Department of Experimental and Health Science Universitat Pompue Fabra Genetic Analysis of the prehistoric peopling of Western Europe: ancient DNA and the role of contamination TESIS DOCTORAL Mª Lourdes Sampietro Bergua Evolutionary Biology Unit Experimental and Health Science Department Pompeu Fabra University Barcelona, Noviembre de 2006 Department of Experimental and Health Science Universitat Pompue Fabra Genetic Analysis of the prehistoric peopling of Western Europe: ancient DNA and the role of contamination Mª Lourdes Sampietro Bergua Memoria presentada por Mª Lourdes Sampietro Bergua para optar al titulo de doctor en Ciencias de la Salud y de la Vida. Esta tesis doctoral ha sido realizada bajo la codirección del Dr Carles Lalueza-Fox (Universidad de Barcel ona) y del Dr Jaume Bertranpetit i Busquets (Universitat Pompue Fabra) en la Unidad de Biología Evolutiva, Departamento de Ciencias Experimentales y de la Salud, Universitat Pompeu Fabra. PhD Program in Health and Life Science (2002 -2004). Carles Lalueza-Fox Jaume Bertranpetit i Busquets Mª Lourdes Sampietro Bergua Director Director Barcelona, Noviembre de 2006 A mis padres, a mi hermana A Oscar ABREVIATIONS aDNA: ancient DNA A: adenina AMH: anatomically modern humans AP: apurinic site Asp: Aspartic Bp: base pairs BP:Before present BSA: bovine serum albumine C: Citosina Ct: Cycle threshold parameter CRS: Cambridge Reference Sequence DNA: Deoxyribonucleic acid. EDTA: ethylenediaminetetra-acetic acid dNTP´s: deoxiribonucleotides triphosphate. ddNTP´s:dideoxiribonuclleotides triphosphate G: Guanina GC: Gas Chromatrography H strand: heavy strand H2O2: hydrogen peroxide HX:hypoxantine HVR: Hyper variable region KYA: kilo years ago L strand: Light strand MS: Mass spectrometry mtDNA: mitochondrial DNA MYA: million year ago MW: molecular weight NA: Avogadro´s number Nfe: Effective population size NUMTs: nuclear mitochondrial sequences ·O 2: peroxide radicals ·OH: hydroxy radicals PC: Principal components PCA: Principal Component Analysis PCR: polymerese chain reaction Ppm: parts per million PTB: N-phenacyltiazolium bromide RT-PCR: Real Time PCR SAM: S-adenosylmethionine SDS: sodium dodecyl sulphate SNP: Single Nucleotide Polimorphism T: Timina TE: Tris-Edta Taq polymerase: Thermus aquaticus DNA polymerase TMCR: Time Most Recent Common Ancestor UNG: Uracil N-Glycosylase UV: Ultraviolet light X-gal: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside 1 INTRODUCTION............................................................................................... 1 1.1 HISTORY OF ANCIENT DNA....................................................................3 1.2 DNA PRESERVATION................................................................................7 1.2.1 DNA MOLECULE .................................................................................7 1.2.2 DNA STABILITY IN VIVO .....................................................................8 1.2.2.1 HIDROLYTIC DAMAGE.................................................................9 1.2.2.2 OXIDATIVE DAMAGE ...................................................................9 1.2.2.3 NONENZYMATIC DNA METHYLATION ...................................10 1.2.3 DNA REPAIR ...................................................................................... 10 1.2.3.1 DIRECT REVERSAL OF DNA DAMAGE:....................................10 1.2.3.2 EXCISION REPAIR:.......................................................................10 1.2.4 DNA DAMAGE AFTER CELL DEATH................................................ 11 1.2.4.1 DNA FRAGMENTATION..............................................................11 1.2.4.2 NUCLEOTIDE MODIFICATION...................................................11 1.2.5 DNA SURVIVAL.................................................................................. 13 1.3 DNA RETRIEVAL FROM FOSSIL REMAINS ........................................14 1.3.1 DNA COPY NUMBER ......................................................................... 16 1.3.2 DNA FRAGMENTATION .................................................................... 20 1.3.3 JUMPING PCR ................................................................................... 21 1.3.4 INHIBITORS ....................................................................................... 23 1.3.5 CONTAMINATION.............................................................................. 24 1.3.6 MISCODING LESSION ....................................................................... 26 1.3.7 MOLECULAR CLONING.................................................................... 28 1.4 AUTHENTICITY CRITERIA.....................................................................30 1.5 ORIGIN AND MAINTENEANCE OF THE CURRENT HUMAN GENETIC DIVERSITY ...........................................................................................................32 1.5.1 MUTATION......................................................................................... 32 1.5.2 NATURAL SELECTION ...................................................................... 33 1.5.3 GENETIC DRIFT ................................................................................ 33 1.5.4 MIGRATION ....................................................................................... 34 1.6 THE MITOCHONDRIA .............................................................................35 1.6.1 HUMAN MITOCHONDRIAL DNA...................................................... 36 1.7 HUMAN POPULATION HISTORY...........................................................41 1.7.1 HUMAN AS A PRIMATE SPECIES ..................................................... 41 1.7.2 THE FOSSIL RECORD OF HOMINIDS.............................................. 41 1.7.3 THE ORIGINS OF MODERN HUMANS ............................................. 45 1.7.3.1 MULTIREGIONAL MODEL..........................................................45 1.7.3.2 OUT OF AFRICA MODEL.............................................................46 1.7.4 THE GENETIC FINGERPRINT IN THE HISTORY OF HUMAN POPULATIONS .................................................................................................. 47 1.7.4.1 EVIDENCE FOR THE MITOCHONDRIAL GENOME: “The mitochondria Eve”........................................................................................... 48 1.7.4.1.1 DISTRIBUTION OF THE MtDNA LINAGES IN THE HUMAN POPULATIONS..........................................................................................50 1.7.4.2 EVIDENCE FOR Y CHROMOSOME ............................................51 1.7.4.3 EVIDENCE FROM AUTOSOMIC LOCUS....................................52 1.7.4.4 EVIDENCE FROM ANCIENT DNA..............................................52 1.8 PEOPLING OF EUROPE ...........................................................................55 1.8.1 UPPER PALEOLITHIC: THE NEANDERTALS................................... 55 1.8.2 THE NEOLITHIC PERIOD ................................................................. 58 1.8.2.1 EVIDENCE FROM THE ARCHAEOLOGY...................................59 1.8.2.2 EVIDENCE FROM THE GENETICS .............................................61 1.8.2.2.1 EVIDENCE FROM AUTOSOMIC LOCUS..............................62 1.8.2.2.2 EVIDENCE FROM mtDNA ......................................................64 1.8.2.2.3 EVIDENCE FROM Y-CHROMOSOME...................................66 1.8.2.2.4 EVIDENCE FROM ANCIENT DNA ........................................66 1.8.3 POST-NEOLITHIC PERIOD............................................................... 67 2 OBJETIVES...................................................................................................... 71 3 MATERIALS AND METHODS ...................................................................... 75 3.1 DNA EXTRACTION..................................................................................77 3.1.1 DNA ISOLATION FROM TEETH........................................................ 77 3.1.2 DNA ISOLATION FROM SOIL SEDIMENTS ...................................... 78 3.1.3 DNA ISOLATION FROM HAIR........................................................... 80 3.2 PCR AMPLIFICATIONS............................................................................80 3.2.1 ANCIENT TEETH PCR ....................................................................... 82 3.2.2 SOIL SEDIMENT PCR ........................................................................ 83 3.3 DNA PURIFICATION................................................................................83 3.3.1 PCR PRODUCTS PURIFICATION ..................................................... 84 3.3.2 GEL BAND PURIFICATION............................................................... 85 3.4 CLONING OF PCR PRODUCTS ...............................................................86 3.5 QUANTIFICATION OF MT DNA BY MEANS OF RT-PCR ......................87 3.6 SEQUENCING ........................................................................................... 89 3.7 SEQUENCING ANALYSIS .......................................................................90 4 RESULTS.......................................................................................................... 91 4.1 CHAPTER 1: T RAKING DOWN HUMAN CONTAMINATIONS IN ANCIENT HUMAN TEETH 93 4.2 CHAPTER 2: D IRTY NEANDERTAL DNA................................................... 102 4.3 CHAPTER 3: N EANDERTAL EVOLUTIONARY GENETICS : MITOCHONDRIAL DNA DATA FROM THE IBERIAN PENINSULA .....................................................................