Systematics of Dirca (Thymelaeaceae) based on its Sequences and ISSR Polymorphisms

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Horticulture Publications

Horticulture

12-28-2004

Systematics of Dirca (Thymelaeaceae) based on its

Sequences and ISSR Polymorphisms

James A. Schrader

Iowa State University, [email protected]

William R. Graves

Iowa State University, [email protected]

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Systematics of Dirca (Thymelaeaceae) based on its Sequences and ISSR

Polymorphisms

Abstract

The genus Dirca consists of three disjunct species of shrubs. Dirca palustris is found in the eastern United

States and adjacent Canada; D, occidcntahs is Umited to six counties near the San Francisco Bay in California;

and the recently discovered D, mcxicana is known from one isolated population in northeastern Mexico. The

three species have been described and classified according to morphological characters, but the morphological

evidence does not provide a clear assessment of the relationships among the species. Morphologically D.

mexicana most closely resembles D. occidenlalis., but known biogeographical trends raise doubt regarding

how the three species are interrelated. We used molecular techniques to examine and clarify phylogenetic

relationships among the three species of Dirca. Evidence from Internal Transcribed Spacer (ITS) sequences

and Inter-Simple Sequence Repeats (ISSR) polymorphisms confirms the species-level divergence of Dirca

mexicana and reveals that, despite their morphological similarity D. mexicana and D. occidenlalis -cue the

most divergent of the three species genetically while D, me.xicana and D. palustris are the most closely related.

The molecular evidence also demonstrates that Dirca occidenlalis was the first of the three species to diverge

and has undergone the greatest degree of differentiation since divergence.

Disciplines

Agricultural Science | Horticulture | Nature and Society Relations | Plant Biology

Comments

This article is from SIDA, 21(2):2004: 511-524. Posted with permission.

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SYSTEMATICS OF DIRCA

(THYMELAEACEAE)

BASED

ON

ITS

SEQUENCES

AND

ISSR

POLYMORPHISMS

James

A.

Schrader

and

William

R.

Graves

Departmentof Horticulture

IowaState University

Amesjowa50011-1WO,USA.

[email protected]

ABSTRACT

The genusDirca consistsofthree disjunct speciesofshrubs.Dirca palustrisisfoundintheeastern United StatesandadjacentCanada;D,occidcntahsisUmitedto sixcounties near theSanFrancisco

Bayin California;andtherecently discoveredD,mcxicanaisknownfromoneisolatedpopulationin

northeastern Mexico.Thethree species have been describedandclassifiedaccordingto

morphologi-calcharacters, but the morphological evidence does not providea clearassessmentofthe

relation-shipsamongthespecies.MorphologicallyD.mexicana mostclosely resemblesD. occidenlalis.,but

knownbiogeographical trendsraisedoubt regardinghowthe three species areinterrelated.

We

used molecular techniquestoexamine andclarifyphylogenetic relationshipsamongthe three speciesof

Dirca.EvidencefromInternal Transcribed Spacer (ITS) sequencesandInter-SimpleSequence Re-peats (ISSR)polymorphismsconfirms the species-level divergenceofDircamexicana andreveals

that,despitetheirmorphological similarityD.mexicana andD.occidenlalis-cuethemostdivergent

ofthe three species genetically whileD,me.xicanaandD.palustrisarethemostcloselyrelated.The

molecular evidence also demonstrates that Dirca occidenlaliswasthefirst ofthe three speciesto

divergeandhasundergonethe greatest degreeofdifferentiation since divergence.

RESUMEN

Elgenero Diixa consta detresespeciesde arbustos separadas geograficamente. Dirca palustrisse

encuentra enlaparte Este delosEstadosUnidosylaparte contigua deCanada;D. occidcntalisse

limitaa seiscondadosalrededordelabahia deSanFrancisco en California, EE.UU.; ylarecien descubiertaD.mexicanaseconocesolamente deunapoblacion aislada enlaparte noreste de Mexico. Lastresespecieshansido descritas y clasificadas segun sus caracteristicas morfologicas, perolas

pruebas morfologicashansido consideradascomonoconcluyentes para hacerunaevaluacion clara delasrelaciones entrelasespecies.Morfologicamente, D.mexicanaseparecemasa D.occidenlalis, perolastendencias biogeograficas establecidas creandudassobreelgrado de parentesco entrelas

dosespecies.Hemosusadotecnicas moleculares paraexaminaryclarificar lasrelacionesIilogeneticas entrelas tresespeciesde Dirca. Porlaspruebas queencontramosenlassecuencias del Espaciador Transcrito Interne (ETI) [Internal Transcribed Spacer(ITS)|ypolimorfismosde Repeticiones de Secuencias Intra-Simple (RSIS) [Inter-SimpleSequenceRepeats(ISSR)],Uegamosa lassiguientes conclusiones;1.Dircamexicana merecesercatalogadacomoespecie;2.Dirca occidenlalis fuela

primeradelasespeciesen evolucionar, y haexperimentadoelmayorgrado de diierenciacion desde su divergencia; y3.Contrarioasus morfologias.D.mexicanatieneungrado de parentescomasalto,

en terminos geneticos, conD.palus(n.squeconD.occiclentali.s.

INTRODUCTION

The

genus DircaL.iscomprisedof three species ofslow-growing, understory shrubs found almostexclusively

m

natureonsteep, west- ornorth-facingslopes

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512 BRIT.ORG/SIDA21(2)

abovea

waterway

(Johnson1994;

Nesom

&

Mayfield1995;Graves2004).

While

D.palusllis L.isthe

most

common

ofthe speciesandisfoundinsparsely

dis-tributed colonies overmostofeasternNorth America,the othertwospecies,D. occidcnUilisGrayandD.mcxiaina

Nesom

&

Mayfield, are rare

and

endemic. Direeloccidcntalisisisolatedtoasix-countyregion

surroundmg

theSan

Fran-ciscoBayin Calilornia,

and

D.mexicanuis

endemic

toonly onevalleyinthe Sierra

Madre

OrientalMountainsofTamaulipas,Mexico

(Nesom

&rMayfield 1995;Graves2004).Althoughthereisinterestinthegenus duetothe obscurity of plantsinthe wild, the vulnerability of plants

m

California(Johnson1994), thediscoveryofthe

new

species inMexico

(Nesom

&

Mayfield1995),andthe potentialto utilizeplants ofthisgenusasshade-tolerantshrubsfor

managed

landscapes(Dirr1998),noresearchhas been donetodeterminethe

phyloge-neticrelationships

among

the three species ofDirca.

InthemostrecenttreatmentofthegenusDirca,

Nesom and

Mayfield(1995)

showed

the three disjunctstobemorphologicallydistinct

and

foundthat D.

mcxicana

more

closelyresembledD.oaulcntahs thanitdidD.paluslns.But,

they alsoquestionedthe relative similarity of D.mcxiaina

and

D.Oiadcntalis

"inviewoithewell-knownpattern of close relationship

and

disjunction

be-tweenspecies of the easternand southeastern UnitedStates

and

the sierra of

northeastern Mexico"

(Nesom

&

Mayfield1995).Considering biogeographical

trends,

Nesom and

Mayfield(1995)believed the bestexplanationforthe origin of three disjunct species was,first,the isolation of the ancestors of D. occidentalis

Iromthose of D. palustns, and,

more

recently, the isolation of the ancestors of

D.mcxicana tromthose of D. palustris.

Our

goalsweretoresolvetheapparent inconsistencybetween morphological and biogeographical evidence by

exam-ining thegenotypicrelationships

among

the three species of Dirca,to recon-struct thephylogenyofDircaspp.byutilizing

methods

ofmolecular

system-atics,

and

todetermineifmolecular evidence supportsthe classification of D.

mcxicana

asadistinct species.

We

usedtwoclasses ofmolecularmarkers, InternalTranscribed Spacer(ITS)

sequences

and

inter-SimpleSequence Repeats(ISSR),toquantifythegenotypic

variabilityotDirca.ITS techniques

compare

the internal transcribedspacer sequencesofthe 18S-5.8S-26Snuclear ribosomal

DN

A.

They

hold

many

advan-tagesover othermethods, includingbiparental inheritance,intergenomic vari-abilitysuitablelorphylogeneticinferenceatthespecific,generic,

and

family

levels(Baldwinf992;Baldwinetal.1995),and easy amplibcation with

univer-salprimers(Whiteetal.1990).ITSsequence dataarcabundant

and

easily ac-cessibleinpublic databases,enablingdirectcomparisons

among

taxa

and

thus

areusedextensivelytorbotanicalphylogeneticsatgeneric

and

inlrageneric levels(Alvarez

&

Wendel

2003).

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SCHRADERANDGRAVES,SYSTEMATICS OF DIRCA 513

capacityto resolvemoleculardifferences

below

the specificlevel,but ISSRsare alsovalued because they samplea largeportionofthe

genome and

therefore

avoidthe bias

accompanying

phylogenies based

on

thesequenceofonly oneor afew genes (Schrader

&

Graves 2004).

Used

together, these

two methods

can provideexcellent resolution of genetic variabilityat

and

belowthefamilylevel

and

provedeffectiveforassessing infrageneric differenceswithinthegenus

Dirca.

MATERIALS

AND METHODS

Samplesof

genomic

DNA

were extracted fromleaftissue of

24

seedlingsby

utilizing thetemplate preparationservice of the

DNA

Sequencing

and

Synthe-sisFacilityatIowaStateUniversity

The

seeds

had

beencollectedfromplants ofallDircaspp. in their native habitats (Table1).Eight samplesof D. palustris

and

seven samples eachof D.

mexicana and

D. occidentaliswere usedto

com-paregenetic variation

m

Dirca. In addition,two samplesof

Daphne mezereum

L.(Thymelaeaceae)

and

one sequence

from

Hibiscus rosasinensisL.,alsofrom withintheOrderMalvales,were usedasoutgrouprepresentativesfor

phyloge-neticanalyses(Table1).

Daphne mezereum and

H.rosasinensis

(GenBank

sample,Shi

&

Yuan

2001) were chosentoestablish ancestral-character polarity

atthegeneric

and

familylevels,respectively.

ITS

methods.—

We

amphfied

the entireITS region(ITS1+5.8S+ ITS2) of

each sample by usingtheuniversalprimers ITS4

and

ITS5 (Whiteetal.1990),

separatedtheITS

bands

by useof agarose-gel electrophoresis, cut outbands,

and

eluted the purifiedsamples

from

theagarose withthe

GenElute™

Gel

Ex-traction Kit

(SIGMA,

St.Louis, Mo.).For ITSamplification,

we

used25-|tL reac-tionmixesthatcontained 50 ngoftemplate

DNA,

0.8 |aM ofeachprimer,

600

|LtM

dNTP

mix

(SIGMA),

Ix

reaction buffer thatcontained Mg(0Ac)2,

and

1.5

units of

KlenTaq

LA

DNA

polymerase (SIGMA). Thermocycler conditions were 94°

C

for5

min

(initialdenaturing),94°

C

forI

min

(denaturing),45°

C

for1

min

(annealing),

and

72°

_C

for2

min

(extension),for35cycleswiththe final

extensionat72°

C

for5min.

The

purifiedsamples were sequenced

on

an

Ap-pliedBiosystems (ABI) 3100 Genetic Analyzer by usingtheforward primer

(ITS5)

and

thelong-readservice of the

DNA

Sequencing

and

SynthesisFacility

atIowaStateUniversity

We

used

CLUSTAL

X

Multiple Sequence

Alignment

Program

(version1.8)to alignsequencesforphylogenetic analyses

and

to con-firmthepresenceofthe plant-conserved, 5.8S

rDNA

motif(Jobes

&

Thien1997) inallsamplesequences.

ISSR

methods.—

ISSR fragmentsforeachof the

24 DNA

samples were

am-plifiedforthree replicationswith eachof eight fluorescent3'-anchored ISSR primers [(CA)6RG,

(AOsG,

(

AG)8YT

(CT)8TG,

(GTG)3GC,

(CA)6RT,

(CAObRC,

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Table1.Origins ofthe 25 individualssampledfor ITSandISSRanalysis.Allplantssampledarefrom theSchraderandGraves DIrca collectionatIowa State University except Hibiscusrosa-sinensis,which wasobtainedthroughaBLASTsearch(Shi& Yuan2001).Latitudeandlongitude are according to Global PositioningSystem(GPS)andareincludedwhenknown.

Species Plant # Accession^ Origin Latitude Longitude Analysis

Dircamexicana D.mex1 DMTA02 TafTiaulipas 23°59'16r'N 99°28'635"

_W

ISSR

D.mex2 DMTA02 Tamaulipas 23°59'161"N 99°28'635"W ITS7ISSR

D.mex3 DMTA02 Tamaulipas 23°59'161"N 99°28'635"W IT5/ISSR

AllinMexico D.mex4 DMTA02 Tamaulipas 23°59'16r'N 99°28'635"W ITS/ISSR

D.mex5 DMTA02 Tamaulipas 23°59'161"N 99°28'635"W ITS7ISSR

D.mex6 DMTA02 Tamaulipas 23°59'161"N 99°28'635"W ITS/ISSR

D.mex7 DMTA02 Tamaulipas 23°59'161"N 99"28'635"W ITS/ISSR Dircaoccidenralis D.occ 9 DOFT02 Contra Costa Co. 37°49'555"N 122°10775"WITS/ISSR

D.occ1 1

DOAV02 Contra Costa Co. 37°56'015"N 122°18'030"WITS/ISSR D.occ 12DOAV02 Contra Costa Co. 37°56'015"N 122°18'030"WITS/ISSR

AllinCalifornia D.occ1 3

DOAV02 Contra Costa Co. 37°56'015"N 122°18'030"WITS7ISSR D.occ 14DOAV02 Contra Costa Co. 37°56'015"N 122°18'030"WITS/ISSR D.occ15 DOAV02 Contra Costa Co. 37°56'015"N 122°18'030"WISSR D.occ 16DOAV02 Contra Costa Co. 37°56'015"N 122°18'030"WITS/ISSR Dirca palustns D.pal17 DPLSPOl BooneCo. 41°59'586"N 93°53'058"W ISSR

D.pal18 DPLSPOl BooneCo. 41°59'586"N 93°53'058"W ITS/ISSR D.pal19 DPLSPOl BooneCo. 41°59'586"N 93°53'058"W ITS/ISSR

AllinIowa D.pal20 DPLSPOl BooneCo. 4r59'586"N 93°53'058"W ITS/ISSR

D.pal 21 DPIAOl BooneCo. 4r56'316"N 93°5r595"W ITS/ISSR D.pal22 DPIAOl BooneCo. 4r56'316"N 93°51'595"

_W

ITS/ISSR D.pal23 DPRMFOlClayton Co. 42°48'838"N 9r20'437"W ITS/ISSR D.pal24 DPRMFOlClayton Co. 42°48'838"N 91°20'437"W ISSR Dapline DAPH25DMEZ03 Purchased ForestFarm Williams, ITS/ISSR

mezereum Nursery Oregon

DAPH27DMEZ03 Purchased ForestFarm Williams, ITS/ISSR Nursery Oregon

l-libiscusrosa- Blast AF460187 NCBI website^ ITS sinensis search

'^Voucher speclrTiens:D/rcomex/cano,AccessionDMTA02.Tamaulipa5,Mexico,Sc/irader;2'^ (ISC).D/rcaocc/denfofo,

AccessionDOFT02, FrenchTraitContra CostaCo.,Calif.,Sc/7rader;25(ISC),D;rcaoccidenfo/;s,AccessionDOAV02,

AquaVista,Contra CostaCo.,CaliCSchrader 126(ISC).Dirca palustris,AccessionDPLSPOl,LedgesState Park,

Boone Co,Iowa, 5c/irac/ef?27(ISC).D/rcopa/asfr/,5,Accession DPIAOl, Iowa Arboretum, BooneCo.,lQwa,Sc/)ra*A

;28(ISC).Dirca po/(jstm,AccessionDPRMFOl,Ret/MemorialForest,ClaytonCo.,lowa,Schrader/29(ISC),Daphne

mezereum. Accession DMEZ03, Purchased fromForestFarm NurseryWilliams,Oregon,Sc/irader130(ISC),

^NationalCenterforBiotechnology Information(NCBI).www.ncbi,nlm.nih,gov/entrez/query,fcgF'cmd = Retrieve&db=Nucleotide&list_uids=32364883&dopt=GenBank

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FacilityatIowaStateUniversity.Optimizationreactionswereruntodetermine proper reaction conditions

and

reagentconcentrationsforconsistent

PCR

amplification.Thermocycler conditionsfor

ISSR-PCR

were 94°

C

for5

min

(ini-tialdenaturing),94°

C

for30s(denaturing), primer-specifictemperatures(see

below)for

45

s(annealing),

and

72°

C

for2

min

(extension),for30-33cycles

withthe finalextensionat72°

C

for5min.Annealing temperaturesforthe eight

primerswere 47°

C

for(CA)6RG, 52°

C

for

(AOsG,

56°

C

for

(AOsYT,

52°

C

for

(CDsTG,

56°

C

for

(GTOsGC,

48°

C

for(CA)6RT,52°

C

for

(CAC)3RC,

and

52°

C

for

(CTObSG.

Inour25-)aLreaction mixes,

we

used 50

ng

oftemplate

DNA,

1.2

^iMofprimer,

300

\iM

dNTP

mix

(SIGMA),

Ix

reaction buffer thatcontained Mg(0Ac)2,

and

1unit ofKlenTaq

LA

DNA

polymerase (SIGMA).

Amplificationproductswere processedatthe

DNA

Sequencing

and

Syn-thesisFacilityatIowaStateUniversity.AppliedBiosystems(ABI)377 automated

DNA

sequencingsystems separatedthe

DNA

byelectrophoresis

and

collected the gelimage.

Image

data were analyzedby

usmg ABI

PRISM™

GeneScan®

soft-ware

that resolves

DNA

fragment lengthdifferences assmallasone basepair

ISSR

bands

(loci)were scoredas"1" for

band

presence

and

"0"for

band

absence.

Only bands

thatappearedinatleasttwoofthe three replicationswere consid-eredpresent.

A

locus

was any

fragment lengththat

was

presentinatleastone sample.

The

resultingtwo-state(1•0)data matricesforthe eightprimerswere

combined

toformacumulative dataset forassessingmolecularrelationships

among

the three species of Dirca.Data

from

three of the primers,(CA)6RG,

(AOsG, and

(AG)8YT,were

compared

withthe results ofSchrader

and

Graves (2004)tohelpassessthe relativetaxonomicdistancesexpectedforspecific

and

subspecific hierarchical levelsaccordingtoISSR

methods

(Tables2

and

3). Dataanalysis.

—

Cladisticanalyses were performed by using

PHYLIP

(Phy-logenyInferencePackage;Felsenstein 1995).

We

usedthe

Dnapars

program

for

Wagner parsimony

(Kluge

&

Farris1969) analysis ofITS data

and

the

Mix

pro-gram

for

Wagner parsimony

analysis ofISSRdata.

The

Seqboot

program

was

usedforbootstrap(Felsenstein1985)

and

jacknife (Farrisetal.1996)analyses (1000 resamplingseach),

and

theNeighbor

program

forneighbor-joining analy-ses (Felsenstein 1995).GeneticdistancesforITS analyses were generated under

the

Kimura

2-parameter

model (Kimura

1980)by usingtheDnadist

program

ofPHYLIP,

and

geneticdistancesforISSR analyses were Euclideandistances

(Sneath

&

Sokal1973).

We

compared and

contrastedour ITS

and

ISSR

phylog-enies, aproceduretermed"crossmatrixdisparity"by

Bateman

(1999),then

merged

thetwo datasets fora"simultaneousanalysis"(Nixon

&

Carpenter1996)

using unweighteddistances(Sneath

&

Sokal 1973)fromthetwo datasets.

RESULTS

ITS.

—

SequencingoftheITSregionprovided complete sequencesforITS1,the

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nucle-516 BRIT.ORG/SIDA21(2)

otidcs)

and

26S(22 nucleotides)

rRNA

genes.

The

ITSregionvaried inlength

among

the tour species

we

evaluated (630

bp

forD.mexicana,617

bp

lorD.

occidentalis,625

bp

lorD. palustris,

and

596

bp

lorD.mezcreu

m) and

contained

ample

sequencevariationforspecies-levelphylogeneticanalysis.

The

5.8S

rRNA

gene

was

165basepairslongin allsamples

and

thesequence

was

identicalin D.

mexicuna

and

D. palustris,with onlyonesitedillcrenceiorD.occidentahsand

foursitedifferencesforD.

mczcreum,

oneof

which was

common

to D. occidcnlalis.There

was

nointraspecificsequencevariation

among

ITSsamples fromD.occidentalism D. palustris,norD.mczcrcuin.There

was

variationat six sites

among

tliesixITSsamplesol D.rncxicana(fiveinsertion/deletionsand onetransversion,all inITS1),but theconsensus sequence

was

identicaltothe

sequenceofoneofthosesamples(D.mex2).

Inour phylogeneticanalysis,exhaustive searchesproducedsingle most-parsimonioustreeswith eachotthetwo chosen outgroups,D.niezereum

and

H. rosa-sinensis,

showing

196

and

496 evolutionarysteps,respectively.

The

trees

agreedintopology

and

revealedthat,withinthegenus Dirca,the ancestralline olD.occidentalis

was

thefirsttodiverge(Fig.1

and

2).Bootstrapandiacl<nlfe

percentages

(100%

lor allcfades)

showed

verystrongsupportforthistopofogy,

and

ourresultsusingthe family-level root(outgroupH. rosa-sincnsis)support

thechoiceofD.

mczcrcum

asasuitableoutgrou]?lorphylogenetic reconstruc-tionofDura.

Sequence

divergence

(Kimura 2-parameter

distance)

between

D. occidcnlaliScindthe oihertwoDircaspeciesioccidcntalistorncxicana= 0.0592, occidentalistopalustris =0.0560)

was

much

greaterthanthedivergence

be-tweenD.mexicana andD. palustris (0.0074).Results of theneighbor-joining analysesrevealbotha

much

earlierdivergenceolD. occidentalisthanthe

di-vergenceoltheothertwospeciesandgreater differentiationthantheothertwo

species since theirtimesofdivergence(Figs.1and2).Thisfeatureis particu-larlyapparent

when

thephylogenyisconstructedloyusingageneric-level root

(Fig.1),butisstillevident

when

usingafamily-leveloutgroup(Fig.2). ISSR.

—

Amplificationwiththe eightfluorescent3'-anchoredprimers

yielded709 ISSRloci(fragmentlengths) across the lour species.

The

fine reso-lutionof LSSR techniquesisillustratedbythehigh degreeof

polymorphisms

found

among

the threespecies ofDirca

and

thehigh

number

of

species-spe-cific foci(Table2).

Comparing

these resultswiththe results ofanearlier,

sub-specilicstudythatinvolvedthe

same methods

andthree of the

same

ISSR

prim-ers(Schrader

and

Graves 2004j,conlirmedspecies-leveldivergence

among

the threetaxaotDirca.Forthe threeprimers used

m

bothstudies,the three species ofDircahad nearly doublethepercentageofpolymorphicloci

and

taxon-spe-cific lociofthoseseen

among

the threesubspeciesofAlnus maritima(Marsh.)

Muhl.ex Nutt. (Table2).

The

differentiation revealedinthe

number

of taxon-specificlocifound

among

the three species otDirca

was

consistentwiththe

(9)

SCHRADERANDGRAVES, SYSTEMATICS OF DIRCA 517 0,0046

\^Dirca

mexicana

0.0333 (100)

"y^Dirca

palustris 0.(X)28 0.214 0.0206 'Dirca occidentalis

Daphne mezereum

Fig. 1. ITSneighbor-joiningdendrogram showingthe inferred phylogenetic relationshipamongthe three species of

D/ffa.Topologyindicatesthat of the singlemost-parsimonioustree.Numbersindicate relativebranch lengths;

num-bersinparentheses areboththe bootstrapandjacl<nifepercentages,which wereidentical.Dahne mezereum, another

memberofThymelaeaceae,was usedas theoutgroupinorder to establish ancestral-character polarity of theITS

se-quence. 0.405 0.056 (100) 020 0.016^

Dirca mexicana

(100) ixiiinDirca palustris

Dirca

occidentalis

Daphne mezereum

Hibiscusrosa-sinensis

Fig.2.ITSneighbor-joiningdendrogram showingthe inferred phylogenetic relationshipamongthe three species of Dircaand anothermemberofThymelaeaceae,Oo/inemezereum.Topology indicates that of the single most-parsimoni-ous tree.Numbersindicate relativebranch lengths;numbersinparentheses are both the bootstrapandjacknife per-centages,whichwereidentical.W/Mcusroia-sinensis,anothermemberofOrder Malvales,wasused as theoutgroupto establish ancestral-character polarity of theITSsequence.

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Table2.Percentage ofpolymorphiclociand percentageandnumberof taxon-specificbands

re-solvedby using three(DircaandAInus)andeight(0/rco),3'-anchored I5SR primers. ResultsforDirca arefromthepresent study;results for /^/nu5wereobtainedbySchraderandGraves (2004) by using thesame methods andthree of thesameprimersasthepresentstudy

Primers

(CA)6RG (AGlsYT (AOsG Three primers

Alleight primers

%

ofpolymorphicloci

Dircaspecies 87 95 88 90 83 AInusmanlimasubspecies 41 55 46 48

%

Taxon-specificloci

Dircaspecies 52 73 47 57 55 AInus maritimasubspecies 15 31 14 22 # Taxon-specificloci Dircamexicana 7 17 13 37 112 Dircapalustris 9 10 6 25 67 Dirca occidentalis 16 16 11 43 164 AInus maritima subsp.oWo/iomens/s 1 3 4 subsp.georg;ens/5 1 2 3 subsp. mor/f/mo 2 4 6 AInusjaponica 9 16 12 37

species-leveldillerentiationintaxon-specificlocioftheoutgroupA/nu.s

japonica(Thunb.)Steud.foreach

and

affprimers

and

was

over fourtimesthat

shown

arnongthesubspeciesofA.maritima. Further evidenceofspecies-level

divergence

was

revealedbyanalysis of the genetic distancesbetweenthetaxa (Table3).

The

distancebetweentheleastdivergentpair,D.mexicanaandD. palustris,(i03)isoverfivetimesgreaterthanthat ofthemostdivergent

sub-speciesolA.maritima(19).

Afteran exhaustivesearch,phyiogeneticanalysis ofISSR dataproduceda singlemost-parsimonioustree of813 evolutionarysteps(Fig.3),and bootstrap

and

jacfcnifepercentages

(100%

torboth)

showed

very strongsupportforthis

topofogy ConsistentwiththeITSresults,ISSR dataverifiedthat D. occidentalis

was

thelirstofthe threeDircaspeciestodiverge(Fig.3).Euclideandistances

betweenspeciesbasedon data fromalleightprimerswere 410forD.occidentalis

and

D. palustris,392forD.Occidents/i.sandD.mexicana,

and

302forD.palustris

and

D.mexicana.Thesegeneticdistances

and

branchiengths derivedfrom ISSR markers

showed

greater relative distancebetweenD.

mexicana

and

D.palustris

than obtained fromITS,indicating a

more

uniformlevelofdivergence

among

(11)

SCHRADERANDGRAVES, SYSTEMATICS OF DIRCA 519

Table3^ComparativeEuclidean distancesforspecies{Dirca)andsubspecies (Alnus maritima) ob-tainedby thesame methods and samethree I5SR primers. ResultsforAlnusmaritimawerederived

fromSchraderandGraves(2004).

Euclidean distances

Specieslevel {Dirca)

Dircamexicana- Dirca occidentalis 11 Dircamexicana- Dircapalustris 103 Dirca occidentalis - Dircapalustris 11

9

Subspecieslevel{Alnus maritima)

oklahomensis - georgiensis 1 5 oklahomensis - maritima 1 9 georgiensis ~maritima 1 2

ISSRresultsconfirmedthat D.mexicana

and

D. palustris are themostclosely related of the three species.

Our

unweighted, simultaneousanalysis ofITS

and

ISSR data(Fig.4)

shows

the best synthesisforthephytogenyofDircabased on

allavailablemolecularevidence.

DISCUSSION

Based onthe results ofITSalone,

we

might concludethatDirca mexicana and

D. palustriscouldbestbe consideredas the

same

species, aconclusionthat

would

contradict themorphological evidenceof

Nesom

and

Mayfield(1995).

With

ITS, the

mean

genetic distancefromD. occidentalistothesetwospeciesis nearlyeighttimesgreaterthanthedistance

between

D.

mexicana and

D. palustris,

and

theneighbor-joining phylogeny, produced

when

using

generic-levelancestralcharacteras theroot,illustrates

how

closelyrelated theITS

re-gionsof D.mexicana

and

D.palustris are toeachother(Fig.1).Results ofISSR

analysis, however,provideconclusiveevidencethat D.mexicana

and

D.palust ris

have divergedsufficiently tobe consideredseparate species,

and

theyindicate thatdivergenceofthe three speciesis

more

uniform thanindicatedby ITS

analy-sis(Table 2and3,Fig,3).Althougha suitableexplanationfortheseemingly contradictorylevelsofdivergenceindicatedby ITS

and

ISSRscould bethat

dif-ferent geneticmarkers

may

bedifferentiafly affectedby occurrences suchas interspecificgene flowor reticulate evolution

(Comes

&

Abbott1999), a

more

obvious explanation can be foundinacloserexaminationof the

two

ITS

phy-logenies.Includingfamily-level ancestralcharacter (outgroupH. rosasinensis) inthe analysis ledto

two

importantinsights.First,thereisahigh degreeof differentiation (longbranchlength) of D. occidentalis

and

D.

mezereum

thatis evidentafter theirdivergence(Fig.2J,

and

this differentiationskewsthe tree

(12)

520 BRIT.ORG/SIDA21(2) 198

Dirca

mexicana

67 (100)

Dirca

palustris

Dirca

occidentalis

'Daphne

mezereum

Fig 3.ISSR neighbor-joiningdendrogram showingthe inferred phylogenetic relationshipamongthe three species of

O/rra.Topologyindicatesthat ofthe singlemost-parsimonioustree.Numbersindicate relativebranch lengths;

num-bersinparentheses are both the bootstrapandjacknifepercentages,whichwereidentical.Do/j/jemezereum, another

memberofThymelaeaceae,was usedastheoutgrouptoestablish ancestral-character polarity of ISSRbanding pat-terns. 389 Dirca

mexicana

Dircapalustris Dircaoccidentalis

Daphne mezereum

Fig.4.SimultaneousITSandISSR neighbor-joiningdendrogramusingunweighted Kimura andEuclidean distances, respectively.Dahne mezereum, anothermemberofThymelaeaceae,was usedastheoutgroupto establish ancestral-character polarity.

(13)

and

D. palustris tobe placedfurther

from

D. occidentalisthan they

would

be

otherwise. Secondly, thephylogeny produced withfamily-level ancestral

char-acter in the root correctsforthisdisproportionaldifferentiation

and shows

a

more

uniformspecies-leveldivergence betweenthe threeDircaspecies,

making

thephylogeny

more

consistentwiththe results of the ISSRs. Together, theISSR

and

ITS analyses supportthe speciesdesignationforD.

mexicana

established

by

Nesom

and

Mayfield(1995).Theseresults also reinforce the principle that

more

thanonegenetic

marker

should be usedinmolecular systematics inves-tigations(Hollingsworthetal.1999)

and

that analysis ofcross-matrix

dispar-itycan beavaluable

method

of clarifyingphylogeneticfeatures

(Bateman

1999).

Contrarytothemorphologicalevidence,both ITS

and

ISSR analyses indi-cate thatDirca

mexicana

is

more

closely related, genetically, to D. palustristhan itisto D. occidentalis.Thisfinding,

and

the findings that D. occidentalis

was

boththefirstofitsgenustodiverge

and

the

most

differentiated of the three species(Figs.1-4),helptoexplaintheapparent inconsistency betweenthe

mor-phological

and

biogeographical evidence noted by

Nesom

and

Mayfield(1995).

Although

thereisa greatermorphologicalsimilarity

between

D.

mexicana

and

D. occidentalis,theyarethe

most

divergent of the three species genetically(Figs. 1-4).Thesefindings are consistentwiththehypothesisof

Nesom

and

Mayfield

(1995)based

on

biogeographythatDirca

was

probably continuousacrossNorth

America

as early as theEocene,

and

disjunction

between

D.occidentalis

and

D. palustris

may

have takenplace before oraroundthe

same

timeas the

move-ment

ofDircaintoMexico. This hypothesis seemsespecially wellsupported

by

our ITS

and

simultaneous phylogenies(Figs.1,2,

and

4).

The

typical ecologicalnicheforthegenus Dircaisconsideredtobethe cool

moistslopes of

mature

temperate deciduousforests.BothD. palustris

and

D.

mexicana

arefound almostexclusively insuchsettings,

and

even

though

ithas adaptedtoa differentenvironment,D.occidentalisisstrugglingtosurvive amidstadryingclimate,removalof overstorytrees,

and

competition

from

ev-ergreentrees

and

shrubs (Johnson1994;Graves 2004).

As mentioned

by

Nesom

and

Mayfield(1995),paleobotanical evidence suggeststhatDirca

was

continu-ousacrossthemidlatitudesof

North

America

as part of theEocene/Oligocene expansionoftemperate deciduousvegetation that took place after the partial regression of theCretaceousepeiricsea

(Graham

1993). Infact,

members

of

Thymelaeaceae

are

documented

inthe lateEocenefloraof centralColorado (MacGinitie1953;

Graham

1993). In geologic time, this extensiverange

was

prob-ably shortlived.Cooling duringthemiddle

Miocene

broughtthe

encroachment

of coniferous forestsfromthenorth (Leopold

&

Denton

1987),

and

bythemiddle

to lateMiocene,colderwinter temperatures

and

reduced

summer

rainfallin the area of theGreatPlains

had

initiated thedevelopmentof prairiegrasslands

(Graham

1993).

While

climaticcooling broughtthe isolation of eastern

and

(14)

representa-522 BRIT.ORG/SIDA21(2)

tives ofDirca,italsoenabledthe

movement

ofeasterndeciduousforestelement

intoeasternMexicofollowing rapidsouthwardretreat ofthelingering Missis-sippi

Embayment

bythemiddletolateMiocene

(Graham

f973, 1993).Finally theprecursorsto

modern

D.pcj/ustn'.sand D.mexicona probably

became

disjunct asprairieand coniferouscommunitiesinwesternand southwestern North America spread

durmg

the Pliocene

and

elementsofthebroad-leaveddeciduousforest

thathad extendedintoeasternMexico

became

isolated

(Graham

1973, 1993).

One

phenotypic manifestationofthehighlevelof differentiationseenin

D.occidcntalisis itsobligate

summer

dormancy.

Only

D.occidcnlalis under-goesthisdrought-deciduous

summer

dormancy,

which

coincideswiththedry seasonintheSan Francisco Bayarea.Trials

we

haveconductedin agreenhouse

indicate thatthisannual phenological event cannotbeovercome by

manipula-tionoftheenvironment.

The

absenceofthisdrought-deciduoustraitintheother

twospeciesoiDirca,and,tothe best ofour knowledge,therestofThymelaeaceae,

suggests thatconsiderablechange has takenplace

m

D.occidcntaiis sinceits divergencetoensuresurvivalinitsMediterranean

chmate

(Freitas f997).Such adaptationisthemostplausibleexplanationforthecontinuedexistence of this disjunct species ofDura. Withouta

means

ofdroughtavoidance,itislikely that the precursors ofD.occidcntaliswould have expired along withtherestof thewestern Dirca elementaswesternclimates

became

increasinglydr\'.Itis

believed thatduringthe Pliocene, theCascade-Sierra

Nevada

andtheCoast Ranges reachedsufficientheightstocreateaneffective rain

shadow

over the

Basin

and

Range

Province, resultinginachange from mcsic and

summer-wet

tothe xeric

and

summer-dry

conditionsthat existtoday (Axelrod1986;

Gra-ham

1993).ThistrendmostlikelyforcedD.occidcntalis intoitsmeagercoastal distribution,whilepromotingselection of the

summer-deciduous

habit.

The

lowerlevelof differentiationinDirca palushisrevealedbyallfour

phylogcncticdendrograms shouldbeconsideredconsistentwithits

much

larger potentialgenepooland, until recentlyitslaidyuniformhabitat.Becausethe

fundamental nicheolD.palustnsismature-forest understorv,itislikelv that

exceptforthetemporaryintrusion of glaciersandtheiradjacent borealforest

biome

(Delcourt

&

Delcourt1993),the geneticaggregateof D. palustris

was

continuousacross easternNorth

America

untilthe harvest ofold-growth

for-estswithinthelast300to

400

years.Futureexaminationofthe genetic varia-tionwithinD./^ci/u.stn.sshonldbeperformedto testthishypothesisandto pro-videan evenclearerpicture of thesystematicsofgenus Dura.

ACKNOWI.HLX^jMENTS

We

thank

Gary

Polkingandthestaffofthe

DNA

Sequencingand Synthesis

FacilityatIowaStateUniversityfortheir assistancewithITSand ISSR

(15)

manu-SCHRADERANDGRAVES, SYSTEMATICS OF DIRCA 523

script

and

toSusan Bennerand Fredy

Romero

forassistancewithtranslation of the abstract to Spanish.

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