The evolutionary history of Antirrhinum suggests that ancestral

12 Pages·2011·1.34 MB·English

ThePlantJournal(2011)66,1032–1043 doi:10.1111/j.1365-313X.2011.04563.x
The evolutionary history of Antirrhinum suggests that
ancestral phenotype combinations survived repeated
hybridizations
YvetteWilsonandAndrewHudson*,†
InstituteofMolecularPlantSciences,UniversityofEdinburgh,King’sBuildings,MayfieldRoad,EdinburghEH93JH,UK
Received29November2010;revised25February2011;accepted1March2011;publishedonline6April2011.
*Forcorrespondence(fax[phone];[email protected]).
†Presentaddress:DivisionofPlantScience,UniversityofDundeeatSCRI,Invergowrie,Dundee,DD25DA,UK.
SUMMARY
The model species Antirrhinum majus (the garden snapdragon) has over 20 close wild relatives that are
morphologicallydiverseandadaptedtodifferentMediterraneanenvironments.HybridsbetweenAntirrhinum
species havebeenused successfullyto identifygenesunderlying theirphenotypic differences,andtoinfer
howselectionactsonthem.Tobetterunderstandthegeneticbasisforthisdiversity,wehaveexaminedthe
evolutionaryrelationshipsbetweenAntirrhinumspeciesandhowtheserelatetogeographyandpatternsof
phenotypic variation in the genus as a whole. Large population samples and both plastid and multilocus
nuclear genotypes resolved the relationships between many species and provided some support for the
traditionaltaxonomicdivisionofthegenusintomorphologicalsubsections.Morphometricanalysisofplants
grownincontrolledconditionssupportedthephenotypicdistinctionofthetwolargestsubsections,andthe
involvement of multiple underlying genes. Incongruence between nuclear and plastid genotypes further
suggestedthatseveralspecieshavearisenafterhybridizationbetweensubsections,andthatsomespecies
continuetohybridize.However,allpotentialhybridsappeartohaveretainedaphenotypesimilartooneof
their ancestors, suggesting that ancestral combinations of characters are maintained by selection at many
differentloci.
Keywords: Antirrhinum,snapdragon,phylogeny,morphologicalevolution.
INTRODUCTION
Much of our understanding of the genes involved in mor- genus Antirrhinum – in which between 17 and 27 distinct
phologicalevolutionandspeciationhascomefromtaxathat species and subspecies have been recognized in different
aresufficientlydifferenttoberegardedasseparatespecies, taxonomicaccounts(Rothmaler,1956;Webb,1971;Sutton,
butthatretaintheabilitytoformfertilehybrids.Bothnatural 1988). Although morphologically diverse and adapted to
andartificialhybridshavebeenusedtodetectlociunderly- different, often extreme, environments, all Antirrhinum
ingdifferencesbetweentheparentalspecies.Insomecases, species can form fertile hybrids with each other and with
thegenesandmutationshavethemselvesbeenidentified. A.majus when artificially cross-pollinated. Such hybrids
This has been particularly successful when the research haveidentifiedgenesunderlyingdifferencesinmorphology
infrastructuredevelopedinacloselyrelatedmodelspecies and flower colour between their parents (Hackbarth etal.,
is available, for example in Drosophila (McGregor etal., 1942;Langladeetal.,2005;Schwinnetal.,2006;Fengetal.,
2007;Jeongetal.,2008). 2009). Natural Antirrhinum hybrids have also identified
Thegardensnapdragon,Antirrhinummajus(Plantagina- genes involved in flower colour variation, and have sug-
ceae)hasbeenusedasamodeltostudyinheritance,andthe gested how selection acts on them (Whibley etal., 2006).
geneticcontrolofdevelopmentandflowercolour(Schwarz- The genus Antirrhinum can therefore provide a model for
Sommer etal., 2003). Its close relatives are native to the understandingthegeneticbasisforpatternsofphenotypic
westernMediterraneanregion,mostlytheIberianpeninsu- diversity and adaptation around the species level, which
lar, and comprise a monophyletic group – the traditional may be typical of many recently evolved Mediterranean
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Constrained evolution inthe genus Antirrhinum 1033
taxa (Thompson, 2005). However, it has been difficult to
relate the genetic differences between pairs of parental
species to variation in the genus as a whole because the
relationships between Antirrhinum species have not been
resolved.Forinstance,itiscurrentlynotpossibletoinferthe
ancestral state of a character and whether similar pheno-
typesmighthaveevolvedmultipletimeswithinthegenus.
One obstacle to resolving evolutionary relationships
withinAntirrhinumisreflectedintheuncleartaxonomyof
itsspecies,manyofwhichhavenotbeenreportedtohave
a unique, fixed character (a synapomorphy; Webb, 1971).
Therefore, species might not represent discrete genetic
entities because they have been delimited artificially. Figure1. ThethreemorphologicalsubsectionsofAntirrhinum.
Nevertheless, support for the genetic distinction of some A representative of each subsection is shown in situ and in cultivation.
Subsection Antirrhinum is represented by A. pseudomajus, subsection
recognized species has been provided by allozymes and
StreptosepalumisrepresentedbyA.braun-blanquetiiandsubsectionKick-
DNA sequence variation (Mateu-Andres and Segarra- xiellaisrepresentedbyA.pulverulentum.Scalebars:150mmforcultivated
Moragues,2000,2003;Jimenezetal.,2005a;Mateu-Andres plants,andtherulershownwithplantsinthefieldis35mmwide.
and de Paco, 2005). Relationships above the species level
are also unclear. The genus has been divided into three
morphological subsections– Antirrhinum, Streptosepalum
majorsubsections,andtheinvolvementofmanyunderlying
and Kickxiella – but no subsection has been defined by a
genes. However, all putative hybrid species appeared to
synapomorphy,andallhavebeensuggestedtooverlapin
resemble one of their parents, suggesting that ancestral
phenotype(Rothmaler,1956;Webb,1971).Thesubsections
suitesofphenotypeshavesurvivedhybridizationasaresult
also correlate with ecology: most members of subsection
ofselectionatmultipleloci.
Kickxiella are small prostrate alpines or xerophytes that
growon rockfaces,whereassubsectionsAntirrhinumand RESULTS
Streptosepalum comprise larger, more upright plants that
Antirrhinumtaxonomyreflectsdiscontinuousphenotypic
are able to grow in competition (Figure1). This raises the
variation
possibilitythateachsubsectionrepresentsanecotype,and
that its species have evolved similar characters indepen- Antirrhinum populations were sampled from across the
dently as adaptations to a particular environment, rather geographic range of each recognized species and subspe-
thansharingcharactersthroughcommondescent. cies, so that the depth of sampling in each taxon broadly
AttemptstoresolveaspeciesphylogenyforAntirrhinum correspondedtoitsabundance(Figure2;Tables1andS1).
from DNA sequences have been unsuccessful. Relatively Theonlyrecognizedspeciesthatremainedun-sampledwas
little sequence variation has been found in the genus, Antirrhinum martenii, which could not be found at its ori-
consistent with its recent origin, and the variation is not ginalcollectionsitesintheMoroccanRif.Forconvenience,
distributed consistently between taxa, so that different wetreatedalltaxaattherankofspecies,includingthosethat
genes support different relationships between species areoftenregardedassubspeciesofA.majus.
(Jimenezetal.,2005b;Vargasetal.,2009).Sparsesampling To assess phenotypic variation within Antirrhinum we
ofthetaxausedforDNAsequenceanalysismightalsohave grewplantsfromarepresentativesubsetof98populations
contributedtoalackofphylogeneticresolution,giventhat together in a glasshouse,and recorded phenotypesfor an
youngspeciesarelikelytosharemanyunfixedallelesthat average of 5.8 plants from each population (Table2). We
mightnotberepresentedinsmalltaxonsamples. chosephenotypesthatdifferedbetweenpopulations,with-
Here, we examine evolutionary relationships within the out regard to their differences between species or sub-
genus Antirrhinum by comparing populations sampled sections, to avoid any bias towards characters that might
from across the geographic range of each species. Plastid automaticallysupporttraditionaltaxonomicdivisions.Char-
andmultilocusnucleargenotypesresolvetherelationships acterswerethenselectedforfurtheranalysisontwogenetic
between many species, and further suggest that the tradi- criteria.Thefirstattemptedtoreducetheuseofcharacters
tional morphological subsections largely correspond to that were strongly influenced by environment. It assumed
separate evolutionary lineages. They also suggest that that members of the same species are genetically most
hybridization has occurred repeatedly between the two similar to each other. Therefore, a comparison of the
major ancestral lineages where they overlap in range. variation within species and between species gives an
Morphometricanalysisofplantsgrownincommongarden estimate of the extent to which a phenotype is genetically
conditionssupportedthephenotypicdistinctionofthetwo determined (approximating its broad-sense heritability).
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1034 YvetteWilsonand AndrewHudson
Figure2. ThedistributionofrecognizedAntirrhinumspecies.
Speciesrangeswereestimatedfromcollectionsitesofthisandpreviousstudies.TherangeofA.tortuosumextendsfurthereastwardsthanisshownhere.The
namesofmembersofsubsectionAntirrhinumareshowninred,membersofKickxiellaareshowninblueandmembersofStreptosepalumareshowninorange.
Eachspeciesrangeiscolouredtorepresentitsgeographiclocation,sothatspecieswithsimilarlocationsareshownincolourswithsimilarhues.ForA.tortuosum
andA.linkianum,subpopulationsthatoccurindistinctgeographicregionsareshownwithdifferentcolours.
Phenotypesforwhichmorethan35%ofthetotalvariance flower size, is accompanied by a narrower leaf shape and
occurredwithinspecies(stomatalindicesinleaves,stemsor more reflexed dorsal petals. PC2 and PC3 capture other
petals, and leaf epidermal cell size) were excluded from aspects of shape and size variation in both leaves and
furtheranalysis. flowers,whereasPC4mainlydescribestheextenttowhich
Thesecondcriterionforscreeningphenotypesattempted flowers vary independently of leaves (Figure3b). Because
to minimize repeated sampling of the same genetic differ- thePCsareuncorrelatedtoeachother,eachwastreatedasa
ences.Differencesinleafandflowersizeandshape(allom- separatephenotypiccharacter.
etry), for example, which have been used as taxonomic We also avoided using other phenotypes that high
characters in Antirrhinum, do not vary independently of correlations suggested might be developmentally con-
eachotherbecausetheyareaffectedbythesamegenes:i.e. strained (e.g. the lengths of the style and stamens), and
the characters are developmentally constrained (Langlade choseflowercolourphenotypestorepresenttheeffectsof
etal., 2005; Feng etal., 2009). Instead of treating them genes known to be involved in interspecies differences
separately,wethereforequantifiedvariationintheallome- (Schwinnetal.,2006;Martinetal.,1987;seeExperimental
triesofleavesandflowerstogetherusingacomputershape proceduresfordetails).
model of flattened node-4 leaves, flattened dorsal petals For the 22 remaining characters, average values were
andsideviewsofintactflowers,inwhichotheraspectsof calculatedforeachofthe98populations,andtherangeof
variationareapparent(e.g.theanglesatwhichpetallobes themeanvalueswereadjustedtoacommonlinearscaleof
are presented). The shape model captures co-variation in 0–22.Theserescaledvalueswereusedinde-trendedcorre-
thepositionsofpointsplacedaroundtheorganoutlinesas spondence analysis (DCA) to position each population in
orthogonal principal components (PCs; Figure3). The first phenotypic space, defined by the two major axes of
fourPCsaccountedfor93%ofthevariancewithinthegenus, co-variationinthedataset(ParnellandWaldren,1996).
allowing the leaf and flower allometry of each plant to be A notable feature of the distribution of populations in
describedaccuratelywiththesefourPCvalues.Thetypesof phenotypicspacewasthatmembersofsubsectionKickxiella
variationcapturedbythePCsareshowninFigure3(b).PC1 formedaclusterdistinctfromsubsectionsAntirrhinumand
describesmainlysize variation,showingthatorgansize is Streptosepalum,withKickxiellapopulationsmainlysharing
themajordifferencebetweenplants,althoughthisiscorre- higher values along DCA axis 1 (Figure4a). Kickxiella
latedwithotheraspectsofshapevariation.Forinstance,an therefore appears to be phenotypically distinct from
increasealongPC1,whichinvolvesanincreaseinleafand the other two subsections. The only exception involved
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Constrained evolution inthe genus Antirrhinum 1035
Table1Antirrhinumspeciesnamesandabbreviations populationsofthesamespeciestendedtoclustertogether,
although many overlapped. The overlap between species
Numbers
in a space involving 22 phenotypes does not rule out the
usedin
Species Abbreviation morphmetricsa possibility that individual species might be defined by
fewer phenotypes or by characters that were not consid-
SubsectionAntirrhinum
eredhere.
A.australeRothm. au 8/48
The DCA allows co-variation between characters to be
A.barrelieriBoreau ba 3/30
A.boissieriRothm. bo 1/12 representedinthesamephenotypespaceasthepopulations
A.cirrhigerumFilcahob ci 4/33 (Figure4b), illustrating their effects on the separation of
A.graniticumRothm. gr 7/33 populations. The spread of characters along axis 1 sug-
A.grosiiFontQuer go
gested that the phenotypic distinctiveness of subsection
A.latifoliumMiller la 5/10
Kickxiella,whichhashigheraxis-1values,doesnotdepend
A.linkianumBoiss.&Reuterb li 6/25
A.litigiosumPaub lt 7/29 solelyononetypeofcharacter,forexamplesizeorflower
A.majusL.b ma colour. This was further supported by the finding that
A.pseudomajus ps 3/15 subsection Kickxiella differed significantly from the other
Ferna´ndez-Casasb
twosubsectionsforallphenotypes,exceptPC2,PC4andVe
A.siculumMillar si 7/16
(P£0.05inStudent’st-tests).Therefore,subsectionKickxi-
A.striatumFerna´ndez-Casasb st 6/17
A.tortuosumVent.b to 9/99 ellacanbedefinedbyacombinationofphenotypesthatare
SubsectionKickxiella likelytoreflectvariationinanumberofdifferentgenes.
A.charidemiLange ch 1/4 Incontrast,allsubsectionsoverlappedalongDCAaxis2,
A.hispanicumChav. hi 3/16
which also reflects variation in a number of different
A.hispanicum Mh 3/16
characters,althoughmembersofthesamespeciestended
(Moroccanaccessions)
A.lopesianumRothm. lo 1/3 toclusterwitheachotheralongthisaxis.
A.microphyllumRothm. mi 1/3
Plastidhaplotypessupportdistinctevolutionarylineages
A.molleLange mo 6/47
A.mollisimumRothm. ms 3/30
OneexplanationforthepartialoverlapofAntirrhinumspe-
A.pertegasiiRothm. pe 1/6
cies in phenotypic space is that the genus behaves as a
A.pulverulentumLazaro pu 2/24
A.rupestreRothm. ru 5/20 single interbreeding population, in which the genetic
A.sempervirensLapeyr. se 2/11 differences between individuals reflect their geographic
A.subbaeticumGuemes su 1/3 separation (isolation by distance). In this case, the similar
A.valentinumFontQuer va 1/3
phenotypes of Kickxiella species should either reflect their
SubsectionStreptosepalum
closergeneticrelatednessandgeographicproximitytoeach
A.braun-blanquetiiRothm. bb 1/9
A.meonanthum me 1/6 other, or may be independent of both relatedness and
Hoffmans.&Link geography if similar phenotypes have evolved indepen-
dently.Wetookthisscenarioofasinglepopulationasthe
aThenumberofpopulationsofeachspeciesusedinmorphometric nullhypothesisagainstwhichtotestalternatives,including
analysis is shown, followed by the total number of individuals
thepossibilitythatmembersofsubsectionKickxiellashare
sampled.
bThesetaxahavealsobeenconsideredsubspeciesofA.majus,e.g. similar phenotypesbecause they descend from a separate
A.majusssp.striatumorA.majusssp.majus. evolutionarylineage.
We first examined the relatedness of individuals from
their plastid haplotypes. Because previous studies had
populations from Morocco that had previously been detected relatively little sequence variation in Antirrhinum
includedinAntirrhinumhispanicum(subsectionKickxiella; plastids(Jimenezetal.,2005b),wecompared19non-coding
Rothmaler, 1956). These mapped in the same phenotypic lociacross10Antirrhinumspecies,andidentifiedthethree
space as subsections Antirrhinum and Streptosepalum, most variable (trnD-trnT, trn-S-trnR and trnS-trnFM; Shaw
whereasSpanishpopulationsofA.hispanicum,fromwhich etal.,2005,2007).Theirsequenceswerethenobtainedfrom
thespecieswasoriginallydescribed,fellwithintheKickxi- 90 populations that represented the geographic range of
ella cluster. Because Moroccan populations could not be eachspecies,andwerefoundtocontain54polymorphisms
classified as A.hispanicum on grounds of morphology, arranged into 34 different haplotypes. The relationships
theyweresubsequentlytreatedasaseparatetaxonwithin betweenhaplotypeswereinferredbyparsimony.Toallow
subsectionAntirrhinum. fortheresolutionofapurelybranchinghaplotypenetwork
Although DCA supported the phenotypic distinctiveness (Figure5), 12 single-nucleotide polymorphisms that were
ofKickxiella,subsectionsStreptosepalumandAntirrhinum presentinrarehaplotypesandnotfixedinanyspecieswere
were not separated from each other. At the species level, assumed to be homoplasious to mutations supporting
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1036 YvetteWilsonand AndrewHudson
Table2Phenotypesusedinde-trendedcorrespondenceanalysis(DCA)
Character H2a Description Scaleb
PC1 81 FirstPCfromtheallometrymodelofleavesandflowers C
PC2 83 SecondPCfromtheallometrymodelofleavesandflowers C
PC3 75 ThirdPCfromtheallometrymodelofleavesandflowers C
PC4 73 FourthPCfromtheallometrymodelofleavesandflowers C
Anth 91 Intensityofredanthocyaninpigmentationinthecorolla D,5
Branch 67 RatioofthelengthofthelongestaxillarybranchtoHt(below) C
CarpL 88 Carpellength(fromovarybasetostigmatip) C
El 73 Presenceofanthocyaninoutsidethecorollafaceanddorsalpetalsinus D,2
FlNode 67 Numberofthenodeatwhichflowerswerefirstproduced D,29
Ht 73 Lengthofthestemfromcotyledonstofirstflower C
InfNode 84 Maximumnumberofnodesbearingopenflowersatanyonetime D
IntHairs 88 Densityofstemtrichomesbetweennodes1and2 C
LfHair 90 Densityofadaxialepidermaltrichomesinnode-4leaves C
PeCell 65 Densityofadaxialepidermalcellsindorsalpetals C
PedL 75 Pedicellength C
StemW 60 Diameterofthestemmidwaybetweennodes1and2 C
StRatio 73 Ratioofventraltodorsalstamenlengths C
Sulf 87 Presenceofwidespreadyellowauronepigmentinthecorolla D,2
Ve 68 Presenceofdarkeranthocyaninpigmentationovercorollaveins D,2
YelFace 78 Extentofyellowauronepigmentationinthecorollaface D,4
YelHair 75 Extentofyellowtrichomeswithintheventralcorollatube D,5
YelTube 68 Extentofyellowpigmentationwithintheventralcorollatube D,4
aPercentageofthetotalvarianceineachcharacterthatiscontributedbydifferencesbetweenspecies,whichapproximatestotheheritability(H2)of
thecharacterinthebroad-sense.
bCharacterswereeithermeasuredonacontinuousscale(C)orscoredindiscretecategories(D),inwhichcasethenumberofcharacterstatesis
given(e.g.D,2referstoabinarycharacter).Unitsarenotgivenforcontinuouscharactersbecausetheyweresubjecttolinearrescaling,which
madethemdimensionless.
deeper branches. Sequences from Misopates and Chae- (subsection Antirrhinum) has only clade-IV Kickxiella
norrhinum,eachofwhichhasbeensuggestedtobesister haplotypes, and A. sempervirens, A. lopesianum,
to Antirrhinum (Ghebrehiwet etal., 2000; Oyama and A. pertegasii and A. molle in subsection Kickxiella appear
Baum,2004;Vargasetal.,2004),bothrootedtheAntirrhinum fixed for clade-II haplotypes usually found in subsections
haplotypenetworkinthesameposition. AntirrhinumandStreptosepalum. Secondly,A.tortuosum,
The network contained four main clades (Figure5). A. barrelieri and A. australe from subsection Antirrhinum,
Clade I was restricted to Antirrhinum siculum. Clade II and A. pulverulentum from subsection Kickxiella, carry
occurred mainly in subsections Antirrhinum and Strepto- both clade-II and clade-IV haplotypes.
sepalum, and Clades III and IV occurred mainly in Thedepthoftaxonsamplingwasincreasedbygenotyp-
subsection Kickxiella. This distribution of haplotypes inganother273individualsfromanadditional156popula-
cannot be explained solely by geographic separation, tions(TableS1).Clade-IVhaplotypeswereidentifiedbythe
and therefore is inconsistent with the null hypothesis that presence of an MseI site in trnD–trnT, and clade-II haplo-
the genus behaves as a single, unstructured population. typeswereidentifiedbyaTfiIsiteintrnS–trnR.Individuals
Forinstance,membersofsubsectionAntirrhinumcarrying lacking both these sites were assumed to carry clade-III
a clade-II haplotype occur throughout the geographic haplotypes, unless they were accessions of A.siculum
range of the genus, and overlap with Kickxiella species (cladeI).Theadditionalsamplesconfirmedthedistribution
carrying clade-IV haplotypes. A more plausible explana- of haplotypes identified by DNA sequencing, and also
tion is that the distribution of plastid haplotypes reflects revealed that populations of A. litigiosum (subsection
the evolutionary relationships between species: i.e. clade- Antirrhinum) from the Valencia region carried clade-IV
III and -IV haplotypes were present in the Kickxiella (Kickxiella) haplotypes, whereas those from central Spain
ancestor; clade-II haplotypes were present in the separate carriedclade-IIhaplotypesonly.
lineage leading to subsections Antirrhinum and Strepto- One explanationfor the cases of incongruence between
sepalum; and clade-I haplotypes were present in the plastid haplotype and morphology is that ancestral poly-
lineage represented by A.siculum only. However, several morphisms have persisted in the lineages, leading to
taxa have haplotypes that are incongruent with their different subsections (lineage sorting). However, this can-
phenotypes, in one of two respects. First, A. latifolium not easily account for the presence of highly diverged
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Constrained evolution inthe genus Antirrhinum 1037
(a) Nucleargenotypessupportspeciesdelimitationsand
evolutionarylineages
Becausematernallyinheritedplastidgenomesprovideonly
limited information about the relationships between indi-
viduals,particularlyforhybrids,wealsoexaminedvariation
in nuclear genes. Previous studies had found relatively
(b)
little nuclear sequence variation and inconsistent distri-
bution between Antirrhinum species (Gu¨bitz etal., 2003;
Vargasetal.,2009).Wethereforesampledmultiplenuclear
loci as amplified fragment length polymorphisms (AFLPs)
fromthesetof293accessionsthathadbeenusedforplastid
haplotype analysis. Removal of potentially homoplasious
markersleft381informativeAFLPs.
The AFLPs were used to infer the population structure
within Antirrhinum using a Bayesian model, STRUCTURE,
whichassignsindividualstoaspecifiednumberofpopula-
tions by minimizing genetic disequilibria within each pop-
ulation(Falushetal.,2003).Thismethodisnotdependent
on prior taxonomic assumptions, and is able to represent
hybridsasgeneticadmixturesofotherpopulations.
When STRUCTURE was used to assign each accession to
Figure3. Models describing organ shape and size variation in the genus oneoftwopopulations(K=2inFigure7),onepopulation
Antirrhinum. comprised subsection Streptosepalum and most of sub-
(a) Points were placed around node-4 leaves, flattened dorsal petals and section Kickxiella, and the second consisted of subsection
imagesofintactcorollas.Greenpointswereplacedmanuallyandredpoints
AntirrhinumandtheKickxiellaspeciesfromsouth-eastSpain
werespacedautomaticallybetweenthem.Variationinthepositionsofallthe
pointswithinthegenusweredescribedbyprincipalcomponents(PCs).The (A.hispanicum, A. mollissimum, A. rupestre and A. chari-
effects of variation of (cid:2)2SDs in the first four PCs are shown relative to demi).Thisdivisionthereforesupportedthegeneticdistinc-
themeanorganoutlinesinblack.Varshowsthepercentageofthevariationin
tionofStreptosepalumandnorthernKickxiellaspeciesfrom
thegenusthatiscapturedbyeachPC.
subsection Antirrhinumandsouthern Kickxiella. A.latifoli-
um and A.molle, which showed incongruence of plastid
haplotypesandmorphologyconsistentwithhybridorigins,
haplotypes within the same species. For example, the weresuggestedtohaveadmixednucleargenomes,aswas
clade-II and clade-IV haplotypes found in A.tortuosum A.siculum.
(subsectionAntirrhinum)differbyupto21mutations,and Amaximum of 14 different populations could be identi-
thoseinSpanishA.hispanicum(subsectionKickxiella)differ fied by STRUCTURE (Figures7 and S3). Only two of these –
by up to 17 mutations, although only 24 mutations distin- A.graniticumandA.latifolium–consistedalmostentirelyof
guish the most dissimilar haplotypes within the genus as uniquehaplotypes,suggestingahighlevelofcoalescence
a whole (Figure5). Such cases of incongruence are more within these species. The two Streptosepalums remained
consistent with transfer of haplotypes by hybridization withinthesamepopulationasA.grosiiandA.lopesianum
between lineages. Further evidence for hybridization is from subsection Kickxiella. The central Spanish Kickxiella
provided by the location of populations with incongruent species, which are all local endemics and do not overlap
haplotypes. They occur where Kickxiella species overlap in range, were assigned to another population, which
with members of the other subsections: for example,with we subsequently refer to as core Kickxiella. Many of the
subsectionAntirrhinumintheSierraNevadaofsouth-east remainingspeciesfellintogeographicgroupswithsimilar
Spain(Figure6).Incontrast,haplotypesarecongruentwith genetic compositions. In south-east Spain, for example,
morphology where subsections grow in isolation (e.g. A.mollissimumwasdesignatedanadmixtureofgenomes
subsection Antirrhinum in Morocco, western Portugal and from neighbouring A.charidemi and A.rupestre, and the
northern Spain, and Kickxiella in central Spain). The only A.rupestregenomewasalsofoundadmixedinA.hispan-
notable exception is that of A.latifolium, which carries a icumandA.barrelierinearby.
clade-IV haplotype, is restricted to the Alps and does not CultivarsofA.majuswereassignedtothesamepopula-
currently co-occur with a Kickxiella species, although this tion as A. pseudomajus and A. striatum from around the
does not rule out the possibility of hybridization with a easternPyrenees,supportingthedomesticationofA.majus
previouslysympatricKickxiella. fromthispopulation.
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1038 YvetteWilsonand AndrewHudson
Figure4. DistributionofAntirrhinumspeciesinphenotypespace.
Thetwomajoraxesofco-variationin22differentphenotypiccharacterswereidentifiedbyde-trendedcorrespondenceanalysis.Themeanvaluesfor98populations
wereplottedinthespacedefinebytheseaxes(a).MembersofsubsectionAntirrhinumareshownastriangles,membersofsubsectionStreptosepalumareshownas
squaresandmembersofsubsectionKickxiellaareshownascircles.PopulationsarecolouredasinFigure2,toreflecttheirgeographicorigins.Thelabelmhdenotes
MoroccanaccessionspreviouslyassignedtoA.hispanicum.Thecontributionofeachcharactertothephenotypicspaceisshownin(b).
CharactersandtheirabbreviationsaredetailedinTable2,andaccessionnumbersforpopulationsarepresentedinFigureS2.
Figure5. Relationshipsbetweenplastidhaplotypes.
Sampledhaplotypesareshowninboxescontainingthespeciesnamesandaccessionnumbersoftheplantscarryingthem.
Themostparsimoniousrelationshipsbetweenhaplotypesareshownbylinesconnectingboxeswithcrosslinesthatrepresentthenumberofmutationsbywhich
haplotypesdiffer.Diagonalcrosslinesshowmutationsthatwereassumedtobehomoplasiouswithearliermutations.Branchesinwhichmutationsintroducedan
MseIorTfiIrestrictionsitearealsoshown.ThetreeisrootedwithsequencesfromMisopatesorontium,whichdiffersfromthein-groupby19mutations.
The ability to progressively subdivide the genus into onomy,andsuggestedthatAFLPgenotypesmightresolve
populationswithboundariesthatcorrespondedtoclassical relationships between species, even though extensive
species delimitations provided support for traditional tax- admixture was inferred for all but A.graniticum and
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Constrained evolution inthe genus Antirrhinum 1039
Figure6. Geographicdistributionofplastidhaplotypes.
MembersofsubsectionAntirrhinumareshownascircles,membersofsubsectionKickxiellaareshownastrianglesandmembersofsubsectionStreptosepalumare
shownassquares.Filledshapesrepresenthaplotypesthatarecongruentwithmorphology;unfilledshapesshowincongruenthaplotypes.
A.latifolium. Therefore, genetic distances were calculated Antirrhinum, supporting the phenotypic evidence for their
betweenallpairwisecombinationsofindividuals,andwere membership of this subsection. The third major clade
then used in neighbour-joining analysis with Misopates consisted of A. meonanthum and A. braun-blanquetii
orontium as the out-group. When all individuals were (subsection Streptosepalum) from north-west Iberia,
included in the analysis, members of the same species togetherwithtwooftheirneighbouringKickxiellaspecies,
usuallyclusteredwitheachother,astheydidintheSTRUC- A.grosiiandA.lopesianum.
TURE analysis, but the relationships between species were
DISCUSSION
poorly resolved (FigureS4). One explanation for poor res-
olutionisthatsomeofthetaxahavehybridorigins,andso BycomparingthegenotypesofAntirrhinumpopulationsat
carry alleles from multiple lineages. These shared alleles multiplenuclearlociwehaveidentifiedthreewell-supported
could reduce resolution both within and between the cladesofspecies.Thetwolargestcladesdonotcorrespond
parentallineages.Wethereforeexcludedpotentialhybrids toindividualsfromthesamegeographiclocations,although
–i.e.plantswithincongruentnuclearandplastidgenotypes, they contain geographic subgroups within them, and so
or apparently admixed nuclear genomes – if they reduced cannotbeexplainedbyageneticstructurethatrelatessolely
the overall support. The remaining accessions were to geographic isolation. The clades are therefore likely to
resolvedintothreemajorclades(Figure8).Onecomprised representdifferentevolutionarylineages.
the core Kickxiella, within which A. pulverulentum formed OnecladeconsistsofspeciesfromsubsectionKickxiella,
a supported group with its central Spanish neighbour which was originallydefined by the shared phenotypesof
A. microphyllum, and A.pertegasii grouped with the geo- itsmembers.Morphometricanalysisundercommongarden
graphicallydistantA.subbaeticum.Thesecondmajorclade conditions confirmed the phenotypic distinctiveness of
consisted of species from subsection Antirrhinum, with thissubsection,withcommoncharactersthatincludesmall
A.siculum at its base and A.graniticum sister to the stature,small,ovateandhairyleaves,andsmallpaleflowers
remainingtaxa,whichincludedtwogeographicgroupings: with reflexed petals. The ancestral Kickxiella is therefore
A.pseudomajus,A.striatumandA.litigiosumfromaround likelytohavesharedthesecharacters,and,likeitsdescen-
theeasternPyrenees,andA.linkianumandA.cirrhigerum dants,tohavelivedonrockfaces.Thesecondmajorclade
from the west coast of Iberia. All accessions of Moroccan comprised mainly members of subsection Antirrhinum.
‘A.hispanicum’ clustered together within subsection These are dark-pink- or yellow-flowered species that form
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1040 YvetteWilsonand AndrewHudson
Figure7. Assignmentofindividualstopopulations.
Plantswereassignedtodifferentancestralpopulations,eachshowninadifferentcolour,bytheirnucleargenotypes.Representativeassignmentsareshownfor
modelsinwhichthemaximumnumberofpopulations(K)wassettoeithertwoor17.BlackbarsdenotemembersofsubsectionAntirrhinum,dark-greybarsdenote
membersofsubsectionStreptosepalumandlight-greybarsdenotemembersofsubsectionKickxiella.
Figure8. Aneighbour-joining tree ofAntirrhi-
numspecies.
The tree was made from pairwise nuclear
geneticdistancesbetweeninferrednon-hybrid
accessions.Supportvaluesareshownfornodes
thatwererecoveredinmorethan50%of1000
bootstrapreplicates.
large, upright plants, with larger, more elongated leaves, classical division of Antirrhinum into three morphological
andareabletogrowincompetition.Thethirdmajorclade subsectionsthereforeappearspartlynatural.
contains both of the species from subsection Streptosep- The overlap of the three subsections along DCA axis 2
alum and two species with Kickxiella phenotypes. The impliesthatallsubsectionsvarytoasimilarextentforthe
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Constrained evolution inthe genus Antirrhinum 1041
different morphological and flower-colour characters that Mediterranean (Kropf etal., 2003; Dixon etal., 2007).
contribute to this axis. This is consistent with ancestral Contractiontoalpinerefugiacanalsoexplainwhyallhigh
variation that has not been fixed during divergence of mountainrangesinIberiaandsouthernFranceeitherhave
subsections, although fixation may have occurred during their own endemic core Kickxiella species or, in the case
speciation because members of the same species tend to of the Alps (A.latifolium), the Pyrenees (A.molle and
clusteralongaxis2. A.sempervirens) and the Baetic Cordillera (the south-east
Distinct plastid lineages were also identified, and each Spanishspecies),retainvestigesoftheKickxiellalineagein
was found predominantly in subsections Antirrhinum and theirplastidandnucleargenotypes.PopulationsfromtheRif
Streptosepalum, or in Kickxiella. Many species carry con- Mountains of Morocco have been recognized as a species
gruentnuclearandplastidgenomes,forinstanceA.micro- withinsubsectionKickxiella(A.martenii;Rothmaler,1956),
phyllum,A.subbaeticumandA.valentinumhavegenotypes suggesting that Kickxiella might once have extended into
expected of direct descendants of the core Kickxiella line- North Africa. As Kickxiella species are found on dry rock
age, whereas A.siculum, A.graniticum, A.pseudomajus, faces, mostly at higher elevations, contraction in their
A.striatum, A.cirrhigerum and A.linkianum have both ranges could have occurred during periods of increasing
plastid and nuclear genomes representative of subsection rainfallandwarming.Severalmajoreventsofthiskindhave
Antirrhinum. occurred in the Mediterranean region since the end of the
However, several species appear to have nuclear and lasticeage(Fletcheretal.,2010).Thesameenvironmental
plastid genomes from different lineages. These include changes that reduced the range of Kickxiella could have
A.sempervirens,A.pertegasiiandA.pulverulentum,which allowedsubsectionAntirrhinumtospreadthroughlowland
have subsection Antirrhinum plastids but core Kickxiella regions, perhaps from coastal refugia, and therefore to
nuclei. Such cytonuclear incongruence can be explained hybridizewithKickxiellainregionsofcontact.
ifhybridizationbetweensubsectionswasfollowedbyintro-
Characterevolution
gression involving Kickxiella, leading to the capture of an
Antirrhinum plastid by a pre-dominantly Kickxiella nuclear The genetic relationships between Antirrhinum species
genome.Similarly,A.tortuosumcouldalsohavecaptured suggestthatseveralhybridizationeventsoccurredbetween
its diverse incongruent plastids from multiple Kickxiella subsectionsAntirrhinumandKickxiella,yetthephenotypes
donors. ofthetwosubsectionsremaindistinctfromeachother.This
Theremainingspeciessharetheirnucleargenotypeswith is most apparent in two species that are sympatric in the
bothAntirrhinumandKickxiellalineages,andconsequently Sierra Nevada of south-east Spain: A.rupestre (Kickxiella
cannot be assigned to one of the three major clades. phenotype) and A.barrelieri (Antirrhinum phenotype),
However, they share plastids and nuclear genes with their which have indistinguishable AFLP and plastid genotypes,
geographicneighbours,suggestingthattheyaretheresult consistent with their continuing hybridization. Therefore,
of hybridizations between subsections Antirrhinum and although hybridization has the potential to create new
Kickxiellawithoutsignificantintrogression.This isparticu- adaptivecombinationsofphenotypes(e.g.,Riesebergetal.,
larlyapparentinthespeciesfromsouth-eastSpain,which 2003),evolutioninAntirrhinumappearstobeconstrained.
have similar nuclear genotypes and carry both Kickxiella Two factors suggest that the evolutionary constraints
and Antirrhinum plastids. Hybridization could also have within Antirrhinum are not developmental. Firstly, the
given rise to the nuclear clade consisting of the two phenotypiccharactersthatdistinguishsubsectionsKickxiel-
Streptosepalum species together with A.grosii and la and Antirrhinum were chosen to represent variation in
A. lopesianum from subsection Kickxiella, because these multiplegeneswithindependenteffects.Secondly,hybrids
species are found together in north-west Iberia and carry betweenA.majusandA.charidemihaveshownthatmulti-
divergedplastids.Alternatively,theclademightrepresenta ple genes underlie the differences between their Antirrhi-
third ancestral lineage in which some members have numandKickxiellaphenotypes(Langladeetal.,2005;Feng
capturedincongruentplastidsfromsubsectionKickxiella. etal., 2009). For instance, 10 genes with additive effects
Itisnotpossibletoresolvetheorderinwhichthethree werefoundtoaffectleafsize,sothatF hybridsandalmost
1
major lineages diverged from each other. However, the allF progenyhadleafphenotypesthatwereintermediate
2
distribution of the core Kickxiella species between the between the two parents (i.e. they occupied the gap in
mountains of central Spain suggests that this lineage was phenotypic space between subsections Antirrhinum and
once more widespread, and became fragmented on con- Kickxiella).
tractiontoitscurrentmountainrefugia.Thisfragmentation A more likely explanation is therefore that Antirrhinum
couldhavecontributedtotheformationofdistinctspecies. phenotypesareconstrainedbyselection.Forsomecharac-
Asimilarrange contraction hasbeen proposedto account tersthismightreflectcontrastingadaptationstolifeonbare
for the genetic relationships in several alpine species that rockfacesorincompetition:forinstance,smallorgansand
are currently restricted to mountains around the western densehairsmightbeadvantageousinlimitingwaterlosson
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ThePlantJournalª2011BlackwellPublishingLtd,ThePlantJournal,(2011),66,1032–1043