UNIVERSITÀ DEGLI STUDI DI PAVIA Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani” Employing mitogenomes to reconstruct migration and dispersal events Stefania Brandini Dottorato di Ricerca in Genetica, Biologia Molecolare e Cellulare XXIX Ciclo – A.A. 2013-2016 UNIVERSITÀ DEGLI STUDI DI PAVIA Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani” Employing mitogenomes to reconstruct migration and dispersal events Stefania Brandini Supervised by Prof. Antonio Torroni Dottorato di Ricerca in Genetica, Biologia Molecolare e Cellulare XXIX Ciclo – A.A. 2013-2016 ABSTRACT ABSTRACT Humans have always been characterized by a strong curiosity about the origins of themselves and the forms of life that surround them. In the past, answers were searched in philosophy and religion, but in last decades also empirical sciences have started to provide evidences about the evolutionary history of humans and other species. Important contributions come from genomics that, through the study of the DNA sequence variation, provides information concerning the genetic relationships among different populations. One important tool to track down the history and the migrations of our ancestors is the mitochondrial DNA (mtDNA). The mitochondrial genome is organized as a small circular molecule of DNA, present in hundreds/thousands of copies per cell, transmitted as a non- recombining unit only through the mother, and characterized by a much greater evolutionary rate than the average nuclear gene. Consequently, the mtDNA is not subject to recombination events and its variability is originated only by the sequential accumulation of new mutations along the maternal lineage. During millennia, this process of molecular divergence has given rise to monophyletic units (clades), called haplogroups, which are generally restricted to specific geographic areas or population groups as they arose after the colonization of different regions and continents. The study of the geographical distribution, the internal variability and the coalescence age of each haplogroup is known as ‘phylogeography’. The timescale is provided by converting lineage diversity to age estimates by using a molecular clock. These estimated ages, combined with the information about the geographical distribution of the mtDNA lineages, allow us to make inferences about the demographic history of populations, such as dispersals, range expansions, or migrations. During these three years of doctoral studies, I analysed the sequence variation of the mtDNA at the highest level of resolution, that of complete sequence (mitogenome), in order to reconstruct the migration events of both human and animal populations. In particular, I mainly focused my research activity on three projects aimed to trace the first human peopling events in Sardinia and South America and to study the recent worldwide Aedes albopictus, the Asian tiger mosquito The first project I worked on aimed to date events that brought to the initial peopling in Sardinia and to clarify the genetic history of Europe. Sardinians are "outliers" in the European genetic landscape and, according to paleogenomic nuclear data, the closest to early European Neolithic farmers. To learn more about the genetic ancestry of Sardinians, we analyzed 3,491 modern and 21 ancient mitogenomes from Sardinia and observed that the age estimates of three Sardinian- specific haplogroups are >7,800 years, the archeologically-based upper boundary of the Neolithic in the island. This finding not only supports archeological evidence of a Mesolithic occupation of the island, but reveals a dual ancestral origin of the 1 ABSTRACT first Sardinians. Indeed, one of the Sardinian-specific haplogroups harbors ancestral roots in Paleolithic Western Europe, but the other two are most likely of Late Paleolithic Near Eastern ancestry, and among those that are often assumed to have spread from Anatolia only with the Neolithic. Thus, their ages are compatible with the scenario of a Late Glacial recolonization of Mediterranean Europe from the Near East prior to the migration wave(s) associated with the onset of farming. The second research project aimed to further assess the mitogenome variation of Native Americans origin. Specifically, I focused on Ecuador and Peru, two geographical areas of particular interest because of their location along the Pacific coast, in order to shed light on the peopling of South America. Phylogenetic analyses encompassing both novel and previously reported mitogenomes, allowed the identification of 50 new sub-haplogroups and the finding of a number of sub- clades shared with Native Americans from North and Central America, thus increasing the number of founding mtDNA lineages that entered South America from the North. Our phylogeographic analyses confirmed that the North to South expansion was extremely rapid, and most likely occurred along both the Pacific and Atlantic coasts. The third study was not carried out on humans, but on a species whose spread is associated with human activities: the Asian tiger mosquito Ae. albopictus. Its aim was to acquire information about the diffusion process of this insect by analysing the mitogenome variation of representatives from Asia, America and Europe. Phylogenetic analyses revealed five haplogroups in Asia, but population surveys showed that only three of these were involved in the recent worldwide spread. We also found out that a sub-haplogroup, which is now common in Italy, most likely arose in North America from an ancestral Japanese source. In the course of my Ph.D. studies I also contributed to two additional projects. In the first a rare human mitochondrial haplogroup, named R0a, was analyzed to reconstruct ancient migratory events involving the Arabian Peninsula and Eastern Africa, while the second one assessed the mitogenome variation of Egyptian cattle breeds to acquire new insights on the initial events that brought to the diffusion of domestic cattle (Bos taurus) outside the Near East. Taken together, the data and results reported in this thesis remark the fact that phylogeography analyses continue to be crucial for interpreting mtDNA data, providing a multidisciplinary perspective on human and animal evolution, with major impacts also in other fields such as archaeology, anthropology and linguistics. 2 CONTENTS CONTENTS ABSTRACT 1 CONTENTS 3 INTRODUCTION 6 1. MITOCHONDRIA 7 1.1. ORIGIN AND EVOLUTION OF MITOCHONDRIA 8 1.2. MITOCHONDRIAL GENOME 9 1.2.1. Genome organization 9 1.2.2. Replication 11 1.2.3. Transcription 12 1.2.4. Translation 13 1.2.5. Genetic code 13 1.2.6. Mitochondrial DNA features 14 1.2.6.1. Maternal inheritance and lack of recombination 14 1.2.6.2. Homoplasmy and heteroplasmy 15 1.2.6.3. Mitochondrial genetic bottleneck 16 1.2.6.4. Mutation rate 17 2. HUMAN POPULATION GENETICS 18 2.1. THE MTDNA CONTRIBUTION AND THE PHYLOGEOGRAPHIC APPROACH 18 2.2. THE MOLECULAR CLOCK 19 2.3. MTDNA NOMENCLATURE 19 2.4. MTDNA REFERENCE SEQUENCES 21 2.5. MTDNA WORLDWIDE PHYLOGENY 22 2.5.1. The origin of modern humans 24 2.5.2. The ‘Out of Africa’ exit 26 2.5.3. Human colonization of the world 28 2.5.3.1. The peopling of Australasia 28 2.5.3.2. The peopling of Europe 29 2.5.3.3. Back to Africa 30 2.5.3.4. The peopling of the Americas 31 MY CONTRIBUTION 35 3. THE FIRST PEOPLING OF SARDINIA 36 3.1. BACKGROUND 36 3.2. THE SAMPLE 37 3.3. RESULTS 38 3.3.1. Phylogeny and phylogeography 38 3 CONTENTS 3.3.2. Age estimates 48 3.4. DISCUSSION 53 3.5. CONCLUSION 60 4. MITOGENOME VARIATION IN ECUADOR AND PERU 61 4.1. BACKGROUND 61 4.2. THE SAMPLE 62 4.3. RESULTS 65 4.3.1. The mitogenome phylogeny 65 4.3.2. Phylogeography 74 4.3.3. Haplogroup age estimates 79 4.4. DISCUSSION 84 4.5. CONCLUSION 86 5. THE WORLDWIDE SPREAD OF THE TIGER MOSQUITO 87 5.1. BACKGROUND 87 5.2. THE SAMPLE 89 5.3. RESULTS 91 5.3.1. The mtDNA control region of Ae. albopictus 91 5.3.2. The phylogeny 92 5.3.3. The geographical distribution of Ae. albopictus haplogroups 96 5.4. DISCUSSION 98 5.5. CONCLUSION 100 6. ADDITIONAL PROJECTS 101 6.1. ORIGIN AND SPREAD OF HUMAN HAPLOGROUP R0A 101 6.1.1. Background 101 6.1.2. Results and discussion 101 6.2. THE MITOGENOME VARIATION OF EGYPTIAN CATTLE BREEDS 108 6.2.1. Background 108 6.2.2. Results and discussion 109 7. MATERIALS AND METHODS 114 7.1. DNA EXTRACTION 114 7.1.1. Extraction of human and bovine DNA 114 7.1.2. Extraction of mosquito DNA 114 7.2. DNA AMPLIFICATION 115 7.2.1. Whole genome amplification 115 7.2.2. Long range PCR for Illumina sequencing 116 7.2.2.1. Long range PCR of human mtDNA 116 7.2.2.2. Long range PCR of bovine mtDNA 117 7.2.2.3. Long range PCR of tiger mosquito mtDNA 118 7.2.3. DNA amplification for Sanger sequencing 119 7.2.3.1. Amplification of human mtDNA 119 7.2.3.2. Amplification of Ae. albopictus mtDNA 121 4 CONTENTS 7.3. ELECTROPHORESIS 123 7.4. SEQUENCE ANALYSIS 123 7.4.1. Next Generation Sequencing 123 7.4.2. Sanger sequencing 124 7.4.2.1. Human mtDNA sequencing 124 7.4.2.2. Mosquito mtDNA sequencing 126 7.5. PHYLOGENETIC (AND OTHER) ANALYSES 129 7.5.1. Tree design and haplogroup definition 129 7.5.1.1. Maximum parsimony 129 7.5.2. Coalescence times 130 7.5.2.1. Rho statistics 130 7.5.2.2. Maximum likelihood 131 7.5.2.3. Bayesian approach 131 7.5.3. Population expansion times 132 7.5.4. Frequency distribution analysis 132 REFERENCES 133 LIST OF ORIGINAL PUBLICATIONS 164 5 INTRODUCTION INTRODUCTION 6 1. Mitochondria 1. Mitochondria Eukaryotic cells are organized in compartments. Each cell contains a nucleus and a surrounding cytoplasm in which are suspended organelles enclosed within membranes.