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MediterraneanArchaeologyandArchaeometry,Vol.10,No.2,pp.114Copyright2010MAA

PrintedinGreece.Allrightsreserved.

ANCIENTmtDNASEQUENCESANDRADIOCARBON

DATINGOFHUMANBONESFROMTHECHALCOLITHIC

CAVESOFWADIELMAKKUKH

Salamon,M.1,3,Tzur,S.2,Arensburg,B.3,Zias,J.4,Nagar,Y.,5

Weiner,S.1andBoaretto,E.6,7

1DepartmentofStructuralBiologyandKimmelCenterforArchaeologicalScience,WeizmannInstituteofScience,Rehovot76100,Israel

2InstituteofEvolution,UniversityofHaifa,Haifa31905,Israel3DepartmentofAnatomyandAnthropology,SacklerFacultyofMedicine,TelAvivUniversity,

TelAviv69978,Israel4ScienceandArchaeologyGroup@TheHebrewUniversity,Jerusalem

5IsraelAntiquitiesAuthority,Jerusalem91004Israel6RadiocarbonDatingandCosmogenicIsotopesLaboratory,KimmelCenterforArchaeologicalScience,

WeizmannInstituteofScience,76100Rehovot,Israel7DepartmentofLandofIsraelStudiesandArchaeology,BarIlanUniversity,72900RamatGan,Israel

Received:01/04/2010

Accepted:06/08/2010 Correspondingauthor:[email protected]

ABSTRACT

DNA from fossilhumanbonescanprovidevaluable information forunderstanding intra and

interpopulationrelationships.UsingtheDNApreservedinsidecrystalaggregatesfromhumanfos

silbones containing relatively largeamountsof collagen,wedemonstrate thepresenceof repro

duciblemtDNAcontrolregionsequences.Radiocarbondatesfromeachboneshowthattheburial

caveswereusedforupto600yearsduringtheChalcolithicperiod(5th4thmillenniumBP).Acom

parisonof theancientDNAsequenceswithmodernmtDNAdatabases indicates thatallsamples

canmostlikelybeassignedtotheRhaplogroupsubclades,whicharecommoninWestEurasia.In

fourcasesmorepreciseandconfidenthaplogroup identificationscouldbeachieved (H,U3aand

H6). The H haplogroup is present in three out of the four assigned ancient samples. This hap

logroupisprevalenttodayinWestEurasia.Theresultsreportedheretendtogeneticallylinkthis

ChalcolithicgroupofindividualstothecurrentWestEurasianpopulations.

Abbreviations:aDNA,ancientDNA;CRS,CambridgeReferenceSequence;mtDNA,mitochon

drialDNA;HVSI,Hypervariablesegment1.

Data deposition: The sequences reported in this paper have been deposited in

theGenBankdatabase(accessionnos.EF060054EF060059,DQ020117).

KEYWORDS: aDNA, bone crystal aggregates, population genetics, collagen preservation, hap

logroups,ChalcolithicCaves,WadielMakkukh

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SALAMON,M. etal2

INTRODUCTION

ThesouthernLevant links the continentsof

Africa, Europe and Asia. This hasbeen an at

tractive region for settlement since prehistoric

timesandservedasavitalcorridorofcommu

nication, migration, and trade. Phylogenetic

analysesofmodernhumanmitochondrialDNA

(mtDNA) haplogroups in the Levant have fa

cilitated reconstruction of human migrations

through this corridor during prehistory

(Richards et al., 1996). The objective of this

studywastouseancientDNAtoprovidedirect

informationon thegeneticsof individuals that

once lived in this region, and in this way to

learnmoreaboutpossiblepastmigrations.

TheWadiMakkukhsitecomprises13caves.Thecavesare located in theJudeanDesertjust

north of the Dead Sea (Hirschfeld and Riklin,

2002). In three of the caves (nos. 1, 3 and 13)

humanskeletal remainswere found, including

atleast40individuals.Asingleburialofamale

still in articulation was found in Cave 13

(known as The Cave of the Warrior), together

withgravegoods that included textiles, abas

ket, wooden arrows and a woodenbowl (see

reports in (Schick,1998)).The latterhavebeen

dated to theChalcolithicPeriodabout6,700 to

4,700BP (Jull et al., 1998;Rowan andGolden,

2009).Theotherhumanboneswerenot found

in articulation and there was no well defined

stratigraphic organization. The presence of

these organic artifacts indicates that preserva

tionconditionsareextremelygoodatthis loca

tion.

Mostofthehumanlongbones(femurs)ana

lysedfromthesecavescontainedrelativelyhigh

contentsofwellpreservedcollagen.Thisisconsistent with good preservation conditions for

organicmaterials.Oneof theobjectivesof this

studywas todeterminewhether there isacor

relation between the HCl insoluble collagen

contentofthebonesandthepreservationofthe

mitochondrial DNA, as this parameter could

provetobeausefulprescreeningtechniquefor

identifying fossil bones with well preserved

DNA.Otherpropertiesof fossilbones thatare

usedforthispurposeareracemizationofasparticacid inwholebone(Collinsetal.,1999)and

C/Nratiosofcollagen (Colsonetal.,1997).We

took advantageof another approach to extract

better preserved DNA from fossil bones,

namelytotreatthebonepowderwithastrong

oxidizingagent (NaOCl)and thenextractonly

theorganicmaterial, includingDNA, thatwas

protected inside intergrown crystal aggregates(Salamonetal.,2005;Dissingetal.,2008).This

approach also removes some authentic DNA

andmostofthecontaminatingDNAandinhibi

torsof thePCR reaction (Salamon et al., 2005;

Malmstrmetal.,2007;Dissingetal.,2008).Us

ing these twoapproaches,wewereable toob

tain 12 sequences of mtDNA control region

fromhuman femurs.Salamonetal. (2005)had

previously shown that one femur from Wadi

Makkukh did contain sequenceable DNA preparedinthismanner.

The closed circular mtDNA molecule has

several unique properties, such as high copy

number,maternal inheritance, lackofrecombi

nation andhighmutation rate (Pakendorf and

Stoneking, 2005). These properties make it a

powerful tool in population genetics and for

understanding human history and evolution

(Vigilant et al., 1991;Jorde et al., 1995)on the

regionalscale (Mountainetal.,1995;Pereiraet

al.,2005;Manwaringetal.,2006).Thehighcopynumber of mtDNA in a cell may improve its

chance of survival in an aDNA sample. The

mtDNAControlRegion(CR)isarapidlyevolv

ingnoncoding region (Lopezetal.,1997) that

includes the phylogenetically informative Hy

pervariablesegmentI(HVSI,1602416383)and

Hypervariable segment II (HVSII, 57372) re

gions(Stoneking,2000).TheprocessofmtDNA

genotypingisusuallybasedonthecombination

of HVSI sequencing and the genotyping ofspecific coding region single nucleotide poly

morphisms (SNPs). The phylogenetic informa

tion isobtained from thealignmentofeachse

quencewith themtDNACambridgeReference

Sequence(CRS).Thisprovidesaseriesofmuta

tionscalledhaplotypes.Agroupofsimilarhap

lotypes isnamedHaplogroup (Hg)and isbest

defined by a combination of coding region

SNPs (Behar et al., 2007). The distribution of

mtDNA haplogroups is well documented in

different modern populations. This valuabledemographic informationcanbeusedasaref

erenceforancientDNA(aDNA)studies.

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ANCIENTmtDNAFROMWADIELMAKKUKH 3

MATERIALSANDMETHODS

SamplesWestudied13leftfemorafromcaves1and3.

Basedonanatomicalmeasurementsthe femora

weretakenfrombothfemalesandmales(Table

1). In cave no. 13, known as The Cave of the

Warrior, an articulated skeleton was found. A

femurandahumeruswereanalysed from this

individual.

Table1.Samplesanalysedinthisstudy.Cave1and3samplesarefemurs,whiletheCave13sampleisafemur

andahumerusfromthesameindividual.Genderwasdeterminedbasedonanatomicalmeasurementsofthe

femur(Bass,1995).WM25femurwasfragmented.Theinsolublecollagencontentforeachboneisshown.Inthis

study14CdatingwasperformedonbonesfromCaves1and3(RTTlabel).ForCave1314Cdatingwasdetermined

basedonlinenfabricthatwrappedthearticulatedskeleton(Julletal.,1998).Uncalibrated14Cagesarereported

inconventionalradiocarbonyearsBPandcalibrateddateswerecalculatedbasedonReimeretal.(2009)bymeans

ofthe2010versionOxCalv.4.1.5ofBronkRamsey.Calibratedrangesaregivenfor1 and2.Forsimplicity

thetotalcalibratedrangeisgiven.Samplesareorderedbycaveandbyradiocarbonage.a)ageobtainedfrom

linenfabric(607/75/1)bytheNSFAMSLaboratory,Tuscon(Julletal.,1998),b)samelinensampleasa)measured

bydecaycountingattheRehovotLaboratory(Julletal.,1998).

Cave Sample Gender Collagencontent(wt.%)

RadiocarbonLab#

14

Cage1 yearBP

Calibratedage1 yearBC

Calibratedage2 yearBC

WM10 female 4.9 RTT4889 565540 45404450 45854365

WM11 female 9.5 RTT4890 563040 45054370 45404360

1

WM30 male 10.7 RTT4873 533035 42404065 43154045

WM28 male 8.0 RTT4872 559540 44604365 45004345

WM27 male 12.6 RTT4434 556080 44904335 45854245

WM25 ? 8.5 RTT5149 547540 43604265 44454240

WM6 male 9.0 RTT5147 540040 43304235 43454070

WM1 male 5.5 RTT5146 536040 43254070 43304050

WM19 female 6.3 RTT5148 535540 43154070 43254050

WM2 male 7.2 RTT4874 530040 42304050 42553995

WM29 male 14.6 RTT4435 524065 42303970 42553950

WM3 male 3.0

3

WM8 female 0.5

Notanalyzed

13 CW male Femur10.0

Humerus10.0

AA22235

RT1946

514050a

492550b40353810

37653650

40453795

39053635

PrecautionsregardingcontaminationDealingwithhumanbonesrequirespaying

close attention tomodernDNA contamination

in the laboratory, as ancient DNA is low in

quantity and quality. Even though the use of

DNA from bone powder treated with the

oxidantsodiumhypochlorite(referredtoasthe

aggregate fraction) should alleviate this

problem, we followed references (Cooper and

Poinar, 2000;Gilbert etal., 2005) andused the

moststringentprecautionsforauthenticationof

ancient DNA sequences and added several

more.Theprecautionsusedwereasfollows:

1.Physicalseparationbetweenthededicated

DNAextractionlaboratory,theprePCRprepa

rationareaandthepostPCRprocessinglabora

tory.

2. Sequences were obtained from samples

withmorethan5weight%insolublecollagen.

3.TheDNAwasextractedfromboneaggre

gates after treating thebone powder with so

diumhypochlorite.

4. Two independent DNA extractions were

carriedouton independentpartsofeachbone.Inonecase,sampleCW,itwaspossibletoana

lyze two differentbones from the same indi

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SALAMON,M. etal4

vidual. At least two amplifications were per

formed,oneforeachextract.Twonegativecon

trols for the extraction procedure and three

negative controls for the PCR reaction were

used.Positivecontrolswereneverused.

5.Noreamplificationwascarriedout,toexcludecontaminationbyPCRproducts.

6. The authentic sequence was determined

from cloned sequences using different sets of

primers.

7. Sequences were obtained for amplifica

tionswith >1000 copynumberof targetDNA,

basedonacalibrationcurveusingtherealtime

PCR.

8. We did not observe amplifications of a

human mtDNA 520bp fragment. Such largefragmentsarenotusuallypresent inaDNAex

tracts. Furthermore, no human mtDNA se

quence of 132bp could be obtained from ex

tractsfromacatbone.Ontheotherhand,using

specificprimerstheobtainedsequencesaligned

well with previously published Felis catus se

quence(Yoderetal.,2003).

9.Onlyonebonewasprocessedatatimeand

byasingleresearcher(M.S.).NotethattheHVSI

(1603616498)sequenceofM.S.isidenticaltothe

Cambridge Reference Sequence (CRS) and theHVSII (30429) contains the mutations A263G,

309.1C,309.2C,315.1CcomparedtotheCRS.

Determiningweightpercentinsolublecollageninbone

Powdered bone (150 mg, particle size:

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determinedby a comparison of PCR products

with andwithout aDNA extract.We also ana

lyzedallextractsformodernhumanDNAcon

taminantsby amplifying a fragmentof 520bp

(primers F15998R16498). The PCR products

were cloned (pGEMT Easy vector, Promega),andsequencedonanAppliedBiosystems3700

DNAanalyzermachine.

RadiocarbondatingThecleaningprocedureofthebonecollagen

andthecharacterizationoftherecoveredcolla

gen followed the preparation procedure of

(Yizhaqetal.,2005).All thesampleswerepre

pared as graphite at the Radiocarbon Labora

toryattheWeizmannInstitute(LabelRTT)andmeasured using Accelerator Mass Spectrome

try.Prior to themeasurement, theweightper

centpurifiedcollagenextractedforradiocarbon

dating rangedbetween 2 and 7.7%.300 to600

mgofbonepowderwasused for radiocarbon

dating.Thequalityofthecollagenwaschecked

again after extraction using infrared spectros

copy. All the IR spectra did not indicate the

presenceofothermaterialexceptcollagen.Ra

diocarbondateswerecalibratedusingthelatest

calibrationcurve(Reimeretal.,2009)bymeans

of the software OxCal 4.1.5 Bronk Ramsey

(2010) based on (Bronk Ramsey, 1995; Bronk

Ramsey,2001).

Haplogroupassignmentandacomparisontomodernpopulations

In this study, only partial CR sequences

were available, therefore, haplogroup assign

ment could not be completely validated and

each sequence was analyzed taking into account its given sequence range. In the first

stage,we tried to identifyspecificHVSISNPs

thatareknowntobeindicatorsforspecifichap

logroups,basedonBeharetal.(2007).Wethen

tried to predict a haplogroup affiliationbased

on the fundamentals of the nearest neighbor

method following Behar et al. (2007). The es

senceof thismethod is to search formatching

haplotypes in a large, high quality, reference

database that contains haplotypes with theirsuitablehaplogroupaffiliations.Asa reference

weusedthelargeststandardizedmtDNAdata

base that is available, namely from the Geno

graphicPublicParticipationProject (78,590HVSI

genotypes). This public dataset contains

mtDNAinformationofindividuals(HVSIhap

loypeandhaplogroup),but lacks theirpopula

tionaffiliations (Behar2007).Eventually,whena unique and highly reliable haplogroup was

determined, we searched for its presence in

modernpopulationdatasetsfromtheliterature.

We define the HVSI positions here tobebe

tween1602316569, similar to theGenographic

study(Beharetal.,2007).

RESULTS

The bones in these caves (except for the

skeleton in Cave 13) were not articulated and

were not excavated in a well defined strati

graphicorder.We thereforeonly analysed the

leftfemora inordertoensurethateachbone is

from a different individual. Furthermore, as

bothChalcolithicandRomanartifactswerepre

sentinthesecaves,allthebonesthatproduced

DNA sequences were radiocarbon dated. The

dates prove that they are all from theChalco

lithic(Table1andFigure1).Radiocarbondates

ofthe linen fabricthatwrapped thearticulatedskeleton from Cave 13 showed that it is also

from theChalcolithic (Jull et al.,1998) andwe

assume that thebonesof this individualareof

thesameage.Table1alsoshows the insoluble

collagencontentsofeachofthebonesanalysed

andthesexidentifications.

As contamination is always a seriousprob

lem when analyzinghuman ancientDNA, we

took many precautions (see Materials and

Methods). The strategy used to sequence the

mtDNA involvesanalyzingsuccessively longer

fragments starting with the same forward

primer. We noted a positive correlation be

tween the longest reproducible PCR fragment

thatcouldbeobtained fromeachboneand the

insoluble collagen content of thebone (Figure

2).This shows that the insoluble collagen con

tentof thewholebone is agood indicationof

DNApreservationintheaggregates.

Figure 3 shows the mtDNA sequences ob

tained. Four femora produced fragments of327bp,oneproducedfragmentsof239bpandin

the remainderonlyshorter fragmentscouldbe

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SALAMON,M. etal6

sequenced.Wenotethatalltheblanks inthese

experimentsshowednoevidenceofcontamina

tion.Inordertoexcludethepresenceofmodern

human contamination in our system, we also

analysedthesesamplesforthepresenceofa520

bp fragment from the human HVSI region.

Such a fragment isgreater than the length ex

pected fromancienthumanDNAsamples.No

suchfragmentwasobtained.

Figure1: Probabilitydistributionof thecalibratedradiocarboncalibratedrangesforthesamplesinTable1.The

datesareorderedbycaveandbyradiocarbonage.Notethattheonearticulatedskeletonfound,isalsothe

youngestspecimendated.Thisisconsistentwiththisskeletonremaininginarticulation.

We then analysed the HVSII region of the

mitochondrial DNA in two of thebones that

gave theCambridgeReferenceSequence (CRS)

intheHVSIregion(WM28andCW).Notethat

MS, thepersonwho performed the laboratory

analyses,alsohas theCRSsequence in this region. In both samples, sequences that differ

from theCRSwereobtained (Figure 4).These

sequencesarethereforedifferentfromtheHVS

II sequence of MS, thus excluding contamina

tioninthelaboratoryfromMS.Furthermore,if

modernDNAcontaminationhadoccurred,we

would have expected the longer DNA frag

ments tohave theCRS sequence andnotonlytheshorterfragments.Wewouldalsonothave

expected there tobe a correlationbetween in

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solublecollagencontentof thebonesand frag

ment length. We are therefore of the opinion

that thesequencesobtainedarenot from labo

ratory contamination. Damage to the DNA is

another serious source of confusion in aDNA

studies. In our study we frequently obtainedreproducible sequences, implying relatively

littlesequencevariationintheextract.Afewof

theclones (6%)didhowevercontainvariations

that did not reproduce. We regard sequence

reproducibilityasan importantcriterionofau

thenticity.

r= 0.9, p>0.001

0

50

100

150

200

250

300

350

400

0 5 10 15 20

Collagen content (Wt.%)

LongestamplifiedDNA(bp)

Figure2:Correlationbetweentheamountofinsoluble

collagencontentandthelongestlengthofmtDNA

PCRproductobtained.

The mtDNA sequencing of thebones from

12individuals(outofthe14tested)producedat

leastfivedistincthaplotypesthatexcludedma

ternal relationshipsbetween them (Figs. 3 and

4). In four cases we could confidently assign

themtospecifichaplogroups(H,H,U3a,H6).It

was however impossible to assign a specific

haplogroup for eight of the samples that con

tained the CRS (Wm1, Wm2, Wm6, Wm10,

Wm11,Wm19,Wm25)or16362C(Wm29)inthe

rangeof thesequenced region,since toomany

possibilitiesexistforHgassignments.

Sample CW that was also sequenced for

HVSII (CRS at: HVSI 1621016498 and 263G

309.1C309.2C309.3C315.1Cat:HVSII30429)

didnotcontainanyknownhaplogrouppredict

ingmotif,andbasedonmatchingHVSIhaplo

typesalone toomanyhaplogroupassignmentsare possible. We then searched for matching

jointedhaplotypesofHVSIandHVSII in the

literatureand inour laboratorydatabases (un

published data), and found only two perfect

matches thatbelong toHhaplogroup.The309

C repeat is a hypermutable nucleotide string

that may frequently change in its length. We

thereforealsosearched forhaplotypes thatdidnot contain the rare mutation 309.3C. Most of

thematchingsequences(19/20)alsobelongedto

haplogroup H. Therefore, the sample CW is

most likely assigned to H haplogroup, or its

subclades.

SampleWm28, thatwasalso sequenced for

HVSII (CRS at: HVSI 1621016410 and 263G

309.1C at:HVSII 30429), didnot contain any

knownhaplogrouppredictingmotif,andbased

onmatchingHVSIhaplotypesalone,toomanyhaplogroup assignments are possible. In addi

tion, we could not find any matching haplo

typesforthemutationsofHVSIandHVSIIin

theliteratureandinourlaboratorydatabase.In

thenextsearch,wealternatelydisregarded the

mutable positions of C repeat strings, 309.2C

and 315.1C. We then found 10 similar haplo

types thatwere all assigned tohaplogroupH.

SampleWm28 can thereforemost likelybeas

signedtoHhaplogroup,oritssubclades.

Sample Wm27 (16343G 16390A at: HVSI1621016498) contains the mutation 16343G

which isacharacteristicofhaplogroupU3.We

found 48 matching HVSI sequences in the

Genographicdatabase.Theywerecomposedof

sevendistinctsequencesandallofthembelong

totheU3haplogroup.Inaddition,themutation

16390A istypicalofhaplogroupU3a(Achilliet

al., 2005). Sample Wm27 was therefore confi

dentlyassignedtoU3ahaplogroup.

Sample Wm30 (16311C 16362C 16482G at:HVSI 1621016498) contains the mutation

16482G which is a characteristic of H6 hap

logroup(Roostaluetal.,2007).Inthesearchfor

matching haplotypes among the Genographic

database15HVSIsequenceswere found (con

sisting of two distinctive sequences). All of

thembelongtoHhaplogrouporitssubclades.

For estimating the frequency of 16482G in H

haplogroup, we searched for this mutation in

thisdatabase subset.Among the samples that

contain thismutation,99.2% (n=365)belong toHhaplogroupor itssubclades.Matchinghap

lotypesofH6haplogroup,thatcontainthemu

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SALAMON,M. etal8

tation16311Cwere found recentlyandnamed

as haplogroup H6a1a1 (Behar, 2008). Sample

Wm30wasthereforeconfidentlyassignedtoH6

andmostlikelybelongtoH6a1a1haplogroup.

Figure3:SchemeshowingHVSIsequencesofincreasinglengthwithdotsrepresentingvariationsfromtheCRS

(

constant

variant,

sporadic

variant).

Total

number

of

amplifications

(amps.)

and

cloned

sequences

are

shown.Thepositionandtypeofeachvariationarelisted.Mostofthesequencesobtainedfromamplificationsof

differentfragmentlengthsandfromdifferentextractsareidentical.AnemptyboxmeansnoPCRproduct.The

first5clonesoriginatedfromthefirstamplificationandthesecondfivefromthesecondamplification.Inthe

caseofsampleCWthefirst10clonesoriginatedfromthefemurandthesecond10fromthehumerus.

DISCUSSION

WereportheresequencesofmtDNAcontrol

region from 12 individuals from the Chalco

lithic period. These sequences were obtained

fromDNApreserved incrystalaggregates.We

note a good correlationbetween the insoluble

collagen contents and the DNA fragment

lengths. In 4 cases a reliable haplogroup as

signmentcouldbemade thatshows that these

individuals are related to modern West Eura

sianpopulations.

ThisstudyshowsthattheHClinsolublecol

lagen contentof fossilbones canbeused as arapid and easy prescreening method to iden

tifybones more likely to have well preserved

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DNA in theiraggregate fraction.Thechoiceof

the best preserved bone samples available is

important forsuccessfulaDNAanalysis.Other

methods (amino acid racemization, C/N ratio)

that monitor collagen diagenesis have been

usedtopredictthestateofpreservationofDNAinbones (Colson etal., 1997;Badaetal.,1999;

Collins et al., 1999; Poinar and Stankiewicz,

1999).Collinsetal. (1999)noted thatracemiza

tionisnotareliableaproxyforDNApreserva

tionsincedenaturationofcollagenisnotalways

consistentwithDNAdepurination. In a study

of 34 fossil cattlebones from 4 archaeological

sites,no correlationwasobservedbetween the

D/LratioofasparticacidandmtDNApreserva

tion (Buckley et al., 2008).Theuseof theC/Nratioisproblematic,asitisnotknownwhether

thecalculatedvalue isbasedona lossofnitro

genfromcollagenmoleculesoragainofcarbon

(Tuross, 2002).Gtherstromet al. (2002)deter

mined thestateofDNApreservationbasedon

thepresenceorabsenceofPCRproductsofan

expectedsize inagel. In thisstudyweusere

producible cloned sequences to determine the

stateofpreservationofancientDNA.Usingcol

lagen from fossilbones forradiocarbondating,

Hedges and van Klinken (Hedges and vanKlinken, 1992) characterized bone containing

less than2% collagenaspoorlypreservedand

not suitable for analysis (Hedges and van

Klinken,1992;vanKlinken,1999).Thisvalueis

similartothecutoffwefoundinthisstudyfor

DNA analysis. The cutoff may varybetween

different geographical regions. We therefore

propose that selection of bones for aDNA

analysiscouldfirstinvolveaprescreeningstep

usingtheinsolublecollagencontentinordertoidentify the best preserved bones for aDNA

analysis.Asimilarapproachprovedtobeeffec

tive when choosing fossilbones most suitable

for radiocarbondating (Yizhaq et al., 2005).A

similar proposal was made by Buckley et al

(Buckleyetal.,2008).

MostancientDNAstudiesofhumanpopu

lationassemblagesarecomposedofjustseveral

individuals sometimesobtained from different

periodsof time ina restrictedgeographicarea

(Colsonetal.,1997;LaluezaFoxetal.,2004;Ricaut et al., 2004; Sampietro et al., 2006). In a

study of 22 skeletons from Mycenae, Greece,

authenticmtDNAwasidentifiedin4individu

als,twoofwhomwereidentifiedasbrotherand

sister (Bouwmanetal.,2008).Inasite inLaco

nia, Greece, 8 bones out of 20 from Middle

Bronze graves yielded authentic mtDNA

(Chilversetal.,2008).

Figure4:Schemeshowingtwoshortoverlapping

fragmentsoftheHVSIIregion.Dotsrepresentvaria

tionsfromtheCRS( constantvariant,xsporadic

variant).Totalnumberofamplifications(amps.)and

clonedsequencesareshown.Thepositionandtypeof

eachvariationarelisted.Mostofthesequencesob

tained

from

amplifications

of

different

fragment

lengthsandfromdifferentextractsareidentical.

The 3 caves in Wadi Makkukh contained

bonesfromatleast40individuals.Thequestion

thereforearisesastowhetherthese individuals

wereburiedinthecavetogetheroroverafairly

longperiodoftime.Basedonthecalibratedra

diocarbonresults themaximumperiodof time

thatthecavewasusedasaburialsiteisaround

600years,namelyfromthemiddletolateChal

colithicperiodinthisregion.Thesecavescould

therefore conceivably contain individuals from

onegeneticallyrelatedfamily,orthatunrelated

individualswereburiedthere.OurmtDNA in

formation contains at least five distinctive se

quences, therefore itcanbeconcluded thatnot

allof the individualsareof thesamematernal

lineage.

HVSIdataaloneoftendonotcontainsuffi

cient information for confident assignment of

haplogroupaffiliation.Inourstudy itwaspos

sible toassigna reliablehaplogroup affiliationforonly4ofthe12samples(haplogroups:H,H,

U3a,H6).

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SALAMON,M. etal10

All 12 samples most probably fall into R

haplogroupsubclades,whicharecharacteristic

of theWestEurasiaregion,andencompass90

95% of the European population (Kivisild,

2000).Wealsonote thatall12samplescontain

theSNP16223CthatistypicalofRhaplogroupsubclades,andtheymightbelongtoanyof its

subclades,namelyhaplogroups such asH,U,

T,J,K,Vetc.(Finnilaetal.,2001).Itwasprevi

ously shown that only 2.5% of the pre

haplogroupRmtDNA genomeshave the SNP

16223C, and only 1.1% of R haplogroup sub

cladeshavetheSNP16223T(Beharetal.,2007).

Theassignmentofoursamples into theRhap

logroup subclades is consistent with the cur

rentworlddistributionofthesehaplogroupsinmodern West Eurasian populations. The com

mon haplogroups of WestEurasia are sorted

into several major lineages. Most of them are

subcladesof themajorRhaplogroup:H,J,U,

K,T,V(encompassing9095%oftheEuropean

lineages),andothersareoutside thisbranch:I,

W, and X (Kivisild, 2000). These haplogroups

incorporateabout95%ofthemtDNAvariation

in European populations (Torroni et al., 1996;

Richardsetal.,2000;Simonietal.,2000;Torroni

et al., 2001; Richards et al., 2002). Most of theprevalent haplogroups of Europe are most

likely of Near Eastern origin (Achilli et al.,

2004). The haplogroups are thought to have

formedtensofthousandsofyearsago,andcor

respond to early human migrations and are

usually associated to specific geographic re

gions(Beharetal.,2007).SampleCWandWm28weremostlikelyas

signedtohaplogroupH.Thishaplogroupisthe

most commonhaplogroup inWestEurasia; itsfrequency in Europe is over 40%. In the Near

East, the Caucasus and Central Asia this hap

logroup frequency is about 1030%. The CRS

sequence is the prevalent haplotype (25%) of

haplogroup H (Roostalu et al., 2007). Hap

logroup H divergence time in Europe was

20,00030,000BP(Kivisild,2000).Itisthusinter

estingthatHhaplogroupwasprominentinthis

group of individuals from the Chalcolithic as

well. (Inourstudy3of4haplogroupassigned

samplesbelongtoHhaplogroup).Sinceitissowidespread inWestEurasia, it is impossible to

locate this common haplotype in a specific

modern population. For final H haplogroup

validation,itisnecessarytogenotypetheSNPs

2706A or 7028C.Hhaplogroup canbe further

divided into its subclades (such H1, H2, H3,

H4etc.)(Roostaluetal.,2007).

Sample Wm27 was confidently assigned tohaplogroup U3 and it most likely belongs to

haplogroupU3a.The frequencyofhaplogroup

U3 inEurope is1%and intheNearEast is6%

(Richard 2007). Few matching haplotypes of

U3ahaplogroupwere found indifferentmod

ernpopulations thatarepresentalloverWest

Eurasia (Iran, Syria, Lebanon, Morocco,

Ukraine, Russia, Poland, Germany, Iceland),

and3sampleswerefoundinJewishcommuni

ties fromPoland,Romania andTurkey (Behar2008). The estimated coalescence time of hap

logroupU3 is about 1627,000 BP in the Near

East and about 1227,000 BP in Europe

(Richards et al., 2000). We therefore conclude

that theU3haplogroupwasalreadypresent in

theWestEurasiangenepoolaround 6,000BP,

andprobablywithin itssubbranch,U3ahg,as

well. For final haplogroup validation of this

sample, it will be necessary to genotype the

SNPs:14139Gor15454C forHgU3and2294G

or4703CforU3aHg(Achilli2005).Sample Wm30 was confidently assigned to

haplogroup H6 and it most likelybelongs to

haplogroupH6a1a1.HaplogroupH6 is largely

restricted to theNearEastand theSouthCau

casusand is found invery low frequenciesall

over Europe (Roostalu et al., 2007). It is most

frequent in Central and Inner Asia (Loogvli

2004).Itssubclade,haplogroupH6a1,isoneof

theoldestcladesintheNearEastwithacoales

cencetimeof20,20010,900BP,butinEuropeithas an extremely young expansion age of

18001300BP(Roostalu2007).Remarkably,H6a

haplotypes that contain the mutation 16311C

were detected recently in 12 individuals from

different Jewish communities from Turkey (5

samples),Morocco(3samples),Poland,France,

Tunisia and Algeria. The mtDNA of the H6a

samples fromTurkishJewswascompletelyse

quenced and assigned as haplogroup H6a1a1,

withcoalescencetimeof1293BP(Behar,2008).

Although position 16311 is relatively mutableand therefore could appear independently in

distinct haplotypes (Stoneking, 2000), its pres

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ence in separated and distant communities of

thesameethnicitymay indicateacommonan

cestry.Onlydeeper clade analysisof complete

mtDNA sequences of this haplotype can con

firm thispossibility.For finalhaplogroupvali

dation of this sample, it is necessary to genotype the SNPs: 2706A and 7028C for H hap

logroup, 3915A or 4727G for H6a haplogroup

(Loogvali 2004), and 7325G or 11253C for

H6a1a1haplogroup(Behar2008).

CONCLUSIONS

The information that isembedded inaDNA

samplesmayprovidenovel insights regarding

demographichistory,migrations,introgressions

and genetic connections between ancient and

modernpopulations.This informationcanalso

validate accepted hypotheses based only on

modern data. In this study of human bones

fromaround6000BP,wefound12mtDNAsequences that most probablybelong to R hap

logroup subclades that are typical of West

Eurasia.We also foundahigh frequencyofH

haplogroup, which is the most prevalent hap

logroup today West Eurasia. The results re

ported here thus tend to genetically link this

Chalcolithic group of individuals to modern

WestEurasianpopulations.

ACKNOWLEDGEMENTS

WethankthedepartmentofAnatomyandAnthropologyattheTelAvivUniversityandtheIs

raelMuseumformakingavailablethebones.WethankEugeniaMintz(TheWeizmannInstituteof

Science) for her help with the sample preparation for radiocarbon dating and we thank Israel

Hershkovitz forhis comments.This studywas fundedby theKimmelCenter forArchaeological

ScienceattheWeizmannInstitute.S.W.istheincumbentoftheDrWalterandDrTrudeBorchardt

ProfessorialChairinStructuralBiology.

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