The use of genetic markers to assess population structure and relationships among species of the genus Prosopis (Leguminosae) - Sociedad Argentina de Botánica

ISSN0373-580 X

Bol. Soc. Argent.Bot. 35(3-4):315

-

324.2000

THE

USEOF

GENETIC

MARKERS

TO

ASSESS POPULATION

STRUCTURE

AND

RELATIONSHIPS

AMONG

SPECIES

OF

THE

GENUS PROSOPIS

(LEGUMINOSAE )*

BEATRIZ O. SAIDMAN12, CECILIA F.BESSEGA13, LAURA I.FERREYRA1,

NORMAJULIO4,and JUANC. VILARDI12

Summary:The genusProsopis constitutesapromissoryresourcefor reforestation ofarid and semiarid regions ofthe world.Itsmain diversity center islocatedinSouthAmerica.These plantsarealsoaninterestingmodel for evolutionary studies, involving matingsystem andpopulation structure analysesaswellasthe evaluationof

genetic differentiation among species.This work isanup-to-date revision of theinformationaboutgenetic

structureof populationsand therelationships amongseveral speciesofProsopis.Somepreliminaryresults and the perspectiveof futurework using modernmoleculartoolsare commented.

Keywords:Prosopis, isoenzymes,RAPD,cpDNA, RFLP,DNA sequencing, matingsystem.

Resumen: Eluso de marcadoresgenéticosparaevaluar la estructura poblacionaly las relaciones entre especies delgéneroProsopis(Leguminosae).El género Prosopis constituyeun recurso promisorioparala

reforestaciónderegiones áridasysemiáridasdelmundo. Suprincipalcentrode diversidad estásituadoen

Sudamérica.Estos vegetalessontambiénun modelo interesante para estudios evolutivos, incluyendoanálisis del sistema de apareamientoylaestructurapoblacional,comoasí tambiénlaevaluaciónde la diferenciación genéticaentre especies.Estetrabajoes una revisión hasta la actualidad dela informaciónacerca delaestructura genéticade laspoblaciones ylas relacionesentrevarias especies de Prosopis. Se comentan además algunos

resultados preliminaresylas perspectivas detrabajos futurosusando herramientasmolecularesmodernas. Palabrasclave:Prosopis,isoenzimas, RAPD,cpDNA, RFLP,secuenciaciónde ADN, sistema de apareamiento.

The rationaluseofpromissoryspecies and the development of programs of germplasm conservation andprotection of endangered species require a deep knowledge of their biological

characteristics,adaptive strategies, and evolutionary . relationships.

Decisions on themost efficient methods for sampling and improvingprofitable characteristics of these species should be largely based on information of biologicalcharacteristics. Studieson taxonomy,demography, anddistribution of genetic variabilityareveryimportant in thisrespect.

Themethodology appliedtopopulation genetic studies provides genetic markers useful for taxonomy.They also allow analyzingreproductive systemandpopulation structure,which constitute important information for demographic studies.

In a wide sense, a genetic marker is any morphological, chromosomal, biochemical, or molecular trait thatcanbe accurately identified in anyindividual of thepopulation and is genetically determined. Historically, morphological traits are themostusedintaxonomic studies for they usually reveal different adaptive responses linked to the

INTRODUCTION

The genus Prosopis includes about 44 species groupedin five sections. Some sectionsaresubdivided

makingatotalof eightseries. Thisgenuscomprises shrubs andtreesthat exhibit ahigh economic and ecologicalpotential in semiaridareasofthe Americas. Some of themareconsideredmultipurpose natural resourcesbecause they may be used for reforestation ofarid and semiarid regions, production of wood,

charcoal,forage,human food,etc.Unfortunately,the extensiveexploitation ofsomespecies of this group is leadingtothe destruction and total eradication of Prosopis forests(Hunzikeretal. 1986).

*Dedicated to Prof.Dr.JuanH. Hunzikeron the ocassion of his75'" anniversary.

'DepartamentodeCiencias Biológicas,Facultad deCiencias Exactas yNaturales,Universidadde Buenos Aires, 1428

Buenos Aires,Argentina,e-mail:[email protected].

"Memberof CarreradelInvestigadorCientífico, Consejo Nacional

de Investigaciones Científicas yTécnicas(CONICET). "Fellow of CONICET.

4GenéticadePoblacionesy Evolución,FacultaddeCiencias

Exactas, Físicasy Naturales, Universidad Nacionalde

Córdoba.

Usually they canbe detectedindependently of

developmental or environmental conditions. Polymorphisms for the length of restriction fragments (RFLP) aremostoftencodominant,but thetechniqueis rather laborious and sometimes their

study requires previousgenetic informationofthe

speciesunder study, whichare notalwaysavailable. Markers basedonthe polymerase chainreactiqn (PCR), mainly RAPDs, are dominant, but the technique is simple and doesnotrequireprevious

genetic knowledge. This technique

is

usefulto analyze closelyrelatedpopulationsorspecies.

Themostdirectapproachtogeneticvariation is

DNA sequencing. However,its

application

at population level is expensive. Dependingonthe relationship between thepopulationstobe compared, morevariable or moreconserved regions of the genome should be chosen for the study,in orderto provide useful markers for evolutionary studies.

Inspecies of genusProsopis.,isozyme, RFLP of

mitochondrialandchloroplastDNA, RAPD, and

DNA

sequencingtechniquesarecurrentlyunderuse toaccomplishdifferent purposesinvolving: speciationprocess. Chromosomal characteristicsare

also frequently associatedtoselective effects, the rearrangements may affectthe cell cycle and the

gametogenesis,andareassociatedtopositioneffects ofthe genes they bear. Among biochemical and molecular markersbothneutral and selectivevariants canbe found. The usefulness of each of theseclasses dependsonthe kind of analysistobe performed.

Neutral markerstypicallyvary randomlyinsuch away that differentiationamongpopulationsis

relatedto divergencetime and degree of isolation (gene flow). Thedistributionofgenotypicfrequencies ofthese markerswithin populationsmainlydepends onthereproductivesystemandpopulationstructure. Incontrast,selective markers reveal adaptationto different environmentalconditions and/oradaptive

strategies.Sometimesthe distinction between these two groups is complicated because ofphysical

linkage among essentiallyneutral genes with others of selective importance. Insuch cases,neutral markers may be usefultoselect indirectly beneficial

traits whose expression is affected by the environmentoroccursin látestagesof

development-1.Estimation of genetic distances andcluster

analysis.

2.Analysis ofgeneticstructureand distribution of geneticdiversityatdifferentlevels:individuals, populations, regions,and species.

3.Analysis of matingsystemparameters. 4.Phylogenetic analysis.

BIOCHEMICAL

AND

MOLECULAR

MARKERS

Theraw data forpopulation genetic studies

consist ofthenumber of individualsinthesamples withacertainphenotypeor genotype.Phenotypicor genotypic frequencies are inferred from this information forthepopulationsas awhole.

Allozymemarkers areusuallyeasy toanalyze

and in many cases are codominant. Asgenotypes canbe inferred directly fromthe observed band patterns, they are especially useful to study population geneticstructure.However,theremaybe some shortcomings associated with isozymic analysis. Although allozymic variation has been

largelyconsidered essentially neutral (seeKimura,

1982),thereareexamples of enzyme polymorphisms

where selective effects have been demonstrated

(McDonald & Kreitman, 1991).In such cases, inferences.ongenetic structure are notreliable. Another limitation ofisozyme analysisis that the expression of many isoenzyme systems

varies

throughout ontogenetic stages and sometimes the

environment

alsoaffects it.

Thereareseveraltechniques for theanalysisof genetic markers basedon

DNA

polymorphisms.

GEOGRAPHICAL

DISTRIBUTION,

TAXONOMIC PROBLEMS, AND POPULATIONS SAMPLED

The genusProsopisis distributedinall arid and semiaridregions of the world. Its distribution

involves

South EastAsia (3 native species),tropical

Africa (T species),andAmerica(40species). The highest numberofspeciesoccursinSouthAmerica,

in

thebiogeographicprovinces(Cabrera &Willink,

1980)of Monte, Espinal, and Chaco, that belongto the Chaqueño Dominion of theNeotropical Region. Theseprovincesareimportant centersofspecies

differentiation

(Burkart,1976; Hunziker etal.,

1986). The genus occupiesmostofthe Argentinean

territoryfromthe annual isohyetal line of 1500mm inthe northup tothe 12°C isothermas theSouth

B. O. Saidman et al., Genetic markers, population structure andrelationshipsinProsopis

distribution limit (Hunziker et al., 1986). The Important differenceswere detectedbetween these ChaqueñoDominion(Argentina,Bolivia,and Pa- sections regarding several population genetic

raguay)constitutesamaincenterof morphological parameters. polymorphism ofProsopis with about 28species,

13ofwhichareendemic (Burkart, 1976).

The species ofAlgarobiaexhibited ahigher

genetic variabilitywithinpopulations,measured in There is another importantcenterofpolymorphism termsofpercent ofpolymorphicloci (meanP=51.5)

inNorthAmerica,the Mexican-Texan, with nine andheterozygosity (mean H=0.2l)than those of

species,three of them endemic (Burkart, 1976).

Speciesbelongingto twoofthe five sections of differentiation among species withinsection

Algarobiameasuredthrough Nei’s(1972)distance wasrelativelylow with valuesrangingwithin the expectedàccordingtoAyalaetal. (1974) for subto semispecies.

Thegeneticsimilarities among species of section Algarobia obtained from isoenzyme data donot agree with the expected according to the morphologicalevidence. The phenogramsobtained

from geneticdistance matrices (Saidman &Vilardi, 1993;Saidmanetal., 1998a)are notconsistentwith the series defined by Burkart (1976).

The lack of consistence,ofmolecular and

Strombocarpa (P=15.31; H=0.06) (Table 1). The Prosopis,AlgarobiaandStrombocarpa, have been

studied using isoenzyme electrophoresis and DNA markertechniques(Saidman &Vilardi, 1987, 1993;

Saidmanetal.,1996, 1998a,b).

The section Algarobia involves the most important species from the economic andecological points of view,and is themost deeply studied

genetically. The taxonomy of this group is complicated because, despite the important

morphological

differences, the speciesare verysimi¬ larbiochemicallyandgenetically.(Saidman,1985,

1986, 1993; Saidman & Vilardi, 1987, 1993;

Saidmanet al. 1997, 1998a,b), andinterspecific. hybridization is very frequentinzones

of

sympatry (Hunziker etal., 1986).The distinction ofsomeof the series proposed by Burkart (1976) maybe

questioned onthe grounds of different lines of

evidence, including morpholdgy, natural

hybridization,chromatography ofphenol compounds, seedprotein,and isoenzyme electrophoresis(see Hunzikeretal.,1986),and RAPD (Saidmanetal., 1998b;Ramirezetal.,1999; Bessegaetal.,2000a). The frequentinterspecific hybridizationcreates intermediatephenotypesthat difficult morphological

determination,

and

the consequences of this

phenomenoninAlgarobiaare notclear. Palacios & Bravo(1981)proposed that agroup of sympatriq species of this section that undergo frequent

hybridization would constitute asyngameon,as defined by Grant (1981).

The understanding of causes and genetic

consequences of hybridization in this grouprequires *

the.useof geneticmarkers.

morphologicaldatawasalso

observed

inaRAPD

analysis by Ramirez etal. (1999), in whichthe

phenetic relationships among species donotsupport Burkart’s (1976)series,

In section Strombocarpaspecies areclearly

differentiated

byisoenzymes,with manydiagnostic loci. The differentiationbetween the sections

Algarobia andStrombocarpaat the isoenzymatic levelwas so strongthat the ancestral homology of bahdpatternscouldbe observedinonlyone(SOD) oftheseven systemsanalysed (Hunzikeretal.,1986;

Saidmanetal., 1996).

RFLPanalysis

of

mitochondrial(mtDNA)and

ribosomalDNA (rDNA)analyzedby Southernblot,

andthe different Sizes of the transcript spacer of rDNA obtained by PCR yielded similar conclusions about the differentiation between these sections (Saidmanetal.,1998a).The phenogram basedon both mtDNA andrDNA variationwereconsistent andshowed higher differentiationamong species belongingtoStrombocarpa thanwithinAlgarobia. Thespeciesof Algarobiaassociate inasingleclus¬ ter,and distances betweenspecies withinthis section are

lower

than any recorded distance betweenspecies belonging to different sections. The distances

betweenspecies within Strombocarpaareinsome

' _casesas high_as those observed betweenspecies

belongingtodifferentsections.

BIOCHEMICAL

AND MOLECULAR

STUDIES IN SPECIESOF

PROSOPIS

Isoenzymeelectrophoresiswasappliedtospecies

belongingto twosections,Algarobiaand Strombocarpa.

F,s=

00 _cn

Section Series Species Population N°loci H P FIS Reference O

P

Algarobia Ruscifoliae P. ruscifolia Herrera,Sgo.delEstero 24 0.232 52.2 0.29 Montoyaetal. (1994)

Sarmiento, Sgo. del Estero 24 0.223 60.9 0.33 Montoyaetal. (1994)

_>

<3

Rivadavia, Salta 24 0.222 60.9 0.3 Montoyaetal. (1994)

CD

Pinto.Sgo. del Estero 24 0.239 60.9 0.48 Montoyaetal. (1994)

3-P. vinalillo Tucumán 24 0ÿ253

0.170

62.5 0.31 Ferreyraetal. (1998)

P. hassleri Dept. Patiño, Formosa 24 45.0 _* Saidman (1985) _CD

Chilenses P.nigra La banda, Sgo. delEstero 24 0.210 48.0 _* Saidman(1985) O

Paraná,Entre Ríos 24 0.220 48.0 _* Saidman(1985)

GO

La Merced,Salta 24 0.244 58.3 0.54 Montoyaetal. (1994) _cn

Huillacatina, Sgo.del Estero 24 0.298 66.7 0.18 Ferreyraetal. (1998)

GO P.caldenia SantaRosa,La Pampa 23 0.200 48.0 _* Saidman (1985)

4ÿ P.tlexuosa Quilmes,Tucumám 24 0.261 60.9 0.29 Bessegaetal.(1996)

0.1

LaAmarga,LaPampa 24 0.222 60.9 Ferreyraetal. (1998) K> O

P.alpataco Chacharramendi, La Pampa 24 0.170 45.0 _* Saidman(1985) _O

P.alba

.

Trancas,Tucumán 24 0.140 41.7 0.31 Montoyaetal. (1994) O Chicoana, Localidad 24 0.169 50.0 0.41 Montoyaetal. (1994)

LaMerced,Salta 24 0.163 37.5 0.33 Montoyaetal. (1994)

Sumalao,Salta _* 24 . 0.168 41.7 0.41 Montoyaetal. (1994) Burruyacu, Tucumán 24 0.190 50.0 0.24 Ferreyraetal.(1998) P.glandulosa Weslaco,Texas 27 0.217 52.2 0.38 Bessegaetal. (2000a) LaCopita,Texas 27 0.222 52.2 0.16 Bessegaetal. (2000a) Bell Co, Texas 27 0.183 47.8 0.27 Bessegaetal. (2000a)

FrioCo., Texas 27 0.210 52.2 0.07 Bessegaetal. (2000a)

P. velutina Santa Rita,Arizona 27 0.127 33.3 0.31 Bessegaetal. (2000a)

P.chilensis VillaDolores,Córdoba 1.7 0.248 58.8 0.29 Julio(2000) Patquia, LaRioja 17 0.262 64.7 0.25 Julio (2000) Belén, Catamarca 17 0.296 64.7 0.16 Julio (2000) Las Talas, LaRioja 17 0.252 64.7 -0.12 Julio (2000) Talampaya, LaRioja 17 0.198 52.9 0.62 Julio(2000) La Higuerita,Sanluis 17 0.219 52.9 0.3 Julio (2000) Media Naranja, Córdoba 17 0.276 64.7 0.31 Julio(2000)

ChacabucoSantiago,Chile 17 0.258 64.7 0.26 Julio (2000) MontePatria, Limari, Chile . 17 0.241 52.9 0.15 Julio (2000) Soto; Córdoba 17

-

0.195 41.2 0.28 Julio (2000) Contara, Córdoba 17 0.262 58.8 0.1 Julio (2000) Astica,SanJuan 17 0.258 52.9 0.03 Julio(2000) P.juliflora Cartagena, Colombia 20 0.130 30.0 _* Saidmanetal. (1997)

Altamira, Colombia 20 0.050 13.0 _* Saidmanetal.(1997)

Sericanthae P. kuntzei Tacana,Tucumán 18 0.139 33.3 _* Saidmanetal. (1998a)

Strombocarpa Strombocarpae P.strombulifeiCopacabana, Catamarca 23 0.050 13.0 _* Saidman(1985) Conesa,Rio Negro 23 0.020 8.0 _* Saidman (1985)

'23

P.reptans Herrera,Sgo.delEstero 0.090 17.4 _* Saidmanetal.(1996) Icaño, Sgo.del Estero 23 0.090 21.0 _* Saidmanetal. (1996)

Famatina,LaRioja

P.torguata 17 0.080 18.0 _* Saidman (1985)

P.pubesccns Bell Co,Texas 14 0.036 7.1 _* Saidmanetal. (1996) Saidmanetal. (1996) | Cavenicarpac P. ferox Los Cardones, Salta 22 0.088 22.7 _*

B. O. Saidman et al., Geneticmarkers,population structure and relationships in Prosopis glandulosa,P.velutina,P.chilensis,P. nigra,P.alba,

Pflexuosa,and P.

_ruscifolia

(Bessegaetal.,2000b). Theestimated valuesofsingle and multilocus

ISOENZYMATIC

AND MOLECULAR

STUDIES IN THE SECTION

ALGAROBIA

_{outcrossingrates variedfromabout 0.72to 1.00,}

indicatingthat thespeciesaremostlyoutcrosserbut upto28% of selfingcanoccur, withanaverageof

15%. These outcrossingrateestimatesweresimilar population genetic standpoint. The studies conducted _{t0the values obtained by Keys & Smith (1994) for} in species of this section involve the distribution of _{populationsof P.velutina. Ther estimates obtained}

genetic variability,population structure,mating _{by Bessegaetal.(2000b)werehigh,indicatingthat} system,genetic differentiation amongpopulations _{outcrossing rates vary among trees within}

andspecies, and relationships between genetic

differentiationandgeographicisolation.

Algarobiais the best studiedsection from the

populations.Finally, high

_rp

estimates werealso

obtained,whichmeansthat many individuals (seeds) withinafamilyarefullsibs.

The estimatedinbreeding coefficientsformother plantgenotypes

(F,SJ

wereinallcasessignificantly lower than the values obtained for the whole seed Populationstructureinspecies of Algarobia

Populationstructure wasanalyzedbymeansof Wright’s (1951) F statisticsin31populationsof 8

species:P. nigra, P.alba,P.flexuosa,P.chilensis,P.

ruscifolia,

P glandulosa, P.velutina,and P.vinalillo

(Table 1).

Observed and Hardy-Weinberg expected

genotypic frequencies werecomparedthroughthe

FIS

coefficient (Table 1). In all populations but one, mean

_F[s

estimates werepositive. These results indicate a general trend towards significant homozygoteexcesswithinpopulations, whichmight

be dueto acertaindegree

of

endogamy.

Since these species were largelyconsidered obligateoutcrosser(Burkart, 1976; Simpson,1977;

Simpsonetal.,1977),the explanation of this result requiredananalysis ofthe matingsystem.

population

(F/s).

A possible explanation for such

differencemight be that selective forces favor

heterozygousseeds,reducingtheproportionofselfed individuals

in

theadult population(Bessegaetal.,

2000b).

Anadditionalobservationwasthat pollen and ovule allele frequencies donotdiffer significantly. This result is compatible with thehypothesis of limited pollen dispersal.

Partial selfing andlimitedpollen dispersalwould explain satisfactorilythe homozygoteexcessdetected in allpopulations ofspecies of Algarobia studiedso far. Selection against homozygoteswouldbe thecau¬ sefortherelativelyhigh variability inthese species (Bessegaetal.,2000b).

Estimation

_of

matingsystemparameters

Isoenzyme

_{differentiation}

within andbetween species

of

SectionAlgarobia

Matingsystemanalysis using isoenzymaticdata

isbasedonthe mixed model and Ritland & Jain (1981)estimationprocedure.The methodassumes that progenyarederivedfromeither random mating (outcrossing) orself-fertilization.

Thesample is subdivided into families. Each familyconsistsofseeds collectedfrom thesame motherplant. Consequently,each familyinvolves.

full and/or halfsibs,dependingontheoriginofthe fertilizingpollen grains. Thegenotypeofthe female parentofeach groupcanbe assessed by the 'method

ofBrown &Allard (1970).

Usingthe program MLTR (the improvedversion

ofMLT;Ritland,1990)multilocus

(Jj

andsinglelocus

(t)

outcrossingrates,the correlation oft

(r)

within progenyarrays,the correlation of outcrossedpaternity

(rp),

and fixation index ofmaternalparents

(FISm)

were estimated insevenspecies of Algarobia (Fig.

1):

P.

Oneofthemostremarkable

characteristics

of the section Algarobia is- thehigh genetic similarity . among thespecies so far studied (Saidman, 1985, 1986, 1993; Saidman & Vilardi, 1987, 1993; Saidman et al., 1997, 1998a,b;Bessega et al., 2000a,b,unpubl).Almost all alleles ofpolymorphic

isoenzymelociareshared by allspecies. Diagnostic

lociaremostlyabsentand speciesdiffer onlyinallelic

frequencies.

At first glance this among-species similarity

mightbe the consequence of the highnatural interspecific hybridizationrates recorded inthis section (see Hunzikeretal.,1986).However,some evidenceareagainstthisinterpretation.

TetraploidP.juliflora populations showedhigh

i

1

life

pEf

m

—

0.8 0.6 0.4 0.2 0

P.alb P.nig P.chi P.fle P.rus P.vel P.gla

UrtBrp

A

II

ii

n

i

1 0.8 0.6 0.4 0.2 0

P.alb P.nig P.chi P.fle P.rus P:vei P.gla

Qtm |ts

B

Fig. 1

.

Parameters ofmatingsysteminsixspecies ofProsopis.P.alb= P. alba,P.nig-P.nigra,Pcbi=P.chilensis,P.fle=P.flexuosa,P.rus= P. ruscifolia,P.vehP.velutina, P.gla=P.glandulosa,A=tmandtsdenotemultilocusandsinglelocusoutcrossingratesrespectively.B=

rtandrp denote correlationoftmwithinprogenyarrays and correlationofoutcrossedpaternity.

geneticsimilaritywithtwoallopatricdiploid species, Texasand P.ruscifoliaandP. fíexuosa fromArgentina

P. ruscifolia and P.caldenia(Saidmanetal,1997). showed that thegenetic differentiationbetweenthe Inthis case,hybridizationand introgression isnot a SouthAmericanspecieswas notdifferentfromthe

plausible explanationforthe geneticsimilaritysince

differentiation

between any of them andP.glandulosa. thegeographicdistribution and thedifferent ploidy

levelareexpectedtopreventanygeneticexchange hybridizationand introgressiondoesoccurbetween between P. julifíora and the othertwospecies.

The conclusion of these studies is that although species

of

sectionAlgarobia,the high similarity

Furthermore,in surveys involving Argentinean amongspecies can notbeexplainedsolelyonthe

populationswith differentspecificstatusandawide basis of interspecific gene flow. Sincemostalleles

geographicalrange indicated thatWright’s(1951) areshared by almost allspe.cies and their genetic

Fsr

estimates among allopatric conspecific variabilityis also verysimilar,it isconceivablethat populations aremuch lower than those recorded selection may haveplayedarole inpreservingthe amongparapatricpopulationsof differentnominal genétic variabilitydetectedisoenzymatically. speciesthat hybridize frequently (Saidman etal.,

1998a).

Finally,recentresults by Bessegaetal. (unpubl.) ' _chilensis

studyingNorth and South American species of

Algarobiaalso indicate that highgeneticsimilarityis

independent of the opportunity ofhybridizationto speciesofthis genus. Burkart (1976) suggested that take place. They found thatinterspecific genetic it should beincluded

in.

reforestation programs

distances

arenot correlatedtogeographicdistances, because of itsgreatmorphological variability and

Theanalysis of populations of P. glandulosa from relativelyfastgrowing.Further studies (Roig, 1993; Distribution of genetic variabilityinProsopis

Prosopischilensisisoneof themostpromissory

B. O. Saidman et al., Genetic markers, population structure andrelationshipsinProsopis Ramirezetal.(1999) studied 15species belonging

Dalmasso, 1993;Cony, 1993; Karlinetal.,1997)

encouraged the development of selection programs to five sections (Prosopis, Anonychium,

based on the high morphological variability Strombocarpa, Monilicarpa, and Algarobia). In this

associatedtoclimatic andedaphicdiversityand the caseDNA obtainedfrom 10individuals of each specieswaspooled andusedastemplatefor PCR high fruit productivityofthisspecies.

Rational programs to improve beneficial reaction.The RAPDpatternsobtainedby this method characteristicsofthis speciesrequire information yieldedaphenogramthat separatesspeciesbelonging aboutthe distributionofgenetic variability.Recent todifferent sections,but it givesno support to the

studiesonthe genetic structureofthisspecies (Ju- series defined by Burkart (1976). Since eachspecies

lio,2000) evaluated the distribution of genetic was represented by onepooled sample of DNA

variability. Ahierarchical analysis of population obtained fromdifferentindividualsnoinferenceon differentiationwasperformedusingthe formula of withinspeciesvariationwereobtained.

Preliminary results byFerreyra et al. (1999) Wright (1978). A total of 12populations were

sampled fromthree geographicalregions:Chaco indicatethatsomecombinations of RAPD bandsare (Córdoba and SanLuis, Argentina) and Monte usefultodifferentiateindividuals of fivespecies (P.

biogeographicalprovinces(LaRioja, Catamarca, and

ruscifolia,

P. alba, P. nigra, P.flexuosa, and P. SanJuan, Argentina), and ValleCentral(Chile).

Results indicated that mostvariation occurs hybrids.

within populations (-87%oftotal variance). The

difieren!

iation amongpopulationswithinregionswas appliedtoevaluatephylogeneticrelationshipsamong verylow(-13%)andnodivergenceoccursamong speciesofProsopis.They

involve

RFLP(Bessegaet

al.,1998)and sequencing (Bessegaetal.,1999)of

cpDNA fragments.

The probes forsouthern hybridizationsaretwo contiguous fragmentscorrespondingtotheregions

IRand SSC. Thesesegmentscombined make about Isozymesprovedtobeexcelldhttools to show ofthe chloroplast genome. The trnT-trnD differencesbetween the sections Algarobiaand intergenic regionwasamplified fromtotalgenomic

Strombocarpaandtostudypopulationstructure.In DNA by the polymerase chain reaction (PCR) using contrast,the lack of diagnosticlocimakesisozymes the universal primersdescribed byDemesureetal. unsuitable for unequivocal species and hybrid (1995).Preliminary resultsonthe relationships

'

recognition within the sectionAlgarobia.

Diversemoleculartechniquesare

produce markers usefultofiilfil these purposes as

flexuosa,

P alpataco, P. nigra, P affinis,and P. wellastoconduct evolutionarystudies.The results kuntzei)andoneofsection Strombocarpa(Preptans) ofrecentanalysesbasedon RAPDs arepromissory, wereobtained by cladisticanalysisofboth datasets.

Saidman et al. (1998b) studied 5 populations Theyshow partialagreement.Tworemarkable facts involvingthe species P. alba,Pnigra,and P.

_flexuosa

maybe pointed:P.reptansis highly differentiated and natural hybrids. They estimated allelic fromthe species ofAlgarobia andP. kuntzeiseparates

frequenciesofRAPD lociusing themethodofLynch early fromthe remainingspecies of Algarobia.The

&Milligan (1994). Aphenetic approach indicated cladograms obtained donotsupportBurkart’s (1976) that hybridpopulationsarenotintermediate between series. Thesepreliminaryresultsareconsistentwith their putativeparents. Bessega et al. (2000a) previous biochemical and molecular evidence

estimated genetic variabilityand differentiation indicatingthat the series within section Algarobia shouldberevised.

vinalillo)andtoidentifyputativeparentsofnatural Othermolecular techniquesarecurrentlybeing

regions.

MOLECULAR

STUDIES

amongtenspeciesofsection Algarobia (P.

ruscifolia,

beingusedto

fl

vinalillo,P. alba,P. glandulosa, P.caldenia,P.

between 4populationsof P.glandulosaandoneof P. velutina using both RAPD and isozyme techniques. Inthis studyRAPDs yielded higher

geneticdistances amongpopulationsofP.glandulosa throw lightontherelationships among the species

thanisozymesdid. °f this genus.

Further studiesincludingmore species of this group and the study ofnewregions of DNA, may

molecular data suggest that the series shouldbe revised.

Theuseofcladisticapproaches using organelle

andnuclearDNAvariation willgiveaninsightonthe

relationships among species with higher

differentiationand itwillgreatlycontributeto the

understandingofevolution trends in this genus.

CONCLUDING

REMARKS

The analysis of therelationshipsamongspecies and the distribution ofgeneticvariabilityatdifferentlevels (amongspecies, amongpopulationswithinspecies and

withinpopulations)inthe genus Prosopis is very important from theoretical and practicalstandpoints.

Classical morphologicalapproaches haveshown

important variation amongpopulations ofsome species,mainly ofsection Algarobia, forwhich varieties and/orecotypeshave been described. Other

species have shown important differences in geographicand environmental range.Finally,their

multipleusesand abilitytogrowinarid and semiarid zoneshave evidenced thepotentialofthese species asnaturalresourcesinareaswheremosttraditional forestspeciesare notabletosurvive.

The studies on the distribution of genetic variabilityusing RAPD and isozyme markers in the most promissory species (section Algarobia) indicated that the majority of withinspeciesdiversity occurswithin populations. This result indicatesthat

fewpopulationsare anadequatesample of the whole withinspecies variability.Studiesontheassociation ofthese markers with selectable traitsare yet not developedand they should be encouragedtodefine the best strategies for improving thesespecies.

Thehigh hybridizationrates among several species of sectionAlgarobiahas ledtotheassump¬ tionthattheyconstituteasyngameon. Inthis group

speciesboundariesare notclearly defined. However, the speciesapparently constitutenatural entities e-ffectivelyisolated fromeachother. Since there¬ productive barriersareincomplete, the speciescon¬ ceptthat best fit theconditionofthese entities would beTempleton’s(1989)cohesiveconcept(Montoya

etal.,1994; Burghardt,1995; Saidman etal.,1998a). RAPDmarkersarethe first molecular tool that allows differentiating qualitatively closelyrelated

speciesof Algarobia: P.

ruscifolia,

Palba, P. nigra, P.

flexuosa,

and P. vinalillo.

Genetic markers have also contributedto the understandingofthematingsystem,an important concernfor thedesignofselection programs.

Thecomparisonofspecies belongingtodifferent sections has shown thattheyconstitute natural well-differentiated groups. ThespeciesofAlgarobiaare more closelyrelatedto each other than those of Strombocarpa. By contrast,the biochemical and

ACKNOWLEDGEMENTS

The authors wishtoexpress their gratitudeto Prof. Dr. Juan H. Hunziker who was apioneer in our countryintheuseof evolutionary thinkingto solve biological issues. Hisenthusiasm,honesty, dis¬ cipline, andcriticalsense was aguidance forhis

followers. Thankstohisvaluable contributionsand permanent supportdifferentresearch lines connected

bythe evolutionaryinterest have been andarestill

growingupinthe Departamento de Ciencias Bioló¬

gicas,Facultad deCienpiasExactas yNaturales,

Universidad deBuenosAires,the institutiontowhich he dedicatedmorethan 40 yearsoffruitfulwork.

Thiswork wascarriedoutthankstothe

financial

"supportofAgenciaNacionalde Promoción Cientí¬ fica y Tecnológica(PICT 01-00000-02269 and 1-6628),Consejo Nacional de Investigaciones Cientí¬ ficasyTécnicas (PIP.0722/98),and Universidad de BuenosAires (TY09).

BIBLIOGRAPHY

AYALA, F.J„'M.L.TRACEY,D. HEDGECOCK& R.C.

RICHMOND.1974.Genetic differentiationduringthe

speciationprocessin prosophila. Evolution28: 576-592.

BESSEGA, C.,B. O. SAIDMAN & J.C.VILARDI. 1996. Alta

similitud genéticaentreespeciesde la secciónAlgarobia

(Prosopis,Leguminosae). ActasXXVIICongresoArgén-,

tino de Genética: 167. Viña del Mar, Chile.

BESSEGA, C., M. CLEMENTE,B.O.SAIDMAN &J.C.

* VILARDI. 1998. Diferenciaciónentrelospatronesde

restricciónde ADNdecloroplastoentreespecies de la Sección Algarobiadel género Prosopis (Leguminosae).

XXVIJomadasArgentinasde Botánicas: 106. Córdoba.

BESSEGA,"C.,M. CLEMENTE,B.O. SAIDMAN & J.C. VILARDI.1999. Análisisde las relacionesentre 11espe¬ ciesde Prosopis(Leguminosae)basado en lasecuencia'de

unfragmentodel espaciador intergénico TrnD-TrnT del cloroplasto.XXIXCongresoArgentinode Genética: 146. Rosario.

B.O.Saidman etal., Geneticmarkers,populationstructure and relationships in Prosopis

KARLIN,U. O., R. COIRINI, L. CATALAN & R. ZAPATA. 1997.Especiesarbóreas y arbustivas para zonas áridas y semiáridasde América Latina. Serie Zonas Aridas y SemiáridasN°12.OEA. Programa de las Naciones Uni¬ das para elMedio Ambiente,FAO. pp. 41-51. BESSEGA, C., B. O. SAIDMAN & J. C. VILARDI. 2000a.

Isozyme and RAPD Studies inProsopisglandulosa and

P velutina (Legúminosae, Mimosoideae). Genet.Molec. Biol. 23: 1-5.

BESSEGA, C., L.I.FERREYRA, N. JULIO, S. MONTOYA,

B. O. SAIDMAN & J. C. VILARDI. 2000b. Matingsystem KEYS, R. N. & S. SMITH. 1994. Matingsystemparameters parametersinspeciesof genus Prosopis (Leguminosae).

Hereditas 132: 19-27.

andpopulationgenetic structureinpioneerpopulations

of Prosopis velutina (Leguminosae). Amer. J. Bot. 81: 1013-1020.

BESSEGA, C.,L.FERREYRA, J. C. VILARDI & B. O.

SAIDMAN.Unpubl. Unexpectedlowgenetic differentiation KIMURA, M. 1982. The neutral theory as abasis for amongallopatric speciesofsectionAlgarobiaof Prosopis

(Leguminosae).

understandingthe mechanismofevolution and variation

atthe molecular level. In: KIMURA, M. (ed.), Molecular Evolution, ProteinPolymorphism and the Neutral Theory,pp. 3-56.Japan Scientific Society Press,Tokyo;

Springer-Verlag,Berlin.

BROWN,A. H. D. &R.W. ALLARD. 1970. Estimation of the

matingsystemin open-pollinatèdmaize populationsusing isozyme polymorphisms.Genetics66:133-145.

BURGHARDT, A. D. 1995. Aproximación al análisis quimiosistemático en la delimitación deespeciesde plantas. Mendeliana 12: 19-24.

BURKART, A. 1976. A monographofthe genusPtvsopis(Leguminosae subfam. Mimosoidae). J. ArnoldArboret. 57: 219-249, 450-525.

CABRERA, A. L. & A. WILLINK. 1980. Biogeografía de Amé¬ rica Latina. Monografías Científicas OEA vol. 13.Washing¬ tonD.C.,USA.

CONY,M. 1993.Reforestaciónracional dezonasáridasysemiáridas

conárbolesdemultiples propósitos.Interciencia20: 249-253. DALMASSO, A. 1993.Selecciónde formasde unápoblaciónde Prosopischilensis Stuntz (Leguminosae) para ensayos de fo¬

restación.ContribucionesMendocinasalaquinta ReuniónRe¬

gionalpara América Latina y el Caribe de la Red de Foresta¬ cióndel CIID. ConservaciónyMejoramientodeEspecies del Género Prosopis.IADIZA-CRICYT-CIID, Mendoza.

DEMESURE,B., N. SODZIN & R.J.PETIT. 1995. Asetofuni¬ versalprimersforamplificationofpolymorphic non-coding regionsofmitochondrial and chloroplast DNAinplants. Molec. Ecol. 4: 129-131.

FERREYRA, L., C. BESSEGA,N. JULIO, F. MOLLARD, B. O. SAIDMAN &J.C. VILARDI. 1998. Estudiosisoenzimáticos enespeciespurasehíbridosinterespecíficosde la Sección Algarobiadel Género Prosopis (Leguminosae). Actas XXXI

Reuniónanual delaSociedadde Genética de Chile: 101. La Serena, Chile.

FERREYRA, L., B. O. SAIDMAN &J.C. VILARDI.1999. Ca¬ racterizaciónde cinco especies de la SecciónAlgarobiadel Género Prosopis (Leguminosae) por medio de RAPD. Actas XXIX CongresoArgentinode Genética: 143. Rosario. GRANT,V.1981. Plant speciation. 2nd. ed. ColumbiaUniversity

Press,New York.

HUNZIKER, J. H.,B. O. SAIDMAN, C. A.NARANJO, R. A.PA¬

LACIOS,L. POGGIO & A.D.BURGHARDT.1986,-Studies

onthetaxonomy,geneticvariationandbiochemistryofArgentine speciesofProsopis. Forest Ecol. Manag. 16:301-315. JULIO,N. B. 2000. Estudios alozímicos sobrevariabilidad,estruc¬

tura y diferenciación genéticaenProsopis chilensis (Leguminosae, Mimosoideae) yespecies'relacionadas.Tesis de DoctoradoenCienciasBiológicas,Universidad Nacional de Córdoba.

LYNCH, M. & B. G. MILLIGAN. 1994. Analysis of population genetic structurewith RAPD markers. Molec. Ecol. 3: 31-99.

MCDONALD, J. H. & M. KREITMAN. 1991. Adaptive proteinevolutionatthe Adh locus inDrosophila.Nature 351: 652-654.

MONTOYA,S., B. O. SAIDMAN, J. C. VILARDI,&C. BESSEGA.1994.Diferenciaciónyflujogénicoentreespe¬

ciesde la sección Algarobia, Género Prosopis (Leguminosae). Actas XXV Congr. Argent.GenéticayIII Jomadas Argenti¬ no-Uruguayas de Genética:17.LaPlata.

NEI, M. 1972. Genetic distances betweenpopulations.Amer. Naturalist 106: 283-292.

PALACIOS, R. A. & L. D. BRAVO. 1981. Hibridación natural en Prosopis (Leguminosae)enlaRegiónChaquefla Argentina.Evi¬

dencias morfológicas y cromatográficas.Darviniana 23: 3-35.

RAMÍREZ, L.,A. DE LA VEGA, N. RAZKIN, V. LUNA & P. J. C. HARRIS. 1999.Analysisoftherelationshipsbetween

species ofthe genus Prosopis revealedbythe use ofmolecular markers. Agronomie 19: 31-43.

RITLAND, K. 1990. A seriesofFORTRANcomputerprograms

for estimatingplant matingsystem.J.Heredity81: 235-237. RITLAND, K. & S. JAIN. 1981. A model for the estimation of

outcrossingrateand gene frequencies usingindependentloci.

Heredity47: 35-52.

ROIG,F. A. 1993.Aportesala etnobotánica del Género Prosopis. ContribucionesMendocinasala quinta Reunión Regional para AméricaLatinayelCaribede la Red de Forestación del CIID, pp.99-121.Unidadesde Botánica yFisiologiavegetal IADIZA, Mendoza.

SAIDMAN,B. O. 1985. Estudio de la variación alozímicaenel género Prosopis.Tesis Doctoral,Fac. Cs. Exactas yNat.,Uni¬

versidad de Buenos Aires.

SAIDMAN, B. O. 1986. Isoenzymatic studiesof alcohol

dehydrogenaseand glutamate oxalacetate transaminase in four Southamericanspecies ofProsopis and their natural hybrids. Silvaegenet.35: 3-10.

SAIDMAN,B. O. 1993. Las isoenzimasenelestudio de la varia¬ cióngenéticaylas afinidadesentreespeciesde Prosopis. Bol. Genet.Inst.Fitotéc. Castelar 16: 25-37.

SAIDMAN, B. O., C. BESSEGA, L.FERREYRA &J. C. VILARDI. 1998b. Randomamplified polymorphicDNA (RAPDS) variationin hybridswarms andpurepopulations

of genus Prosopis. In: BRUNS, S., S. MANTELL, C. TRAGÃRDH & A. V.VIANA(eds.), Recent Advances in

Biotechnology_forTreeConservation and Management, pp. 122-134.International Foundation forSciences, Stockholm. SIMPSON,B. B.1977.Breedingsystemof dominantperennial

plants oftwodisjunctwarmdesertsecosystems.Oecologia 27: 203-227.

SIMPSON, B. B.,J. L. NEFF & A. R. MOLDENKE. 1977. Prosopisflowersas a resource.In: SIMPSON, B. B. (ed.), Mesquite: Itsbiologyin twodesertecosystems,pp. 84-107.US/IBP Synthesis Series, Dowden Hutchinson and Ross, Stroudsburg.

TEMPLETON,A. R. 1989. The meaningof species and

speciation: Ageneticperspective.In: OTTE, D. & J. A. ENDLER (eds.), Speciation and its consequences, pp.

3-27.SinauerAssociates Inc.,Sunderland,Massachusetts.

WRIGHT,S. 1951. Thegeneticalstructure of populations. Ann. Eugen. 15: 323-354.

WRIGHT, S. 1978. Evolution and Genetics ofpopulations,

Vol. 4.Variabilitywithin and among naturalpopulations.

University of Chicago Press, Chicago.

Recibido el 10de Noviembre de 2000, aceptado el 20 de Noviembre de 2000. SAIDMAN, B. O. & J. C.VILARDI.1987. Analysisof the

genetic similaritiesamongseven speciesof Prosopis (Leguminosae: Mimosoideae).Theor. Appl. Genet. 75: 109-116.

SAIDMAN, B. O. & J. C. VILARDI. 1993. Genetic variability and germplasm conservation in the genus Prosopis. In: PURI, S. (eds.), Nursery Technology_ofForest Tree Species_ofArid andSemiaridRegions, pp. 187-198.

Winrock-Oxford& IBH Publishing Co., PVT.,New Delhi, Bombay, Calcuta.

SAIDMAN, B.O.,J.C. VILARDI, M. I.POCOVÍ&N. ACRECHE.1996. Isozyme studiesinArgentinespecies

of the Section Strombocarpa, Genus Prosopis (Leguminosae/ J. Genetics 75: 139-149.

SAIDMAN,B. O., J. C. VILARDI,S.MONTOYA & L. POGGIO.1997.Genetic VariabilityandPloidylevelin

species ofProsopis(Leguminosae). Bol. Soc. Argent. Bot. 32: 217-225.

SAIDMAN, B.O.,J.C. VILARDI, S. MONTOYA, M. J. DIEGUEZ & H.E.HOPP.1998a.Molecular markers:a

tool for theunderstandingofthe relationships among species of Prosopis (Leguminosae, mimosoidae). In: PURI, S. (eds.), Tree Improvement: Applied Research and

Technology Transfer,pp.311-324.SciencePublishers Inc., USA.