingNor-
throughoutthenaturaldistributionrangeofA.millefolium malUniversity(BNU).
agg.andfromthreediploidcongenericspeciesoutsidethe
aggregate.Onthediploidlevel,weinferthedemographic Data sampling
history among species and populations using the newly Total genomic DNA was extracted from ca. 0.02 g silica
generatedDNAsequencedataincomparisontotherela- geldesiccatedleafmaterialsfollowingthe2×CTABpro-
tionships inferred from the previous AFLP data. To this tocol [30] with slight modifications: Before the normal
end,wealsoapplyaprobabilisticmodel(IMa2)[26,27]to extraction process, sorbitol washing buffer was used to
threewidespreaddiploidspecies to shedlighton thekey removepolysaccharidesintheleafmaterials(add 800μL
parameters, i.e.,ancestral effective populationsizes,time sorbitolbuffertothegroundleafpowder®incubate the
of speciation, and rates of gene flow. For the polyploid sample in ice for 10 min®centrifuge at10,000 g for 10
populations,wetrytountangletheirpolytomicandpoly- min at 4°C and then follow the established 2 × CTAB
phyleticrelationshipswhichareprobablycomplicatedby protocol).
geneflowonthesameandbetweendifferentploidylevels Two nuclear genes were sampled and partially
usingtheco-dominantsingle-genehaplotypedata. sequenced for this study. They are the chloroplast-
expressed Glutamine Synthase gene (ncpGS) and the
Methods Sedoheptulose-Bisphosphatase (SBP) gene. The ncpGS
Plant sampling gene has been used in many plant phylogenetic studies
We sampled thirty populations of seven diploid, seven andshowntobesingle-copy inallangiospermspeciesso
tetraploid and four hexaploid taxa or cytotypes of the far studied [31] and especially in Achillea [19]. We
Achillea millefolium aggregate throughout the temperate sequencedpartofitscodingandnoncodingregionsfrom
N Hemisphere (Table 1). On average, two to three exon 7 through to exon 11. The SBP gene has been
Table1Taxa, populations and DNAloci sampled hG
ttpuo
Taxa Taabtaixboonrensvi- Pcoodpe. Ploidy Geographiclocalityofpopulations Canodlledcatoterss Ngnueunmcelebsae:rrof cinpdDivN.Aa:nnaulymzebderof ://wwwetal.B
indiv./ .bM
clones iomCE
analyzed edcvolu
Achilleamillefoliumagg. ncpGS SBP entio
A.asiaticaSerg.s.lat.(=A. asi-2x NM 2xf China:DaqingMt.,41°04’52”N,112°35’56”E;2010m GR, 3/8 3/9 4 trana
sergievskianaShaulo&Shmakov) ARX 2xf China:ArxanMt.,47°17’39”N,120°27’11”E;1130m 2Y0G0,6.08.25 3/14 3/ 3 l.com/1ryBiolo
2007.10.08 14 47gy
SHB 2xf China:Hebei,42°26’N,117°15’E;1500m YG, - - 2 1-220
11
2007.07.27 42
8,
AL1 2xc Russia:Altai,51°02’52”N,85°36’47”E;1100m M20S0,2.07.30 3/9 311/ 3 /12/212:2
A.asiaticaSerg.s.str. asi-4x AL9563 4xc Russia:Altai,49°32’66”N,88°13’35”E;2350m AT, 3/13 1/5 3
2003.08.02
AL3 4xc Russia:SSiberianlowlandnearNovosibirsk;220m MS, 3/13 3/ 2
2002.08.16 15
UT 4xc Uzbekistan:Tashkent,TschimganMt. HG,2002.11 - 3/ 3
15
A.asplenifoliaVent. asp BZ 2xc Austria:Burgenland,ZitzmannsdorferWiesen Tod, 3/9 2/8 2
2001.01.10
NS2 2xc Austria:Burgenland,Rust,nearlake“NeusiedlerSee” FE,JS,YG, 3/9 3/ 3
2003.05.27 15
Ta 2xf CzechRepublic:SouthMoravia,Terezin FE&LE, 3/11 2/ 2
2002.07.12 10
A.borealisBong.s.lat.(A.lanulosa bor-alp US2 4xi USA:Washington,Mt.RainierNationalPark;1350-2070m PS&AT, 2/12 2/ 2
Nutt.var.alpicolaRydb.) 2002.08.18 10
A.borealisBong.s.lat.(A.lanulosa bor-lan US5 4xi USA:Connecticut,HopevilleStatePark JE, 2/12 2/ 2
Nutt.var.?) 2004.07.17 10
A.borealisBong.s.lat.(A.lanulosa bor-lan US6 4xi USA:Utah,ascentfromSnowbirdAltatoLakeSecret KT, 3/15 3/ 3
Nutt.var.lanulosa) 2004.07.31 18
A.ceretanicaSennens.str. cer-2x 10240 2xc France:EPyrenees JS,2001 2/10 2/9 2
A.ceretanicaSennens.lat. cer-4x 10222 4xc France:MassifCentral JS,2001 2/10 2/9 2
A.cuspidataWall. cus ID 2xi Kashmir:34°25.80’N,75°44.80’E;3200m LK, 1/4 1/5 1
2004.09.14
A.distansWaldst.&Kit.exWilld. dis DIKF 6xi Austria,Kaltenleutgeben,Flösslberge JS,s.n. 2/16 2/ 2
13
A.inundataKondr. inu K13 4xi Ukraine:Kiev,SofDesnamouthintoDnjepr;100m FE&YG, 2/9 2/9 2
2003.07.28
A.latilobaLedeb.exNordm. lat Geo 2xi Georgia:Adjara,41°29’55″N,42°31’46″E;2006m DK, 3/12 1/4 3 Pa
g
2004.07.18 e
3
A.millefoliumL.s.lat.(A.apiculata mil-api Ra-c 6xi Russia:Karelia,Louhskiregion,(a)KandalakshaNaturalReserve;(b)Kivbay, OA,2003.08 5/32 5/ 5 o
N.I.Orlova) nearMedvezhijpeninsula;(c)nearcapeIvanovNavolok 37 f1
8
Table1Taxa, populations and DNAloci sampled (Continued) hG
ttpuo
A.millefoliumL.s.lat.(A.sudetica mil-sud STms 6xi Austria:Salzburg,HoheTauern;ca.2300m PS, 2/10 2/ 2 ://wet
OA.prioz)seoalbaEhrend. ros-2x/ros-4x Si3 2x Slovenia:Ljubljana 2FE0,02.08.31 2/8 115/5 2 ww.bal.BM
+4xc 2002.07.31 ioC
mE
Si6 2+x4xc Slovenia:Ljubljana,Podpec FE,2002.7.31 2/8 1/5 2 edcenvolutio
V 2x Italy:Udine,Kanalta,MalborghettoValbruna JS,2002.07 3/15 2/ 3 trana
+4xc 10 l.cry
A.schmakoviiKupr. sch AL5 6xc Russia:Altai,51°02’52’’N,85°36’47’’E;1700m MS, 2/9 2/ 2 omBio
2002.07.30 15 /14log
7y
A.setaceaWaldst.&Kit. set GR 2xi Greece:Thessaloniki,drainfromlakeLimniKoronia FE,2001 1/3 1/5 1 1-220
K4 2xc Ukraine:Kiev,BaldMt.,LisaGora FE&YG 3/7 2/ 2 1412
8,
NS1 2xc Austria:Burgenland,EofSt.Margarethen;ca200m 2FE0,0J3S.0,7Y.2G2, 2/8 120/7 2 /12/212:2
2003.05.27
SeAA 2xc Turkey:Anatolia,Aksaray FE, 3/10 2/ 2
2002.03.26 10
A.styriacaJ.Saukel&J.Danihelka, sty StE 4xi Austria:Styria,Einach,Wald JS,s.n. 1/5 1/4 1
ined.
SpeciesoutsideA.millefoliumagg.
A.acuminata(Ledeb.)SchultzBip. acu CB1 2xf China:Jilin,ChangbaiMt.,HancongValley,680-620m YG&GR, 3/8 3/8
2002.07.24
ARX2 2xc China:InnerMongolia,Arxan,N47°17’39.5”,E120°27’09.9";865m YG, 3/12 2/
2007.10.08 11
TB5 2xc China:Shanxi,TaibaiMt.,N34°01’17”,E107°18’21";1700m. YG, 3/9 3/7
2006.09.09
A.ligusticaVis.exNym. lig SN 2xi Italy:Sicily,NebrodiMts. FE, 1/3 1/5 1
2001.09.21
A.nobilisL. nob ZN 2xi CzechRep.:Znoimo LE&FE, 3/24 1/5
2002.07.13
cploidylevelcheckedbychromosomecounting;fploidylevelcheckedbyflowcytometry;iploidylevelinferredfromFE’spreviousstudiesorfromliterature.
Namesofcollectors:AT=A.Tribsch;DK=D.Kharazishvili;FE=F.Ehrendorfer;GR=G.-Y.Rao;HG=H.Greger;JE=J.Ehrendorfer;JS=J.Saukel;KT=K.Tremetsberger;LE=L.Ehrendorfer-Schratt;LK=L.Klimes;
MS=M.Staudinger;OA=O.Alexandrova;PS=P.Schönswetter;YG=Y.-P.Guo
P
a
g
e
4
o
f
1
8
Guoetal.BMCEvolutionaryBiology2012,12:2 Page5of18
http://www.biomedcentral.com/1471-2148/12/2
studied in representative taxa of the family Asteraceae were eliminated to avoid influence of PCR-mediated
[32]. It was shown to be single-copy by our preliminary recombination [19,35,36]. The final numbers of indivi-
analysesinseveraldiploidspeciesofAchillea.Readersare duals/clones analyzed at each locus for each population
referredto Maetal.[19] forprimersused foramplifying arelistedinTable1.Allthesequencesanalyzedweresub-
the ncpGS locus and to Chapman et al. [32] for the SBP mitted to the NCBI GenBank under accession numbers
locus(itsexon5throughto7,i.e.thelocusB12inChap- HQ601971-HQ602593 (the nuclear ncpGS and SBP
manetal.2007). genes)andHQ450864-HQ451071(theplastidloci).
ThreenoncodingchloroplastDNAregions,trnH-psbA, Theallelicdatasetsofthetwonucleargenes,ncpGSand
trnC-ycf6 (including partial ycf6-psbM) andrpL16 were SBP,wereanalyzedseparately,whereassequencesofthree
sequenced.PCRreactionswereconductedwithuniversal cpDNAfragmentswerecombinedasonelocus.
primerpairs[33]. Gaps in the nuclear data sets were treated as missing
Theamplificationwascarriedoutinavolumeof20μL data, whereas each indel position (no matter how many
withfinalconcentrationof1×PCRbuffer,0.05UexTaq nucleotide sites it contained) of the plastid data set was
(TaKaRa,Shiga,Japan)orHiFi(TransTaqDNApolymer- codedasabinarycharacter(0/1=A/C)usingtheprogram
ase High Fidelity, TransGen Biotech), 200 μM of each GapCoder[37].
dNTP,1%DMSO,0.5μMof eachprimer,andwith 1μL AsA.millefoliumagg.consistsofspecieswithshortevo-
templateDNAandddH Oaddedtothefinalvolume.The lutionaryhistory[10,11],NeighborJoining(NJ),Maximum
2
amplification was conducted on a Peltier thermocycler Parsimony (MP) and Median-Joining network were
(Bio-RAD)initiatedwith5minofpre-denaturingat94°C applied to the present data. For the nuclear sequences,
followed by 30 cycles of 1 min at 94°C, 30s at 48-55°C, Neighbour Joining and Parsimony analyses were per-
and 1.5 minat 72°C. A final extensionwas then taken at formedwithMEGA5.05andPAUP*4.0b10a,respectively.
72°Cfor15minfollowedbyaholdat4°C.ThePCRpro- All nucleotide substitutions were equally weighted.Gaps
ductswereelectrophoresedonandexcisedfromthe1.0% weretreatedasmissingdata.Wefirstanalyzeddataofthe
agarosegelinTAEbuffer.Theywerethenpurifiedusinga diploidspeciestoshowdiversificationofthegenelineages,
DNAPurificationkit(TianGenBiotechorTransGenBio- andthenofallthetaxatoinvestigaterelationshipsamong
tech, Beijing, China). The purified PCR products were thepolyploidsanddiploidswithinA.millefoliumagg.The
either used for direct sequencing (for the cpDNA frag- NJanalysiswasconductedwithKimura’s2-parameterdis-
ments) or ligated into a pGEM-T Vector (for nuclear tances[38]andbootstrappedwith1000replicates.Forthe
genes) withaPromegaKit(Promega Corporation,Madi- MP method, heuristic searches were performed using
son,USA).Forsequencingthenucleargenes,aboutfiveto 1000 random taxon addition replicates with ACCTRAN
eight positive clonesfromeach diploidandtentofifteen optimization and TBR branch swapping. Up to 10 trees
fromeachpolyploidindividualwererandomlyselectedfor with scores larger than 10 were saved per replicate. The
sequencing. The plasmid wasextracted with an Axyprep stabilityofinternalnodesoftheMPtreewasassessedby
Kit (Axygene Biotechnology, Hangzhou, China). Cycle bootstrapping with 1000 replicates (MulTrees option in
Sequencing wasconducted using ABIPRISM® BigDye™ effect, TBR branch swapping and simple sequence
Terminator.Thesameprimersusedforamplification(for addition).
cpDNA fragment) or the vector primers T7/Sp6 (for Median-Joining networkanalysis implemented inNet-
nucleargenes)wereappliedhere.Thesequencedproducts workver.4.5.1.6availableathttp://www.fluxus-engineer-
were run on an ABI PRISM™ 3700 DNASequencer (PE ing.com/sharenet.htm[39]wasappliedtothecpDNAdata
AppliedBiosystems). set.Allvariablesiteswereequallyweightedandthehomo-
plasylevel parameter (ε)wassettozerogiven thatvaria-
Data analyses tionratesofthecloselyrelatedspeciesislow,especiallyin
Sequences were assembled with the ContigExpress pro- theirplastidDNA.
gram(InformaxInc.2000, NorthBethesda,MD),aligned To understandthe populationdemography at the time
with ClustalX 1.81, and then manually improved with ofspeciationofthediploidspecies ofA.millefoliumagg.,
BioEdit version 7.0.1. To prevent possible sequencing weappliedaprobabilisticmodel,theIsolationwithMigra-
errors,singlemutationsinthenucleargenedatasetslikely tionModelformultiplepopulationsimplementedinIMa2
generated by the cloning sequencing method were [27], to three widespread and closely related species A.
excluded from the analyses. Furthermore, unique asplenifolia-2x and A. setacea-2x and A. asiatica-2x.
sequencesinthe nuclear gene data matrix, which donot Thesespeciesarehereregardedasthreedivergedpopula-
fall into any majority-rule consensus sequence group tions which share nuclear sequence variation. Shared
[19,34]orshowinconstantbranchpositionsintreesbased allelescouldreflectancestralpolymorphismorgeneflow
ondifferentsubsetsofdata,i.e.,withpartialcharactersor after separation of the populations or species. Assuming
randomly selected sequences, during the initial analyses neutrality,retentionofancestralpolymorphismsislikelyif
Guoetal.BMCEvolutionaryBiology2012,12:2 Page6of18
http://www.biomedcentral.com/1471-2148/12/2
speciationisfastrelativetodrift,whichisinverseininten- rates(2NM=4Nu ×m/2).The priorswere finally set as
sity to the effective population size. As a rule of thumb, follows: the upper bound of population sizes q = 100,
speciesarewellseparatedwithlittleancestralpolymorph- splittingtimest=5andmigrationratesm=2.0,respec-
ism andthusalmostcompletelineagesorting,ifthetime tively. We ran the Markov-chain Monte Carlo (MCMC)
ofseparationisatleastaslongasfourtimestheeffective simulationswith1,000,000burn-instepsand20,000gen-
populationsize[40].Secondarygeneticexchangebetween ealogies sampled per locus. The analysis was done with
the diverging species can also lead to shared alleles 10independentrunsintheMmode,eachusingidentical
observed [41]. The multipopulation model IMa2 allows priors and 20 Metropolis-Coupled chains with different
bothancestralpolymorphismandgeneflowsubsequentto random number seeds. The genealogies sampled from
divergence. It assumes a known history of the sampled the M mode runs were combined in an L mode run to
populations, whichcanbe representedbyarootedbifur- buildanestimateofthejointposteriorprobabilityofthe
cating tree. In earlier analysis using AFLP data [10], we parameters[26,27].
inferredtherootedspeciestreeas:((A.asiatica,A.aspleni-
folia), A. setacea). We note that Ima2 provides posterior Results
distributions of parameters, such that the confidence in Nuclear gene trees with allelic haplotypesequences
the inference of each parameter can be obtained from Amplification of the partial ncpGS and SBP genes pro-
observingthespreadoftheposteriordistribution.TheIM duced a single clear band for each amplification. This
model also assumes neutral genetic variation, freely and the results from earlier work (see “Methods”) sug-
recombining unlinked loci andno intragenic recombina- gest sequences of the two nuclear loci obtained here
tionorgeneconversion[42].Sequencesofthetwonuclear each as belonging to a set of orthologs.
loci, the ncpGS and the SBP genes, and of three plastid AftereliminatingsomesequenceslikelycontainingPCR-
fragments were used for this analysis. The polymorphic recombination (about 10% of the total), 303 sequences
sitesofthesequencednuclearandplastidlociaremostly (clones) of the ncpGS and 313 of the SBP gene from
of introns or intergenic spacers and thus should fit the broadlythesame70individualsof29populationsbelong-
neutral variationmodel. Usingthe four-gamete criterion ingtoA.millefoliumagg.wereusedforthedata analyses
[43], we do not find intragenic recombination in the (Table 1). In addition, 56 ncpGS and 36 SBP sequences
nuclearsequencesamongthesethreespecies.Thedataof fromfive populationsofthreecongenericspeciesoutside
the three plastid fragments were combined because the the A. millefolium agg., A. nobilis-2x, A. ligustica-2xand
chloroplastgenesaregenerallylinkedandnoevidenceof A. acuminata-2x, were also analyzed here. The ncpGS
recombinationbetweenthethreeregionsisfound. alignmentcontains918nucleotidepositionswithsequence
To run IMa2, one random haplotype per plant indivi- length varying from 795 to 861 bps. The SBP alignment
dual was chosen for the nuclear gene data sets, and the contains 420 nucleotide positions with sequence length
plastid data set was composed of sequences from the varyingfrom386to405bps.
same plant individuals. This avoids bias but decreases Prior to the analyses of all the diploid and polyploid
the amount of information and thus leads to broader taxa, we present the gene trees at the diploid level first
posterior distributions. The IS (Infinite Sites) model [44] (Figures1&2).
of sequence evolution was chosen for the plastid locus, The diploid-only ncpGS data come fromseven species
whereas, the HKY model [45] which allows for multiple withinandthreeoutsideA.millefoliumagg..Theycontain
substitutions was selected for the two nuclear loci 31 haplotypes with 134 substitution sites from 186
because double mutations were found for a few poly- sequences (Additional file 1: S-Figure 1). Out of the 134
morphic sites at both loci. The inheritance scalar was polymorphicsites,122areinintrons.Intragenicrecombi-
set to 1.0 for the nuclear and to 0.25 for the plastid loci, nationamongsomesamplesislikely:Adiscordanceinthe
respectively. alignmentcanberesolvedbypostulatingarecombination
To set upper bounds on the prior distributions of the in three of the four ncpGS haplotypes of A. nobilis-2x
parameters, we estimated for each of the three species aroundthe89thpolymorphicsite(Additionalfile1:S-Fig-
the geometric means of the population mutation rate ure 1). Another discordance can be resolved in the two
4Nu across all three loci using Watterson’s estimator θ haplotypesofA.cuspidata-2x(IIIinFigure1A)bypostu-
(per sequence not per site). The largest mean value was lating a recombination around the 26th polymorphic site
foundwithA.asiatica-2x(anestimateof4Nu=9.8205), betweenhaplotypegroupsofA.millefoliumagg.andofA.
and this was used to set the upper bound on uniform ligustica-2x(Additionalfile1:S-Figure1).
priorforeachofthethreepopulationdemographicpara- Figure1AshowsanunrootedNeighbourJoiningphylo-
meters:populationsize(θ=4Nu),splittingtime(t=Tu, gram based on the diploid-only ncpGS data. The topol-
where T is the time in generations since the common ogy of the MP tree on the same data set is broadly
ancestry,anditisofthesameorderof4N)andmigration comparable,andthusonlybootstrapvaluesfromtheMP
Guoetal.BMCEvolutionaryBiology2012,12:2 Page7of18
http://www.biomedcentral.com/1471-2148/12/2

B A.asplenifolia

A . r o s e o alb a A
. m
A . c e r e tanica ille
fo
liu A
m
A.asiatica agg 3029 31
.
A.acuminata
29=acu (ARX2-3/12; CB1-3/8)
30=acu (TB5-1/2)
31=acu (TB5-3/7)
A.setacea

A . n o b i l is
A .crithmifolia

A . c l y p e olata
A . l i g u s t ica
A . m o s c hata 100/100
A . o x y l o ba

A.acuminata
10=nob (ZN-2/9) 1 3 = a s i ( A L 1 - 2 / 7 )
A.wilhelmsii 11=nob (Zn-1/1) 1 4 = a s i ( A R X - 2 / 8 )
12=nob (Zn-3/9) 15=asp (NS2-1/3)
12 16=asp (BZ-1/1; Ta-1/1);
A.nobilis 10 cer (10240-2/10)
21 A.cusIpIiIdata 11 17=asp (BZ-1/2)
20 18=asi (ARX-1/1)
19=asi (NM-3/5)
2201==ccuuss ((IIDD--11//25)) 100/100 100 /100 15
100/100 1 6 1 9
99/99 1 8 17
100/100 56/80
93/99 A.ceretanica
70/85 A . a s p l e nifolia
23 A.ligustica 0.01 72/74 97/99 14 A . a s i a tica
22 substitutions/site 13 IIb
22=lig (SN-1/1) 88/90
23=lig (SN-1/2) 8
6
72/95
7 31
IIa 542
A.asplenifolia
A . r o s eoalba
A.asiatica
26 Ib A.latiloba
24 25 9 A.nobilis
1=ros (Si6-1/1)
A.setacea 2=ros (Si3-1/5; Si6-1/2)
24=set (K4-1/1) 100/100 3=ros (Si6-1/1)
25=set (K4-2/5) 4=ros (V-1/2)
26=set (K4-1/1; SeAA-3/8) 5=asp (BZ-2/6)
2278==sseett ((SGeRA-1A/-32;/ 2N)S1-2/8) Ia 67==aasspi ( (ANLS12-1-2/2/6; A; TRaX-3-1/1/40;) ;N rMos- 2(/V3-)1/3)
8=lat (Geo-3/12)
27 28 9=nob (Zn-1/5)
Figure1ThencpGSgenetreeandtheAFLPtree.A.UnrootedNeighbourJoiningphylogramof10diploidAchilleaspecies(sevenofand
threeoutsideA.millefoliumagg.)basedonthencpGSgenesequencedata.Thetreecontains31ncpGShaplotypesgeneratedfrom186clones
(sequences)from49individualsof20populations.Bootstrapsupports(>50%)frombothmethods(NJ/MP)areshownnexttothemajor
branches.Labelsofterminalbranchesarewrittenas“taxonabbreviation(populationcode-numberofindividuals/numberofclones)”.Fortaxa
abbreviations,seeTable1.B.TheAFLPtreefromapreviousstudy(Guoetal.,2005[10])forcomparison.
Guoetal.BMCEvolutionaryBiology2012,12:2 Page8of18
http://www.biomedcentral.com/1471-2148/12/2
Ia Ib
A.asiatica, A.asplenifolia,
1=set (NS1-1/1; SeAA-2/8) A.roseoalba, A.ceretanica,
2=set (NS1-1/2) A.asplenifolia, A.roseoalba, 10
3=asp (BZ-2/3; NS2-1/1) A.ceretanica, A.setacea 963/62 A.setacea
4=asp (NS2-2/7) 4 7 7=asp (BZ-2/2; Ta-2/10);
5 = acsepr ((1B0Z2-410/1-;2 N/2S);2-1/2); 1 2 5 97/97 62/62 8 8 = acseir ((A1L012-410/3-1);/2); ros (Si3-1/5)
ros (Si6-1/5);
3 set (GR-1/5; K4-1/5; NS1-1/1; SeAA-1/2)
6 = caseserpt ( ((1KB04Z2--4110//5-11;; /NN2S)S12--11//35)); 71/736 --/62 190==aassi i( (AARLX1--21//22;) ;N cMer- 1(1/10)2; 4a0s-p1 (/B3)Z-1/1)
61/66
79/96
79/96 13
A.ligustica1112 14A.acuminata
11=lig (SN-1/4) 13=acu (ARX2-2/11; CB1-2/6; TB5-1/2)
12=lig (SN-1/1) 14=acu (CB1-1/2; TB5-2/5)
97/99
III
A.asiatica 16 67/91 51/--
17 A.latiloba
15
II
16=asi (ARX-2/10)
17=asi (ARX-1/2)
15= lat (Geo-1/4)
98/98
--/98
A.asiatica 19
A.cuspidata
IV
18
18=asi (AL1-1/2) 97/100
19=cus (ID-1/5);
asi (AL1-2/4; NM-3/8)
A.nobilis
20=nob (ZN-1/1)
0.05 substitutions/site 20 21=nob (ZN-1/4)
21
Figure2UnrootedNeighbourJoiningphylogramof10diploidAchilleaspecies(sevenofandthreeoutsideA.millefoliumagg.)based
ontheSBPgenesequencedata.Thetreecontains21SBPhaplotypesgeneratedfrom163clones(sequences)from35individualsof19
populations.Bootstrapsupports(>50%)frombothmethods(NJ/MP)areshownnexttothemajorbranches.Labelsofterminalbranchesare
writtenas“taxonabbreviation(populationcode-numberofindividuals/numberofclones)”.Fortaxaabbreviations,seeTable1.
Guoetal.BMCEvolutionaryBiology2012,12:2 Page9of18
http://www.biomedcentral.com/1471-2148/12/2
analysis are presented in Figure 1A. In this gene tree, Figure3showsthencpGSgenetree (unrootedNJtree)
haplotypes of A. millefolium agg. fall into two major ofallthediploidandpolyploidtaxaofA.millefoliumagg.
groups, group I corresponding to A. setacea-2x, and andofthreecongenericdiploidspeices.Itisbasedon110
group II those of the other Eurasian diploid species haplotypesgeneratedfrom 359sequenceswith 155 poly-
exceptA.cuspidata-2x.Thisrelationshipagreeswiththat morphicsubstitutionsites.InFigure3,allelesofeachpoly-
fromthepreviousAFLPanalysis(Figure1Binferredfrom ploidindividual,populationortaxon(markedindifferent
Guo et al., 2005). In haplotype group II, both the sub- colors)scatteramonghaplotypesofdifferentdiploidspe-
groupsIIaandIIbharborsequencesofA.asplenifolia-2x cies (all in black letters) of A. millefolium agg. except
and A. asiatiaca-2x. This is not in line with the AFLP A.cuspidata-2x.A.roseoalba-4xandA.asiatica-4xshare
treeandismostlikelyduetoretentionofancestralpoly- some oftheir alleleswiththeir diploidcytotypes,respec-
morphism or secondary contacts between the two spe- tively, whereas A. ceretanica-4x is quite differentiated
cies. A. asplenifolia-2x shares its diverged alleles with fromA.ceretanica-2x.AllelesfromthetetraploidA.bor-
A.ceretanica-2xandA.roseoalba-2x,respectively,corre- ealisvar.alpicolaandvar.lanulosa-4xinNAmerica,the
sponding to the previous AFLP tree (Figure 1B). tetraploid A. asiatica-4x in C Asia and the hexaploid
SequencesofA.ceretanica-2x(relictintheEasternPyre- A.millefoliumsubsp.apiculata-6xinNEEuropearemore
nees) only appear in IIb and those of A. roseoalba-2x oftenassociatedwitheachotherthanwiththoseofother
(from the meadows in the S-Alps) only in IIa. The polyploid taxa. Only the Ukrainian A. inundata-4x and
sequences of A. cuspidata-2x (in the Himalayas) appear the C Asian A. schmacovii-6x share nuclear haplotypes
distanttoothermembersofA.millefoliumagg.butclose withA.setacea-2x.
toA.ligustica-2x(Figure1A).Thesingleplantsampleof The date set of SBP gene of all the diploids and poly-
A.cuspidata-2xwascollectedrecently and thuswas not ploids contains 68 haplotypes generated from 349
includedinthe previousAFLP study.Clearlymore sam- sequenceswith68polymorphicsubstitutionsites.Dueto
ples of this species should be investigated. Out of the the severe conflicts between the SBP gene tree and the
four haplotypes of A. nobilis-2x, one falls into clade IIa species tree inferred from the AFLP and morphological
together with several members of A. millefolium agg., data,sequencesofthisgenearenotsuitableforthephylo-
and three form a group between IIa and IIb. The latter geneticinference,butcouldprovidesomecluesaboutthe
canbe explained by the intragenic recombinationvisible progenitorsofthepolyploidtaxa.Wethereforeonlypre-
inthealignment(Additionalfile1:S-Figure1).Consider- sent the SBP gene tree of all the diploid and polyploid
ingrelationshipsofA.nobiliswithA.millefoliumagg.,the samples in the supplementary materials as Additional
ncpGSgenetreeisneithercongruentwiththeAFLPtree file3:S-Figure3.
(Figure1B)norwiththeSBPtree(seebelow,Figure2).
The diploid-only SBP data also include seven species Phylogenetic networks based on plastid haplotypes
withinandthreeoutsideA.millefoliumagg.Theycontain Thirty populations with broadly the same 70 individuals
21 haplotypes with 60 substitution sites based on 163 analyzed with the nuclear genes were sequenced at
sequences.Outofthe60polymorphicsites, 48belongto three plastid loci, trnH-psbA, trnC-ycb6 and rpl16. The
the intron regions. The alignment of the SBP sequences length variation and number of polymorphic sites of
does not show obvious intragenic recombination (Addi- each fragment are listed in Table 2.
tional file 2: S-Figure 2). The topology of the NJ tree is ForthediploidmembersofA.millefoliumagg.together
broadly comparable with that of the MP tree and thus with their sister species A. ligustica, three plastid frag-
onlybootstrapvaluesfromtheMPanalysisarepresented mentsfrom18populationsand34individualsgenerateda
(Figure 2). The SBP gene tree (Figure 2) is remarkably combinedmatrixwith1855(varying from 1814 to1842)
incongruentwiththencpGStree(Figure1A)andwiththe nucleotide positions and 26 variable sites. Out of the 26
AFLPtree(Figure 1B).InFigure2, haplotypes belonging variable sites, 18 are substitution sites and 8 are indels
tomembersofA.millefoliumagg.donotgrouptogether. (Table 2). The polymorphic sitesallow the identification
SomeofA.asiatica-2x,alloftheCaucasusA.latiloba-2x of 13 plastid haplotypes named as dH1-13, where “d”
andtheHimalayanA.cuspidata-2x(heredefinedasAsian standsfordiploidstobedistinguishedfromthoseusedfor
types)aredistantlyrelatedtotheothersofA.millefolium thediploid-polyploidcombineddata asdescribedbelow.
agg.Surprisingly,sequencesoftheCEuropeanA.nobilis- AsshowninFigure4,polymorphicplastidhaplotypesare
2xareclosetotheAsiantype,whereas,haplotypesofthe foundwithin eachof the three relativelywidespread spe-
EAsianA.acuminata-2xareclosetothemajorhaplotype cies A. setacea-2x, A. asplenifolia-2x and A. asiatica-2x.
group of A. millefolium agg., which is mostly of the Furthermore,distributionoftheplastidpolymorphism is
European members. We thus observe little sorting of not even among the diploid species. Among the three
ancestralpolymorphismsoftheSBPgeneduringthespe- widespreadspecies,theEuropeanA.setaceaandA.asple-
ciationprocessesofthediploidspeciesofAchillea. nifoliaeachharboursarelativelyfrequenthaplotype,dH7
Guoetal.BMCEvolutionaryBiology2012,12:2 Page10of18
http://www.biomedcentral.com/1471-2148/12/2
72/63 alp (US2-1/1); lan (US5-1/1)
alp (US2-1/1)
alp (US2-1/4); lan (US5-1/1)
alp (US2-1/1)
alp (US2-1/3)
ros (V-1/2); api (Rb-1/1); lan (US5-1/1)
dis (DIKF-1/1)
lan (US5-1/1)
api (Rc-1/1)
lan (US6-1/1)
inu (K13-1/1)
lan (US6-1/1)
lan (US6-1/1)
67/-- api (Rc-1/1); lan (US5-1/1; US6-1/5)
api (Rc-1/1)
ros-4x (V-1/1)
sch (AL5-1/1)
asp (BZ-2/6); api (Ra-1/2); asi-4x (AL9563-2/4);
inu (K13-1/2); sud (STms-1/3); ros-4x (V-1/1); sch (AL5-2/3)
inu (K13-1/1)
72/61 ros-4x (V-1/1)
57/-- 53/-- rrooss ((SSii63--11//15); Si6-1/2); ros-4x (Si6-1/3)
ros (Si6-1/1)
inu (K13-1/1)
sud (STms-1/1)
asi-4x (AL3-1/1)
asi (AL1-1/2; ARX-1/4; NM-2/3); asi-4x(AL3-1/3; AL9563-1/2)
ros-4x (Si6-1/1)
63/-- cer-4x (10222-1/1)
78/-- cer-4x (10222-1/1)
api (Ra-1/3; Rc-1/2)
api (Rb-1/5); cer-4x (10222-2/7); sud (STms-1/3); ros-4x (Si3-1/2)
72/67 ros-4x (V-1/1)
sty (StE-1/1)
61/-- asp (NS2-2/6; Ta-3/10); ros (V-1/3); ros-4x (V-1/2)
sud (STms-1/1)
dis (DIKF-1/1)
dis (DIKF-1/1)
66/63 dis (DIKF-1/1)
asi-4x (AL3-1/2); dis (DIKF-1/2)
65/-- dis (DIKF-1/1)
asi-4x (AL3-1/1)
alp (US2-1/1)
dis (DIKF-1/1)
98/8770/-- aassii- (4AxL (1A-L23/7-1);/ 5as) i - 4 x (AL9563-1/3)
70/-- asi (ARX-2/8); asi-4x (AL9563-1/2)
asi-4x (AL9563-1/1)
asi-4x (AL9563-1/1)
api (Ra-1/1)
74/-- api (Ra-1/1)
lan (US6-1/1)
89/87 lan (US6-1/1)
59/55 --/57 93/-- aasspp ((NBZS2-1-1/1/3; )T a - 1 / 1 ) ; c e r ( 1 0 2 4 0 - 2 / 1 0 ) ; ros-4x (Si3-1/1)
api (Ra-1/1)
85/80 54/67 aappii ((RRaa--11//11))
asi (ARX-1/1)
asp (BZ-1/2)
api (Ra-1/1; Rc-1/1)
asi (NM-3/5); api (Ra-1/1); lan (US6-1/1); dis (DIKF-1/3)
api (Rc-1/1)
ros-4x (V-1/1)
sud (STms-1/1)
50/-- ddiiss ((DDIIKKFF--12//12))
alp (US2-1/1)
57/-- sscchh ((AALL55--11//11))
nob (ZN-1/5)
api (Ra-1/2); lan (US5-1/4; US6-1/3); asi-4x (AL3-1/1)
dis (DIKF-1/1)
lan (US5-1/1)
lan (US5-1/1)
dis (DIKF-1/1)
lat (Geo-3/12)
68/68 sscchh ((AALL55--11//11))
cer-4x (10222-1/1)
sud (STms-1/1)
ros-4x (V-1/2)
api (Ra-1/1)
98/7856/52 aappii ((RRaa--11//12)); lan (US5-1/1)
sty (StE-1/1)
99/100 sty (StE-1/1)
53/52 ros-4x (V-1/1)
--/50 73/70 sty (StE-1/2)
inu (K13-1/1)
51/-- api (Ra-1/1)
81/77 lan (US6-1/1)
nob (ZN-1/1)
100/100 54/-- nnoobb ((ZZNN--23//99))
80/-- set (K4-2/5)
96/90 set (K4-1/1; SeAA-3/8)
90/90 sseett ((KG4R--11//13); N S 1 - 2 / 8 ); inu (K13-1/2)
91/97 100/10068/-- sineut ((SKe1A3A-1-2/1/2))
sch (AL5-1/1)
76/-- 99/101000/100 ccliuugss ( ((SIINDD---111///252)))
76/-- 100/100 lig (SN-1/1)
acu (ARX2-3/12, CB1-3/8)
100/10080/-- aaccuu ((TTBB55--13//27))
Figure3UnrootedNeighbourJoiningcladogramofthencpGSgeneofallthediploidandpolyploidtaxaanalyzedinthisstudy.The
treecontains109allelichaplotypesgeneratedfrom359sequenceswith155substitutionsites.TopologyoftheMPtreeonthesamedatasetis
broadlycomparablewiththatoftheNJtree.Bootstrapsupports(>50%)fromNJ/MPanalysesareshownnexttothebranches.Labelofeach
terminalbranchiswrittenas“taxaabbreviation(populationcode-numberofindividuals/numberofclones)”.Fortaxaabbreviations,seeTable1.
Diploidtaxaareinblack,polyploidtaxaindifferentcolours.