Genetic diversity and structure of the tree Enterolobium contortisiliquum (Fabaceae) associated with remnants of a seasonallydry tropical forest.

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ContentslistsavailableatScienceDirect

Flora

jou rn a l h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / f l o r a

Genetic

diversity

and

structure

of

the

tree

Enterolobium

contortisiliquum

(Fabaceae)

associated

with

remnants

of

a

seasonally

dry

tropical

forest

Patrícia

de

Abreu

Moreira

a,∗

_,

_Murilo

_Malveira

_Brandão

b

_,

_Natalia

_Hayashida

_de

_Araujo

b

_,

Dario

Alves

de

Oliveira

b

_,

_G.

_Wilson

_Fernandes

c

a_Departamento_de_{Biodiversidade,}_Evoluc¸_ão_e_Meio_Ambiente,_Instituto_de_Ciências_Exatas_e_Biológicas,_Universidade_Federal_de_Ouro_Preto,_CEP_35400-000

OuroPreto,MG,Brazil

b_Laboratório_de_{Bioprospecc¸}_ão_e_Recursos_Genéticos,_DBG,_Universidade_Estadual_de_Montes_Claros,_CEP_39401-089_Montes_Claros,_MG,_Brazil c_Ecologia_Evolutiva_&_{Biodiversidade,}_DBG,_ICB,_Universidade_Federal_de_Minas_Gerais,_CP_486,_30161-970_Belo_Horizonte,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20January2014

Receivedinrevisedform6October2014 Accepted15October2014

EditedbyBohumilMandák Availableonline1November2014 Keywords: Geneticstructure ISSR Conservation Dryforest Brazil

a

b

s

t

r

a

c

t

Fragmentationoftropicalforestsisoneofthemajorthreatstobiodiversityandviablenatural popu-lations.Brazilianseasonallydrytropicalforests(SDTF)areendangeredbecauseofhumanoccupation, conversionoflandstoagricultureandhighdeforestationratesinthesefertilesoils.Enterolobium contor-tisiliquumhasbeenremovedfromSDTFnaturalareasduetotheadvanceofcattlegrazingandagriculture inBrazilianSDTFs.Toaidconservationeffortsofthisspeciesweusedmolecularmarkerstostudythe geneticdiversityandpopulationstructureofE.contortisiliquuminBrazilianSDTFremnants.Atotalof 263individualsin13forestpatcheswereanalyzedwith103ISSRfragments.Inspiteofbeingfound scat-teredamongtheSDTFpatches,E.contortisiliquumpopulationsexhibitedhighgeneticdiversity(mean Shannon’sindexofdiversity=0.384;meangeneticdiversity=0.280)andgeneticdivergencebetween populationswasdetected(˚ST=0.155,p<0.0001).UPGMAdendrogram,principalcoordinateanalysis

andBayesiananalysisshowedthatE.contortisiliquumpopulationswereclusteredintothreegroupsthat werenotrelatedtogeographicdistance(r=0.119,p=0.197).However,thesepopulationsarespatially structuredintogroupsdistributedintheWest,CentralandEastofthestudyarea.Thisgeneticstructure mayberelatedwithlandscapeandboththeEspinhac¸oRangeandSãoFranciscoRivermayactasa par-tialgeographicbarrierlimitingthegeneﬂow.Becausethehighestratesofgeneticdiversitywerefound outsideofconservationunits,weproposethecreationofprotectedareasindifferentgeographicregions thatincludeE.contortisiliquumpopulationsfromdifferentgeneticclusters.

Introduction

Ongoingfragmentationoftropicalforestsisoneofthemajor threatstobiodiversityconservationandviablenaturalpopulations (Saundersetal.,1991).Thisisespeciallyworrisomeforseasonally drytropicalforests(SDTF)whichareconsideredtobeoneofthe mostendangeredtropicalecosystems(Janzen,1988;Milesetal., 2006;Steiningeretal.,2001).SDTFsoccurinregionscharacterized bypronouncedseasonalityinrainfalldistribution,where precipita-tionis<1600mm/yrwithseveralmonthsofdroughtandaperiod ofatleastﬁveorsix monthsreceiving<100mm(Gentry,1995;

∗ Correspondingauthor.Tel.:+553135591214.

E-mailaddresses:patriciadabreu@yahoo.com.br,pattyabreu13@gmail.com

(P.A.Moreira).

Grahamand Dilcher,1995).SDTFsare dominatedby deciduous treeswithamoreorlesscontinuouscanopy(Mooneyetal.,1995; Sánchez-Azofeifaetal.,2005).Themostdominantplantfamilies areFabaceaeandBignoniaceae(Gentry,1995;Mayle,2004).

Thetropicaldryforestsexhibitadisjunctdistribution through-out the Neotropics (Pennington et al., 2006). The currently discontinuousdistributionofSDTFsrepresenttheremnantsofa morewidespreadandcontiguousformationduringthePleistocene thatformedthe“PleistocenicArc”(Prado,2000;PradoandGibbs, 1993).ThelargestareasofSDTFsoccurinSouthAmerica, speciﬁ-callyinBrazil(Penningtonetal.,2006),wheretheyrepresent3.21% ofitsterritory(Sevilhaetal.,2004).Asinotherlocalities,Brazilian SDTFsareassociatedwithfertilesoilswithamoderatetohighpH andhighnutrientcontent(Espírito-Santoetal.,2009;Pennington etal.,2006).Hence,theBrazilianSDTFs patchesarenowunder severethreatsbecauseofhumanoccupation,conversionoflands http://dx.doi.org/10.1016/j.ﬂora.2014.10.005

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toagricultureand highdeforestation rates inthese fertile soils (Espírito-Santoetal.,2009).

Studiesoffragmentationeffectsinplantshaveshownthat for-estlossmayhavenegativeconsequences onreproduction,gene ﬂowandgeneticdiversity(Aguilaretal.,2006,2008;Ledig,1992; Loweetal.,2005;Templetonetal.,1990).Thus,treesinremnant fragments couldalter matingpatterns,increase inbreeding and decreasegeneticdiversity(Aguilaretal.,2008).Therefore,the dis-junctdistributionofBrazilianSDTFanditsalterationbyhuman activitiesmaydecreasethegeneticdiversityofplantspecies.

Enterolobium contortisiliquum (Vell.) Morong is a Neotropi-caltreespecies consideredasaSDTFspecialist (Särkinen etal., 2011) and it was used to define the Pleistocenic Arc (Prado, 2000;PradoandGibbs,1993).Thespeciesflowersduringashort period,September andOctober, when itis leafless,while fruits ripenbetweenJuneandJuly(P.A.Moreira,personalobservations). ThematurefruitsandseedsofE.contortisiliquumresembleother Enterolobiumspecies.AswithE.cyclocarpum,itsfruitsaresmooth, shiny,indehiscentanddeepbrown(RochaandAguilar,2001a)and theseedsarehard,ovoidandbrown(Janzen,1982).Recently,itwas discoveredthataproteinaseinhibitorofthisspecies,Enterolobium contortisiliquumtrypsininhibitor(EcTI),inhibitedtheinvasionof gastric cancercells, providing a possiblecandidate for therapy againsttumors(Paulaetal.,2012).

Thistreehasbeenremovedfromnaturalareasowingtothe advanceofcattlegrazing andagriculturein BrazilianSDTFsdue toitspoisonousfruits.TheingestionofE.contortisiliquumpodsis harmfultocattle,causingphotosensitivityreactionsand sponta-neousabortion(Bonel-Raposoetal.,2008;Costaetal.,2009).Asa resultmanyfarmershavecutthesetreesneartheirranchesas fruit-ingofE.contortisiliquumoccursduringthedryseasonandcoincides withlowforageavailability.Thislogginghasleadtopopulations of E.contortisiliquumbecomingvulnerable in thedisjunctSDTF matrix.Hence,understandingoftheeffectsoffragmentationand selectiveloggingonthistreeisurgentlyneededforitsconservation. To betterconserve this species we usedmolecular markers to study the genetic diversity and structure in populations of

Table1

GeographicallocationandsamplingdetailsofEnterolobiumcontortisiliquum popu-lationsstudied.

Populations Populationcode Latitude/longitude Samplesize

Unaí UNA 16◦₁₉_S₄₆◦₅₅_W ₂₀

Paracatu PAR 17◦₀₆_S₄₆◦₅₀_W ₂₀

JoãoPinheiro JOP 17◦₄₄_S₄₆◦₁₁_W ₁₆

FelíciodosSantos FES 18◦_04S₄₃◦₁₂_W ₂₀

Gouveia GOU 18◦37S44◦01W 22 Buenópolis BUE 17◦52S44◦10W 20 Manga MAN 14◦₅₀_S₄₃◦₅₇_W ₂₁ Itacarambi ITA 15◦₁₀_S₄₄◦₁₂_W ₂₀ Verdelândia VER 15◦₃₄_S₄₃◦₃₅_W ₁₅ Jequitaí JEQ 17◦₁₅_S₄₄◦₂₇_W ₂₀

VárzeadaPalma VAR 17◦34S44◦44W 29

Beltrão BEL 18◦22S44◦29W 20

MontesClaros MOC 16◦₄₁_S₄₃◦₅₆_W ₂₀

Total 263

E.contortisiliquuminBrazilianSDTFpatches.Byusingintersimple sequencerepeats (ISSRs)weaddressedthefollowing questions: (1)WhatisthelevelofgeneticdiversityinE.contortisiliquum,in selectedBrazilianSDTFpatches?(2)Howisthegeneticvariationof thisspeciesstructuredamongthesepatches?and(3)Arethere geo-graphicbarriersinthelandscapethatlimitedgeneticdistribution betweenpopulationsalongtime?

Materialsandmethods

Plantmaterialssampling

Enterolobium contortisiliquum is a common species in the BrazilianSDTFs,withadensityrangingfromthreetoeight indi-viduals per hectare(Oliveira-Filho, 2006).From 15 to 29 adult trees were sampled in 13 Brazilian SDTFpatches, totaling 263 individuals(Fig.1,Table1).Expandedleaveswerecollectedfrom allindividualtreesandstoredonsilicageluntilDNAextractions werecarriedout.EachSDTFremnantwastreatedasapopulation:

Fig.1.GeographicdistributionoftheEnterolobiumcontortisiliquumpopulationssampledforthisstudyinBrazilianseasonallydrytropicalforestpatches.Populationsare identiﬁedaccordingtospatialgeneticgroupsestablishedbySTRUCTURE(identiﬁedinthelegend).Blacklinesindicatetwobarriersamongpopulations.

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Table2

ISSRprimersusedforPCRampliﬁcationsofEnterolobiumcontortisiliquum.

Primername Sequence(5–3)a _T

a(◦C)b Nc John (AG)7YC 47 10 Manny (CAC)4RC 47 7 Mao (CTC)4RC 47 6 Terry (GTG)3GGTGRC 46 14 UBC810 (GA)8T 47 9 UBC827 (AC)8G 47 10 UBC840 (GA)8YT 47 18 UBC879 (CTTCA)3 47 6 UBC880 GGAGAGGAGAGGAGA 47 11 UBC899 (CA)6RG 47 12 Total 103 a_Y₌_C_or_T;_R₌_A_or_G.

b _Annealing_temperature_of_primers.

c _N₌_number_of_fragments_for_each_primer._All_fragments_were_polymorphic_when

consideringallpopulations.

Unaí(UNA),Paracatu(PAR),JoãoPinheiro(JOP),FelíciodosSantos (FES),Gouveia(GOU),Buenópolis(BUE),Manga(MAN),Itacarambi (ITA),Verdelândia(VER), Jequitaí(JEQ),VárzeadaPalma (VAR), Beltrão(BEL)andMontesClaros(MOC).

DNAextractionandISSRampliﬁcation

GenomicDNAextractionfollowedthestandardCTABprocedure (Doyle and Doyle, 1987). DNA integrity was checked by elec-trophoresison1.0%agarosegelswith1×TBEbuffer,stainedwith ethidiumbromideand photographed under UV light. The DNA obtainedwasdilutedinultrapurewateruptoﬁnalconcentration of5.0ng/␮LpriortoPCRampliﬁcation.

Aninitial screening of 20 primers was undertaken for ISSR analysis.ISSRampliﬁcationswereperformedina 15␮Lvolume containing20.0ngoftemplateDNA,PCRbuffer(50mmKCl,10mm, Tris–HClpH8.4,0.1%TritonX-100,Phoneutria),0.50mMMgCl2,

0.25␮gof BSA,250␮M ofeachdNTP, 10.0␮M ofprimerand 1 unitofTaqDNApolymerase(Phoneutria).Ampliﬁcationswere per-formedusingVeritithermalcontroller(LifeTechnologies)under thefollowingconditions:94◦Cfor4min(onecycle);94◦Cfor1min, 46or47◦Cfor1min(accordingtoeachprimer,Table2),72◦Cfor 1min(37cycles);and72◦Cfor7min(onecycle).Anegative con-trol,inwhichtemplateDNA wasomitted,wasincludedineach PCR.ISSRampliﬁedproductswereseparatedelectrophoreticallyat aconstantvoltageof100Vfor4hin1.5%agarosegelswith1XTBE buffer,stainedwithethidiumbromideandphotographedunder UVlight(GelLogic212Pro,Carestream).A100-bp DNAladder

(Fermentas)wasusedasastandardmolecularweighttoestimate themolecularsizeofthefragments.

Dataanalysis

Fragments amplified by ISSR were scored as present (1) or absent(0)bymanualverificationtocreatea binarymatrix. We assumedthat amplifiedproductsofsimilarsizeusingthesame primerwerehomologous.Toobtainconsistentresults,onlydata fromclearandunambiguousbandswereusedforgeneticanalyses. Besides,thenegativecontrolwasusedineachagarosegel.Genetic diversitywasmeasuredbypercentageofpolymorphicloci(P),the observednumberofalleles(Na),effectivenumberofalleles(Ne) andShannon’sindex(I)(Lewontin,1972),usingthesoftware Pop-Genev.1.32,whichisdesignedfortheanalysisofdominantmarkers (Yehetal.,1999),assumingHardy–Weinbergequilibrium,FIS=0.

The measuresof geneticdiversity included a Bayesiananalysis (Holsingeretal.,2002),implementedinHickoryv1.0.4(Holsinger andLewis,2005).We estimatedHs,whichisanalogoustoNei’s (Nei,1978)unbiasedexpectedheterozygosity(HE).

Toestimatevariancecomponentsand topartition the varia-tionof the specieswithin and among populationsthe Analysis ofMolecularVariance(AMOVA)wasperformedusingArlequinv. 3.01(Excoffieretal.,2005).Thegeneticstructurewasestimated by10,000permutations,assumingHardy–Weinbergequilibrium, computingthedistancematrixandproportionofthedifference. Statisticalsignificanceofthevarianceproportionassociatedwith thefixationindex˚STwasdeterminedthroughpermutationtests

againstanulldistributiongeneratedbythedata.

Thepairwise B _(a _Bayesian_analysis _analogous_to_F ST) was

obtainedwith95%conﬁdenceintervalsusingthesoftware Hick-oryv1.0.4(HolsingerandLewis,2005)forallpopulationpairsand usedtoconstructaUPGMA(unweightedpair-groupmethodwith arithmeticaverages)dendrogramusingNTSYSv.2.2(Rohlf,2000) andtocarryoutaprincipalcomponentsanalysis(PCA)withthe softwareGenAlEx(PeakallandSmouse,2006).Toverifythe iso-lationbydistanceaManteltestwasperformedusingPC-ORDv.6 (McCuneandMefford,2011).

Todeterminethenumber ofgeneticclusters(K),a Bayesian analysisofpopulationstructurewascarriedoutwiththesoftware STRUCTURE2.2.(Pritchardetal.,2007).Aburn-inperiodof100,000 generationsand100,000stepsofMarkovChainMonteCarlo sim-ulationswereusedtoestimatelnPr(X/K).Weusedtheadmixture modelandtheallelefrequenciescorrelatedmodel,withoutprior assumptionsconcerningthepopulation.ForeachKvalue,10runs werecarriedouttoverifytheconsistencyoftheresults.Theaverage Table3

Geneticdiversityparametersof13Enterolobiumcontortisiliquumpopulationsdetectedby10ISSRmolecularmarkers.

Population Na Ne Hs I P(%) UNA 1.952(0.215) 1.571(0.341) 0.339(0.007) 0.492(0.206) 95.19 PAR 1.875(0.332) 1.490(0.345) 0.313(0.008) 0.439(0.231) 87.50 JOP 1.883(0.323) 1.527(0.361) 0.316(0.009) 0.454(0.237) 79.81 FES 1.711(0.455) 1.463(0.388) 0.254(0.013) 0.390(0.283) 71.15 GOU 1.701(0.459) 1.392(0.367) 0.277(0.007) 0.349(0.273) 70.19 BUE 1.721(0.451) 1.342(0.366) 0.255(0.011) 0.315(0.264) 72.12 MAN 1.779(0.417) 1.414(0.374) 0.273(0.008) 0.369(0.264) 77.88 ITA 1.740(0.441) 1.472(0.398) 0.272(0.008) 0.392(0.283) 74.04 VER 1.659(0.476) 1.397(0.393) 0.241(0.008) 0.338(0.289) 61.54 JEQ 1.798(0.403) 1.431(0.366) 0.259(0.009) 0.385(0.258) 79.81 VAR 1.711(0.455) 1.308(0.330) 0.269(0.008) 0.302(0.249) 71.15 BEL 1.769(0.423) 1.406(0.355) 0.279(0.011) 0.370(0.258) 76.92 MOC 1.762(0.427) 1.458(0.361) 0.298(0.008) 0.401(0.265) 71.15 Mean 1.774(0.405) 1.436(0.361) 0.280(0.003) 0.384(0.258) 76.03 Species 2.000(0.000) 1.589(0.299) 0.370(0.005) 0.519(0.162) 98.78

Na=numberofallelesperlocus,Ne=effectivenumberofallelesperlocus,Hs=expectedBayesianheterozygosity(withoutassumingHardy–Weinbergequilibrium),

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Table4

Analysisofmolecularvariance(AMOVA)amongandwithinEnterolobiumcontortisiliquumpopulations.

Sourceofvariation d.f. Sumofsquares Variancecomponents %Totalvariance p-Value

Amongpopulations 12 139.54 0.512 15.54 <0.0001

Withinpopulations 250 612.90 2.785 84.46 <0.0001

ThreegroupsaccordingtoBayesiananalysis

Amonggroups 2 41.472 0.15387 4.58 <0.0001

Amongpops.withingroups 10 98.071 0.41777 12.44 <0.0001

Withinpopulations 240 612.905 2.78593 82.97 <0.0001

Fig.2.UPGMAdendrogramshowingtherelationshipbasedonpairwiseB_values_among_Enterolobium_{contortisiliquum}_populations_in_Brazilian_seasonally_dry_tropical_forest

patches.ColoredcirclescorrespondtogroupsestablishedbySTRUCTURE(seeFig.1forlegend).

ofeachlikelihoodvalueforK,‘logofprobability’[LnP(D)]through allrunsweredeterminedtoinferthenumberofgeneticclusters (Pritchardetal.,2000)andthestatistic_Kwasestimatedaccording toEvannoetal.(2005).Afterward,anotherAMOVAwasperformed accordingtothegroupsdeﬁnedbythisanalysis.

ToinvestigategeneticdiscontinuitiesinspaceMonmonier max-imundifferencealgorithm(Monmonier,1973)wasimplemented bythemethodofDelaunaytriangulationusingBarrierv.2.2(Manni etal.,2004)asproposedbyLegendreand Legendre(1998)and Maneletal.(2003).

Results

From20ISSRprimerstested10wereusedandresultedin103 clearlyidentiﬁablefragments(Table2).Eachprimerampliﬁedfrom sixto18lociandall103scoredbandswerepolymorphicatthe species leveland at populationlevelthe polymorphismranged from61.54to95.19%(Table3).Theaverageeffectivenumberof allelesperlocusatthepopulationlevelwas1.436and1.589atthe specieslevel(Table3).Thegeneticdiversity(Hs)and Shannon’s index(I)were0.370and0.519,respectively,forE.contortisiliquum. PopulationsexhibitedhighgeneticvariabilitywithmeanHsandI equalto0.280and0.384,respectively.TheVERpopulationshowed thelowestgeneticdiversity(Hs=0.241,I=0.338)whiletheUNA populationshowedthehighestdiversity(Hs=0.339,I=0.492).

Mostofthevariationwasfoundamongindividualswithinthe samepopulation(84.46%)andonly15.54%ofthevariationoccurred amongpopulations(Table4),whichimpliessigniﬁcantlyhigh aver-ageﬁxationindices(˚ST=0.155,p<0.0001).TheE.contortisiliquum

populations groupedinto three main clusters(Fig.2).The ﬁrst clustercorrespondedtoUNA,PAR,JOP,ITAandMANpopulations, thesecondclustercorrespondedtoVER,JEQ,BEL,VAR,BUEand GOUpopulations,andﬁnallyathirdclustercorrespondedtoFES

andMOCpopulations(Fig.2).ThepairwiseB_ranged_from_0.054

(betweenUNAandJOP)to0.392(betweenVARandFES).The high-estgeographicdistancewasamongFESandUNApopulationswhich showedB₌_0.231_and_the_nearest_distance_was_among_VAR_and

JEQpopulationswhichshowedB₌_0.271₍_Table₅_)._No_signiﬁcant

correlationwasfoundbetweengeographicaldistanceandgenetic distance(r=0.119,p=0.197).

PCAresultswereconsistentwithUPGMAdendrogram, group-ingthesamepopulationsofUPGMAanalysis.Thefirsttwoaxes explained mostof thetotal variability(axis 1=39.95% and axis 2=26.30%)(Fig.3).TheBayesianapproachshowedthatthe num-berofgeneticgroups(Kvalue)whichbestfitourdatawasK=3 (Fig.4A).ThesegroupsaresimilartotheclusteroftheUPGMA den-drogramand thethreegroupsappear tobespatially structured withthefirstgrouplocatedattheWest,thesecondgrouplocated attheCentralandthethirdgrouplocatedattheEastofthestudy area(Figs.1and4B).IntheAMOVAclusteringthepopulationsin

Fig.3.PrincipalcomponentsanalysisbasedontheEnterolobiumcontortisiliquum populationsdistancematrix.Axes1and2explained39.95and26.30%ofthe varia-tion,respectively.Thecolorsofpopulationsareaccordingtogroupsestablishedby STRUCTURE(seeFig.1forlegend).

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Table5

MatrixofpopulationpairwiseB_(below_diagonal)_and_geographic_distance_in_km_(above_diagonal)_among₁₃_Enterolobium_{contortisiliquum}_populations.

UNA PAR JOP ITA MAN VER JEQ BEL VAR BUE GOU FES MOC

UNA – 98 173 323 361 370 282 348 271 341 397 442 323 PAR 0.073 – 102 356 398 386 253 287 225 295 340 402 312 JOP 0.054 0.082 – 353 393 370 191 194 154 214 251 320 268 ITA 0.194 0.224 0.133 – 49 79 234 358 272 297 380 337 170 MAN 0.178 0.191 0.143 0.251 – 95 275 400 317 338 422 370 206 VER 0.238 0.286 0.175 0.244 0.199 – 210 328 253 264 346 281 129 JEQ 0.202 0.239 0.171 0.253 0.193 0.139 – 125 48 77 160 163 80 BEL 0.271 0.259 0.215 0.326 0.228 0.152 0.061 – 93 67 56 141 196 VAR 0.333 0.327 0.282 0.356 0.275 0.264 0.271 0.213 – 69 139 174 130 BUE 0.279 0.298 0.243 0.333 0.246 0.163 0.229 0.185 0.069 – 88 106 132 GOU 0.243 0.277 0.202 0.325 0.260 0.256 0.179 0.158 0.351 0.273 – 109 216 FES 0.231 0.286 0.234 0.341 0.337 0.365 0.373 0.367 0.392 0.321 0.283 – 172 MOC 0.237 0.248 0.238 0.341 0.332 0.341 0.347 0.339 0.368 0.310 0.261 0.115 –

threegroups(assuggestedbyBayesiananalysis)4.58%ofthetotal variationwasfoundamongthegroupsand12.44%among popula-tionswithingroups.Again,mostofthevariationwasfoundamong individualswithinthesamepopulation(82.97%)(Table4).

TheDelaunaytriangulation,usedtoindicatethegenetic discon-tinuitiesgeneratedbybarriersinthelandscape,revealedthatthere aretwokeybarriers(Fig.1).Themostintense(barrier‘a’)separates populationsbelongingtotheEastgroupandthesecond(barrier‘b’) separatesmostofthepopulationsoftheCentralgroup(VAR,BUE, BELandGOU).

Discussion

TheISSRmarkersrevealedhighlevelsofgeneticdiversityof Enterolobiumcontortisiliquumpopulationsandthisdiversityis sim-ilartootheroutcrossingspeciesanalyzedwithanotherdominant molecularmarker,RAPD which estimates are comparable with ISSR(Nybom,2004).Thehighlevelsofgeneticdiversityfoundin E.contortisiliquumpopulations(Hs=0.280,I=0.384)aresimilarto otherLeguminosaespeciesasDerristrifoliata(Wuetal.,2012)and Ormosiahosiei(Zhangetal.,2012)andishigherthanfortwoother treespeciesfromtheBrazilianCerrado,Dimorphandramollisand Dimorphandrawilsonii(SouzaandLovato,2010).Thehighergenetic diversitycouldberelatedtothepollinationsystemofE. contor-tisiliquumanditslonglifecycle.AlthoughtheE.contortisiliquum matingsystemis still unknown,its congener E.cyclocarpum is predominantlyoutcrossed(RochaandAguilar,2001b).We postu-latethatE.contortisiliquumcouldbeanoutcrossingspecies,which couldexplainthehighlevelsofgeneticdiversityfound.Besides,E. contortisiliquumﬂowersareapparentlypollinatedbymoths,asE. cyclocarpum(Frankieetal.,2004;HamrickandApsit,2004).Some mothsarecapabletoﬂylongdistancesandcanvisitmanytreesin

aforagingroute(HaberandFrankie,1989).Inaddition,mothscan carrylargepollenloads(WillmottandBúrquez,1996)andcover greatdistancesbetweenconsecutivelyvisitedplants(Linhartand Mendenhall,1977).Furthermore,woodyplantssuchasE. contor-tisiliquum mayhavepersisted in remnant populationsin SDTFs patchesforalongtimeandourresultsprobablyreﬂectthe histori-calgeneticdiversityofadulttrees.Inarecentmeta-analysisitwas conﬁrmedthatadultsexhibitedhighergeneticvariationthan off-springinafragmentedlandscapebecauseoflongtermpersistence inhabitats(Vranckxetal.,2011).

ThehighestgeneticdiversitiesareregisteredinUNA,PARand JOPpopulations(mean=0.323),intheWestofstudiedarea,andthe lowestinMAN,ITAandVERpopulations(mean=0.262),located intheupperSãoFranciscoRiver.Theseresultsmaybecorrelated withtheconservationhistoryoftheseareas.TheWestofthestudy areawasoccupiedonlyin18thcenturyinducedbyexploration ofgold.Beforethis,thenorthofthestudyareawasfragmented andaltered.TheregionofSãoFranciscoRiverbasinishighly dis-turbedandwasoccupiedinthe17thcentury.Duetothehighsoil fertilitythisregionhasbeenreceivinggovernmentalﬁnancial sup-portforagribusinessimprovementandirrigationprojects,suchas JaíbaIrrigationproject(seewww.projetojaiba.com.br).Lowgenetic diversitywasobservedpreviously inHandroanthusochraceusin thenorthofthestudiedregion(Moreiraetal.,2009;Moreiraand Fernandes,2013).Thus,lateexplorationoftheUNA,PARandJOP regionmayhavecontributedtothemaintenanceoflarge popula-tionsofE.contortisiliquumovergenerationswhichallowedgreater geneﬂowamongpopulations.Despitethecurrentfragmentation oflandscapecausedbyexpansionofagriculturalareas,these rem-nantpopulationsmaintainhighgeneticdiversity.Asaresult,UNA, PARandJOPpopulationsareimportantreservoirsofgenetic diver-sityofthespeciesE.contortisiliquumandmustbepreserved.Itis

Fig.4. BayesiananalysisofEnterolobiumcontortisiliquumindividualsfromBrazilianseasonallydrytropicalforestpatches.(A)KgraphicindicatingK=3asthemostprobable numberofgroups.(B)GraphicrepresentationofthedifferentgeneticpoolsforK=3.Populationsareseparatedbyverticalbars.

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importanttoidentifypopulationswithhighgeneticdiversityto indicatetheoperatingunits(OU).Theseunitscanguideeffective conservationstrategiesforthespeciesinsituorexsitu(Diniz-Filho andTelles,2002;Maneletal.,2003).Besides,theother popula-tions(JEQ,VAR,BUE,BEL,GOU,FESandMOC,mean=0.270)have lowergeneticdiversitythanUNA,PARandJOP.Theseresultsmay becausedbyfragmentationofthelowerSãoFranciscoRiverand bylandscapefeaturessuchasriversandmountainrangesinthese areas,possiblylimitingthegeneﬂowamongpopulations(seethe Discussionsection).

TheﬁxationindexofE.contortisiliquum(˚ST=0.155)was

con-sistentwithoutcrossing,long-livedperennialspecieswhichhave seedsdispersedthroughingestionbyanimals(Nybom,2004).In addition,thepairwise B _showed_genetic_divergence_among _E.

contortisiliquum populations which is not correlated with geo-graphicdistances. The E.contortisiliquumpods can beregarded asaNeotropicalanachronismwhoselargeandindehiscentfruits wereeatenbytheextinctmegafauna andseedsweredispersed bythemJanzen(1981a,b),JanzenandMartin(1982),Guimarães etal.(2008).Thus,theextinctmegafaunacouldhavepromoted extensivegeneflowthroughseeddispersalinthepastandbecause ofthisthereisnoisolationbydistanceamongE.contortisiliquum populations.Rodents,suchasagoutis,areprobablythepresent-day E.contortisiliquumseeddisperserandtherodentsmaymaintain thegeneticstructuregenerated bymegafauna.Itwasconfirmed thatseeddispersalbyrodentsisefficientandmaybe responsi-blefortheNeotropicalwoodyspeciespersistenceafterPleistocene megafaunaextinctions(Jansenetal.,2012).

The UPGMA dendrogram and PCAresults showthat E. con-tortisiliquumpopulations wereclusteringintothreegroups that werenotrelatedtogeographicdistance(Figs.2and3).However, thesepopulationsarespatiallystructuredintogroupsdistributed intheWest,CentralandEastofthestudyarea.TheWestgroup corresponds to populations UNA, PAR, JOP, ITA and MAN and this last populationexhibited geneticadmixture withthe Cen-tral group,indicative of gene flow (Fig.4B). We estimatedthe populationQ-matrix(resultsnotshown)whichcalculatesthe pro-portionsofmembershipofeach pre-definedpopulationin each of the K clusters, and MAN population representsan admixed populationwith35%ofancestryfromCentralgroup.The admix-tureofMANpopulationmustberesponsiblefortheproximityof thispopulationwiththeCentralgroupobservedinPCAanalysis (Fig.3).VERandJEQpopulationsbelongtotheCentralgroup,but displayedgeneticadmixturewiththeWestgroup.Accordingto populationQ-matrixVERandJEQhave31%and17%,respectively, ofancestryfromWestgroup.GOUpopulationhas16%of ances-tryfromWestgroupand27%fromtheEastgroup.Itisimportant toobservethatVER andJEQ populationsareclosetotheWest groupinPCA,probablybecauseofadmixturewiththisgroup,and themostadmixturepopulationGOUislocatedlinkedallgroups in PCA(Fig.3).Finally, theEast groupwasformed byFES and MOCpopulations.Thisgeneticstructuremayberelatedwith land-scape.Populations ofthe Westgroup,for example, arelocated ontheleftbankoftheSãoFranciscoRiver,whichmayactasa partialgeographicbarrierlimitinggeneflow.Anotherimportant geographicbarriermaybetheEspinhaçoRange,whichmayhave influencedthepatternsobservedfortheEastgroup.These pos-siblebarriersare partiallyconsistentwiththetwo keybarriers detectedusingthesoftwareBarrierv.2.2(Fig.1).Genetic discon-tinuitiescorrespondingtotheEspinhaçoRangeweredetectedfor twoChamaecristaspecies(Leguminosae)(Silvaetal.,2007), Bulbo-phyllumexaltatum(Orchidaceae)(Ribeiroetal.,2008)andVellozia gigantean(Velloziaceae)(Lousadaetal.,2011).Besides,rivers act-ingas geographic barrierstogene flow werealsoobserved for Copaiferalangsdorffii(Pintoetal.,2004)andSilenetatarica(Tero etal.,2003).

Thepresent-day deforestation caused byhumanactivitiesis reducingnaturalhabitatsofE.contortisiliquumandrepresentsan immediatethreattoitssurvival.However,itwasobservedthat the species has high genetic diversity which needsto be pre-served. Only two of 13 populations analyzed in this study are locatedinprotectedareas,respectively,MataSecaStatePark(MAN) andLapaGrandeStatePark(MOC).Thehighestrates ofgenetic diversityanalyzedwerefoundoutsideoftheseconservationunits. We propose the creation of protected areas in different geo-graphicregionswhichincludesE.contortisiliquumpopulationsfrom differentgeneticclusters.

PopulationsUNA,PARandJOPareprioritiesbecausetheyare not inprotected areasand have thehighestgeneticdiversities. Besides,VARis importantsincejointlywiththeprotectedareas (MANandMOC)and populationscitedabove,thethree genetic clusterswillbepreserved,whichcouldmaximizetheretentionof geneticdiversityforthespecies.TheGOUpopulationisalso pri-oritybecauseitislocatedintheextremesouthofthestudyarea andisformedbyallgeneticpools(seeFig.4B)which may rep-resentaconnectionbetweenthosegroups,anditsconservationis necessarytoavoidsomeisolationbetweenthem.Thismanagement willpreservedifferencesingeneticdiversitywhichwillprovidethe evolutionarypotentialforadaptation,andlong-termsurvivalinthe faceofenvironmentalchange.Theincreaseinprotectedareaswill ensurethesurvivalofE.contortisiliquumspeciesandatthesame timecontributetothepreservationofthethreatenedBrazilianSDTF remnants.

Acknowledgments

WethankUniversidadeEstadualdeMontesClarosforlogistical supportduringourlaboratoryworkandF.S.Nevesforfieldhelp.We gratefullyacknowledgeapostgraduatescholarshiptoP.A.Moreira fromCoordenaçãodeAperfeiçoamentodePessoaldeNível Supe-rior(CAPES),theFundaçãodeAmparoàPesquisadeMinasGerais (FAPEMIG/APQ-00372-11)andConselhoNacionalde Desenvolvi-mentoCientíficoeTecnológico(CNPQ/475528/2013-1)forsupport. Wewouldliketothankthetwoanonymousreviewersfortheir suggestionsandcomments.

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