Sexual Size Dimorphism and Sex Determination by Morphometric Measurements in the Coscoroba Swan

ORIGINALARTICLE

Sexualsizedimorphismandsexdeterminationbymorphometricmeasurements intheCoscorobaSwan

CeciliaPerezCalabuiga∗, AndyJ.Greenb,MiguelFerrera,RobertoMuriela HedenMoreirac

aDepartmentofBiodiversityConservationandAppliedBiology,EstaciónBiológicadeDoñana-CSIC,Sevilla,Spain;bDepartment of Wetland Ecology, Estación BiológicadeDoñana-CSIC,Sevilla, Spain; cDepartment of GeneticsandZoology, Federal UniversityofPelotas,Pelotas,RS,Brazil

Theaccuracy ofmorphologicalsexingand theoccurrence ofsexualdimorphism wereanalyzed inmature and immature CoscorobaSwans (Coscorobacoscoroba,Anatidae) near the Estação Ecológica do Taim, southern Brazil.Onthebasisofweightand10linearmeasurements ofexternalmorphology,multivariateanalysisofvari- anceshowedthatmaleswere consistentlylargerthanfemales(sexconfirmedviageneticmarkers)andmaturebirds wereconsistentlylargerthanimmatures.Overall,38%ofimmaturesand14%ofmaturebirdsweresexedincorrectly bycloacalexaminationwhencompared togeneticdata.Therefore,weperformed adiscriminant functionanalysis ofdifferentageclassesbasedonmorphometricmeasurements.Mature birdsweresexedwith96%accuracyusing headandtarsuslengthsaspredictor variables,whereasimmatures weresexedwith90%accuracybasedonhead andforearm lengths.Method validation conducted withdata foradditionalmature sampled inadifferentyear showedthattheuseofheadlengthalonewasasaccurateforsexing(92%correctclassification)thandiscriminant functionsbasedontwocharacteristics (91%).

Keywords:Brazil; cloacal examination; Coscoroba Swan; genetic sexing; morphological sexing; sexual size dimorphism

Introduction

Accurateandeasymethodstodeterminethesex of individualsarevaluableforstudiesof avianevolu- tionaryecologyandgenetics,population dynamics, behavior, migration andconservationmanagementof speciesandpopulations(Clutton-Brock1986;Newton

1998).Inspecieslackingobvioussexualdimorphism, cloacal sexing is often used but can be unreliable (Brown et al. 2003; Odwyer et al. 2006, Volodin etal.2009). Molecularsexing,basedontheamplifica- tionofthechromo-helicase-DNA-binding1(CHD1) gene,isthemostreliablemethod(EllegrenSheldon

1997;Griffithsetal.1998),butisrelativelyexpensive. However, molecular sexing can complement meth- ods based on discriminant function analysis (DFA) inspecieswithweaksizedimorphism (Dumbell etal.

1988; Ackerman etal.2008; Hart etal.2009).In waterbirds,DFAhasoftenbeen used tocombinethe discriminatory powerofsingle charactersintoone formula thatbestdiscriminatesbetweensexes (e.g. WeidingervanFraneker1998;SvageljQuintana

2007;Ackermanet al.2008;Quintanaet al.2008;Hart etal.2009).DFA canbeareliable,fastandinexpen- sivemethod fordiscriminating thesexofindividuals

duringnon-breeding seasonswhensexuallydimorphic characters arenotexpressed(e.g.Zavalaga Paredes

1997;Bourgeoisetal.2007).

The Coscoroba(Coscorobacoscoroba)isan unusualmemberoftheAnatidaefoundin South America,fromtheFalkland IslandsandTierradel Fuego, north through ChileandArgentina, Uruguay and southern Brazil, and as far north as Paraguay (Kear2005).InBrazil,C.coscorobaoccursyear-round inRioGrande doSul(Belton2000) withirregular records in other states (Bornschein et al. 1997). It istraditionallyconsidered to beaswan, but molec- ularstudiessuggestitis morecloselyrelatedtothe aberrantCapeBarrenGooseCereopsis novaehollan- diae(Donne-Gousséetal.2002).Verylittleisknown about itsecology(seeKear 2005forareview).It is amonochromatic species,andpreviousattempts to separatesexesbasedonmorphologyhavebeenunsuc- cessful(Nascimentoetal.2001).DFA haspreviously beenapplied insomeother swanspecies(e.g.Miller etal.1988; Brownetal.2003).Otherstudiesofswans havecompared differentsizemeasures betweensexes without applying DFA (e.g.Scott 1972;Mathiasson

1981;2005;Limpertetal.1987).

∗Correspondingauthor.Email:

The present study describes sexual dimorphism ofimmatureandmatureCoscoroba inBrazil,and compares theaccuracyofalternative sexingmethods. Themainobjectiveswereto:(a)determine theaccu- racyofcloacalexamination,bycomparing theresults withthosefrommolecular sexing;(b)present typical morphometricmeasuresofCoscoroba, anddetermine differencesbetweenthesexesandtwoageclassesiden- tifiable byplumagecharacteristics;(c)developdiscrim- inantmodelstofacilitatethesexingofbirdsbasedon morphometry;and(d)comparetheaccuracyofDFA withthatofsexingbycloacalexamination.

Materialsandmethods

Studyarea

RioGrande doSulStateinthesouthern tipofBrazil hasabig lagooncomplexinthesouthernportionof its coastalplain,formedby Patos,Mirim,andMangueira Lagoons andother smallerlakes.Allbirdswerecap- turednearthe “EstaçãoEcológicado Taim”,anarea located inthesouthern coastal plainofRS,between the counties of Rio Grande and Santa Vitória do

Palmar (∼32◦23 W,52◦32 W).This area wascho-

senbecauseitiswithinthemost importantarea for

breeding and molting ofCoscorobainBrazil(Seijas

2001).

Captureofbirdsandsampling

Birdswere capturedbyhandfromaboatduring flightless wingmoltbetweenAugustandJanuary (Nascimentoetal.2001;Seijas2001)inthreeconsecu- tiveseasons(2005,2006and2007).Birdsweredivided intotwogroups:immatures (fledgedbutlessthantwo yearsold)and matures (2yearsold)according to plumagecharacteristics (Johnsgard1978).Coscoroba Swanshavered eyes,legs,feetand billfrom imma- turetomature ageclassesbuttherearedifferencesin theplumagepattern. Theadult’sdefinitiveplumageis totally whitewiththeexceptionofablacktiponthe firstsixexternalprimaries.

Although plumageis mainlywhite,immature Coscoroba Swanshavebrownishdownfeathersand brightishbrownplumesall overthebodyandhave grayish brown stains on the tail, back and wings (in primaries,secondariesandupperwing coverts). Mature swans have gray down feathers only on the backandunderthewings (axillaries).Theyusually displaymorethansix primarieswithgrayishbrown fleckingsbutveryrarelyonthesecondaries(Calabuig etal.2010).

The immature group consisted of41individuals

(19,11and11for2005,2006and2007,respectively).

Themature group consisted of345individuals (120,

123and102for2005, 2006and2007, respectively).

Morphometricmeasurements

Birdswere weighedwithabalance(tothenearest20g) and10 biometricmeasuresweretakenbythesame person (CPC).Adigitalcaliperwasusedtomeasure thefollowing(tothenearest0.1 mm):headlength (length ofhead to the occipital-tip ofthe bill),bill height, billdepth (maximum width ofthebill),nos- tril(distaledgeofanostriltotheendofthebill),total culmenandtarsuslength(ontheleftsideofthebody). Arulerwasusedtomeasurethefollowing(tothenear- est1 mm):tail(fromthepreengland),winglength without feathers (metacarpophalangealarticulation), forearm(FerrerDeleCourt1992)andnecklength. Beforereleasing,allswanswerebanded withanum- beredmetalCEMAVE-IBAMAring.Recaptureswere readilyidentified,anddatawere onlyusedforeach individualonthefirstcapture.

Cloacal andgeneticsexdetermination

Genital identification

CloacalexaminationwascarriedoutbyCPC,identify- ing“males”bythepresenceofavisibleerectilegroove penis on the ventral wallof the cloaca (Proctor Lynch1993;Brownetal.2003;Mathiasson2005).

DNAidentification

Blood(3ml)wastakenfromeachbirdfromthewing veinandsampleswerestoredinVacutainertubeswith EDTAandkeptcoolinice, untilprocessing.Blood sampleswere analyzedintheBiotechnologyCenter (CenBiot),FederalUniversityofPelotas,Brazil.DNA extraction wasperformedaccordingtotheprocedures ofLahiri and Nurnberger(1991)and DNA samples were amplifiedusingprimersdescribedbyHeetal. (2005)for the Tundra Swan (Cygnus atratus). The PCRamplificationswere carriedoutinthereaction mixture ofIto etal.(2003).After amplification, the PCRproducts wereseparated ona1% agarosegel (synthesizedbySangonCo.,Shangai,China),stained with ethidiumbromide(SangonCo.)andvisualized underUVlight.

Dataanalyses

We usedmultivariateanalysisofvariance(MANOVA) to compare mean differences between age and sex groups for morphological measurements and body mass.Also,one-wayanalysisofvariancewasusedto

determinewhetherindividualmeasuresvariedwithsex ineachagegroup(SokalRohlf1995).Forboth analyses,we usedonlythe225matureCoscorobas sampledduringthe2006and2007seasons(thoseused forDFA, seebelow).Differenceswereconsideredsig-

nificant atp≤0.05.Alldata satisfiedLillieforsand

Levenetestsofnormality.

Forallvariables,we calculatedasexualsize dimorphism index as: SSD ={(xm −x¯f)/x¯f}×100 (WeidingervanFraneker1998;SvageljQuintana

2007);wherex¯m andx¯f arethemeanvaluesofdiffer- entage malesandfemales,respectively.Thecoefficient ofvariation (CV =(SD/x¯)×100)wascalculated for each sex and averaged between them (Fletcher

Hamer 2003)toindicate thedegreeofvariability of eachmeasurement (SokalRohlf1995).

DFAsweredevelopedseparatelyforimmatureand maturebirds.Weexcludedbodymassfromtheseanal- yses sinceitvariesgreatlyovertime(Croxall1995; SvageljQuintana2007).Theperformanceofeach singlemeasurement asadiscriminating variable(uni- variate DFA) was evaluated. Forward DFAs were appliedtoobtaincombinationsofcharacteristics (dis- criminant functions) that bestdistinguished thesexes (seeTabachnickFidell 1996;Phillips Furness

1997).

Forimmaturebirds,theDFAwas appliedtoall individuals. For mature birds, the DFA wasapplied to 225Coscorobassampled in2006and 2007.For both ageclasses,theeffectivenessoftheanalyseswas assessed,firstinterms oftheproportionofbirds of knownsexthatwereclassifiedcorrectly,andsecondby jackknife validation. Correct classification rates tend

immature birds;overall,38%ofimmature Coscoroba weresexedincorrectly. Among mature birds, overall,

14%ofmatureCoscoroba weresexedincorrectly.

Morphometricdifferencesaccordingtosexandage

Analysisof thewholedatasetshowedthatmature Coscorobawerelargerthanimmaturesandmaleswere bigger than females, with no age×sexinteraction

(MANOVA: age:F11,242=4.6,p0.01, Wilks=0.82;

sex:F=29,p0.01,Wilks=0.43;andage×sex:

F11,354 =1.5,p=0.1,Wilks=0.93).Thus, ineach

agecohort, maleswerelargerthanfemalesinallmea-

surements, except in tail length for immature birds

(Tables1and2).

Inimmature birds,characteristicsthatshowedthe highestsexualsize dimorphism weremassandtail, whereasbillheight,wingandtailwerelessdimorphic. Mass showed thehighest within-sexvariation whilst headlengthhadthelowest(Table1).Inmature birds, mass,tarsusandneck lengthshowedthehighestsexual sizedimorphisms whereasbilldepthandtailwereless dimorphic.Massandneck lengthshowedthehighest within-sexvariation whilstbilldepthandheadlength hadthelowest(Table2).

Table 1.Body measurements (mean ±SD and range, inmm),coefficientsofvariation (CV) andsexualsize dimorphismindex (SSD) forimmaturemaleandfemale CoscorobaSwans(Coscorobacoscoroba)fromsouthern Rio

Grande doSul,Brazil.Allmeasuredcharacteristicsdiffered betweenthesexes(p0.05)excepttaillength.CVwasfirst calculatedforeachsexandthenaveraged.

One-way

tobeoverestimated whenDFAsarevalidatedwiththe

same sample used to generate them (Tabachnick

MaleFemale

Body

ANOVA(%)

Fidell1996).Thejackknife validation isaprocessin which eachindividualcaseisclassifiedusingafunction obtained fromthetotalsample,excludingtheindivid- ualcasetobeclassified(TabachnickFidell1996). Furthermore,theaccuracyofDFAs formature birds wasconfirmedbyapplyingtheresultingfunctionstoa noveldataset, composed of120birdscaptured inthe

2005season.

Results

Cloacal andgeneticsexdetermination

Genetic sexingshowedthat therewere14malesand

27femalesamongyoungbirds,and189malesand156 femalesamong mature birds.Somebirdswereincor- rectlyclassifiedbycloacalexamination. Although geneticfemaleswererarelymisclassified as“males”, males were often wrongly identified as “female”. Cloacal inspection was particularly unreliable for

measurementn=14n=27F1.39CVSSD

Mass(g)3760±4603230±40014.414.416.1

2650–44002250–4100

Totalculmen70.6±4.665.4±2.422.76.18.0

62.5–79.262–73

Nostril52.7±2.848.7±2.621.46.68.2

48.6–56.641.3–56.3

Billheight25.7±1.024.5±1.56.764.9

23.6–2722.2–28.8

Billdepth30.2±1.428.5±1.313.75.26.0

27.2–32.724–30.1

Headlength138±4.9129.6±3.146.64.26.5

129–146.3 123.1–135

Tarsus101.6±5.695.4±4.414.65.76.5

87.8–108.285–104

Wing173.8±7.1164.1±9.710.965.9

162–185145–185

Forearm213.1±8.3195.9±8.837.75.98.8

200–225175–215

Tail183.1±14.7 173.2±21.92.511.45.7

161–205107–220

Necklength345.4±24.7315±21.816.18.39.6

305–390260–370

Table 2.Body measurements (mean ±SD and range, in mm),coefficientsofvariation (CV)and sexualsizedimor- phismindex(SSD)formaturemaleandfemale Coscoroba Swans(Coscorobacoscoroba)sampled in2006and2007in

southern RioGrande doSul,Brazil.Allmeasured charac- teristicsdifferedbetweenthesexes(p0.05).CVwasfirst calculatedforeachsexandthenaveraged.

Table3.Accuracyofsexingofimmature CoscorobaSwans usingsinglemeasurements or combinationsinadiscrimi- nant function (DF1 =headlengthandforearm). Identical resultswereobtained bythejackknifemethod(seetext).

Originalsample(n=41)(%correct classification)

Body

measurementMalesFemales

One-way

ANOVA(%)

Variables

Wilks’

lambdaMales Females Overall

Cut-off point (mm)

2006–2007n=125n=100F1.223CVSSD

Mass(g)4000±3353340±360197.212.919.8

3050–5140 2400–4500

Totalculmen70.6±2.665.5±2.3241.55.17.8

62.8–78.859.8–71.8

Nostril52.8±1.649±2.2221.75.37.8

48.4–58.840–59.2

Billheight26.3±1.124.6±1.1136.35.57.1

23.7–2922.2–27.5

Billdepth30.7±0.829.2±0.9152.23.74.9

29–33.326.4–31.3

Headlength140.1±2.8131.2±2.8548.93.96.8

132.2–147123.8–138.8

Tarsus105.8±3.896.1±3.4397.3610.1

93.3–117.2 84.6–104.5

Wing178.7±7.5165±8.6162.86.18.3

152–200130–186

Forearm212.6±8196.4±7.7235.35.58.2

177.0–230168–217

Totalculmen 0.63 64.3 92.6 83 69.3

Nostril 0.65 71.4 89 83 51.9

Billheight 0.85 64.3 74.1 70.7 26.2

Billdepth 0.74 78.6 70.4 73.2 30.1

Headlength 0.45 86 89 88 135.0

Tarsus 0.73 78.6 66.7 70.7 101.0

Wing 0.78 78.6 70.4 73.2 173.8

Forearm 0.51 78.6 81.5 80.5 207.1

Tail 0.94 64.3 66.7 66 202.4

Necklength 0.74 78.6 70.4 73.2 339.0

DF1 0.42 78.6 96.3 90.2

onlytwoCoscorobas differedbetweenclassifications (changing thecorrect classification to86%formales and 85%overall). Validation withanovelsampleof birdsprovidedslightly differentclassificationswhen compared with DFA, decreasing the accuracy for

Tail175.4±6.9166.4±8.576.25.15.4

145–190141–187

all measures except for bill height and

bill

depth

Necklength 355.9±18.6 317.3±20.2 221.1 8 12.2

275–400265–365

Discriminantanalysis

Immatureswans

Head length wasthe most accurate singleindicator ofsex,correctlyclassifying88% ofbirds(Table3). Coscorobaswith head lengths longer than 135mm wereclassifiedasmales.Jackknifevalidation provided exactlythesameclassificationsas thoseproducedby DFAsforallsinglemeasurements.DFAretainedhead lengthandforearmasthebestpredictor variablesand excludedothers. Thismodel correctly classified79% offemalesand96% ofmales(Table3).Onlythree femalesandonemaleweremisclassified.Thediscrimi- nantfunctionobtained forimmatureCoscorobaswas:

DF1 =0.43(head length) +0.13(forearm) –84.6.

Values of DF1 0identified males and values of

DF10identifiedfemales.

Matureswans

Head length was the most accurate single indica- torofsex,correctlyclassifying93% ofCoscorobas (Table 4).Jackknife validation provided exactly the sameclassificationsasthoseproduced byDFAsforall singlemeasurements exceptfornostril length, where

(Table4).DFA retainedheadlengthandtarsusasthe

bestpredictors. Thismodelcorrectlyclassified96% of femalesand95%ofmales(overallsuccess,96%,three femalesandfourmalesmisclassified)withalowvalue for Wilks’lambda (Table 4).TheDF1 obtained for

mature Coscorobaswas:DF1 =0.84(head length)

+0.38(tarsus) –152.6.Thismodel wasrepresented

inFigure 1wheremature Coscorobaswereclassified

accordingto headlengthandtarsusmeasurements. Misclassificationswere unusuallylargefemalesmis- classified asmales.

ADFA withonlyhead length aspredictor vari- able classified93%of cases correctly, with a value ofWilks’ lambdaclosetothebestmodelwithhead length and tarsus together. This alternative discrim-

inant function (DF2) was:DF2 =1.1(head length)

–150.DF2performed slightlybetterthan DF1when

validatedagainstanewsampleofbirds(Table4),with

92%ofindividualssexedcorrectly.

Discussion

Sexdeterminationbycloacalinspection

Althoughcloacalexaminationisawidelyusedmethod forsexingwaterfowl(e.g.Green2000;GrayHamer

2001;Nascimento etal.2001),ithasseveraldisadvan- tages.Thismethod requires observers withconsider- ableexperience,isonlypossible during thebreeding

Table4.AccuracyofsexingofadultCoscorobaSwansusingsinglemeasurements andadiscriminant function (DF1=head lengthandtarsus)for2006and2007(originalsampleincludedindiscriminantanalyses)and2005(usedforvalidation,seetext). Ajackknifemethod produced identicalresultstothoseshownherefortheoriginalsample,withtheexceptionofnostril(see text).

Originalsample(n=225)(%correctclassification)Newsamplecross-validation(n=120)

Figure1.Plotof 329matureCoscorobaSwansfromsouthernRioGrandedoSul,Brazil,accordingtoheadlengthandforearm length.Malesandfemaleswereidentifiedbygeneticsexing.Thestraight linerepresents 50%probabilityofsexclassification accordingtothediscriminant function.

periodforsomespecies,andcancauseinternal dam- agetothebirdduetothepressureappliedduringpenis visualization (e.g.SaxHoi 1998;Lombardo2001; Oliveiraetal.2004).Wefound ittobeanunreliable methodfortheCoscoroba,owingtothedifficultiesof observingthepenis,especially inyoungbirdswhere penisdevelopment isincomplete(Odwyeretal.2006). Similar levelsof inaccuracy have been recorded in petrels(Odwyeretal.2006) andinwhistlingducks (genusDendrocygna,Volodin etal.2009).Wedonot believethat the error insexingcan beattributedto an observer effectin our study because the person who sexed the swans was an experienced specialist whohasbeenringingwaterbirdsfor20years.Previous studiesrelyingexclusivelyoncloacalinspectiontosex Anatidae (e.g.Green2000)mayalsocontainerrors.

Sexualsizedimorphismandageeffects

Fewpreviousdata wereavailableonthemorphomet- ricsofCoscoroba (Kear2005). Ourresultsindicate significantsize dimorphism,withmaleCoscoroba generallybeinglargerthanfemales,bothforimmature andmaturebirds.Ingeneral,largesizemaybeadvan- tageousinmaleswansandgeese becausetheyare responsiblefornestprotection(Scott1972;Veselovsky

1973;Hawkins 1986;Whitehead Tschirner 1990) or may need to defend females against males seek- ingextra-pair copulations (Mineau& Cooke1979; McKinney et al. 1983; Welsh Sedinger 1990; GauthierTardif1991;ChoinièreGauthier 1995). AsintheCapeBarren Goose, both Coscorobasexes care for their cygnets and maintain long-term pair bonds,butthemaleisprimarilyaguardian andhelps withnestbuildingbutnotincubation.

Massandneck lengthshowedthehighestdegree of sexualsizedimorphism inCoscoroba, butheadlength wasthemostusefulsingle variabletodistinguish betweenmalesandfemales,becauseit hadaninterme- diatelevelofdimorphism combined withparticularly lowCV.Inmostbirdspecies,intraspecificvariation is markedly lowerinthebillandotherbodypartsasso- ciatedwithfoodintakethanin therestofthebody, perhaps asaresultofadaptationtoparticularforag- ingbehavioranddiet(Milleretal.1988). However, differentCVsbetweenmeasurescanalsoreflect dif- ferencesinmeasurement error(Yezerinacetal.1992), andnecklengthislikelytohavehadarelativelyhigh measurement errorin ourstudy.Ourfindingswere consistentwithpreviousstudiesthatshowedthathead (e.g.Veselovsky1973;Milleretal.1988;Brownetal.

2003),tarsus(e.g.Veselovsky1973;Brownetal.2003; Mathiasson2005)andforearm(e.g. Mathiasson2005) lengthsarekeycharacteristicstodifferentiate sexes in Anseriformes.

CoscorobaSwans reach maturity when they are two years old and start breeding at three to four years old (Wilmore 1979; Silva-García Brewer

2007).Although birdsaregenerallyassumed tohave determinategrowth,ourresultsshowed thatmature Coscorobadifferedsignificantlyinsizefromswansless thantwoyearsofage,implyingthatgrowthcontinues inthisspeciesforanextendedperiod prior toreach- ingsexualmaturity (Kirkwoodetal.1989;Carrier Auriemma 1992; Tumarkin-Deratzianetal.2006). ANOVAs ofindividual measures showed that mass, billdepth, head length, tarsus, tail and necklength were all significant greater in mature birds (results not shown). Growth rates oflarge, precocious birds suchasswansareparticularly slow (Ricklefs1973; CarrierAuriemma 1992).

Sexdeterminationbydiscriminantfunctionanalysis

Discriminantfunctionsweredevelopedusingonlytwo morphometricvariables: head and forearm lengths for immaturebirds,andheadandtarsuslengthsfor mature birds, resulting in 90%and 96%of correct sexual classification,respectively.Theseclassification ratesaremuchhigherthanthosebasedon cloacal inspection.However,cross-validationwithanewsam- pleofmatureswanssuggestedthatasingle measure (head length) wasthe most reliable sexingmethod, with92% correctclassification.Whilejackknifeval- idationrevealsinfluentialobservations thatcanbias DFA, cross-validationis amorerigorousvalidation processandshouldideallyinvolveanew sampleof individualsmeasured atdifferenttimes,locations and bydifferentobservers(TabachnickFidell1996). We suspect that discriminant functions would provide a betteralternative tocloacalsexinginmanyotherbird species.

More research isrequired tocalibrate theuseof ourdiscriminant functions inotherCoscorobapopu- lationswithotherobservers.Giventheresultsof the validation with noveldata, sexdeterminationbased onheadlengthis likelytobethemostrobust dis- criminant function method. Whencloacalinspection iscarried out without geneticsexing,thesedata can beplotted together withheadlengthdatatoestablish thebestcut-offpoint forsexdeterminationbasedon morphometrics.Forexample,inapopulationinwhich Coscorobawerephysicallysmaller,thecut-offpoints ofTables3and4forheadlengthmightbetoohigh.

Acknowledgments

WewouldliketothankS. Schererforhisvaluable assistanceinthefield.WethankDr.B.Vaz, R.Tavares, J.Camacho and L.Bassinifrom theDepartmentof

GeneticsandZoology, Federal University ofPelotas, RS,Brazilfortechnicalhelpin carryingoutDNA extraction.

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