Genetic Variation Related to the Adaptation of Humans to an Agriculturalist Lifestyle
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Dissertation Genetic variation related to the adaptation of humans to an agriculturalist lifestyle submitted in fulfillment of the requirements for the degree Doctorate of natural science doctor rerum naturalium at the Faculty of Biology Johannes Gutenberg University Mainz by Jens Bl¨ocher born 22.04.1984 Marburg, Germany Mainz, 2019 Abstract This study investigates the influence of climate and subsistence on the genetic variation of pre- historic individuals of Europe. Partial genomes from more than 100 individuals were analyzed, originating from a region spanning from north-western Anatolia, over Germany to Lithuania. The data is spread over a period of more than 10,000 years and thus covers the phases before and after people started to become sedentary. To fill temporal and geographical gaps, 27 individuals were se- quenced specifically for this study. For this purpose, a hybridization-based enrichment process was used in combination with next-generation sequencing to obtain predefined genomic segments from ancient DNA (aDNA). These regions consist of over 400 functional markers, 68 non-recombinant Y markers, and more than 5,000 neutral regions of 1kb in length. The data set was completed by genomes of three modern reference populations. These made it possible to calculate haplotype- and frequency-based neutrality tests such as the integrated haplotype score or Tajima's D and thus to be able to detect signals of past selection events. Using the allele frequencies of the ancient genomes, the timing of possible selection processes could be narrowed down. The analyzes focused on three main topics: 1.) the development of skin, eye and hair color phe- notypes in Europe; 2.) The development of Europe's predisposition to type 2 diabetes and the metabolic syndrome; and 3.) The development of the immune system with respect to autoimmune diseases. The historical focus was on the influence of subsistence and the transition to farming on the evolutionary processes. The results suggest that a high proportion of the genetic variation in genes associated with the energy and fat metabolism has been affected by adaptations to the local climate in Central Europe. Furthermore, clear indications were found that due to the reduced UV radiation in Central and Northern Europe, depigmentation of the skin had already begun before the onset of the Mesolithic. On the basis of the available data, it can be assumed that the the shift in diet and lifestyle that ac- companied the transition to farming partially reinforced already existing selection pressures. Thus, with the Neolithic, a significant increase in allele frequencies associated with lighter pigmentation phenotypes was found. This is probably due to the reduced vitamin D content in the diet. Further- more, evidence was found that indicates a metabolic adaptation to a plant-rich diet. For the later Neolithic and Bronze Age periods, an increase in allele frequencies has been found in genes that enhance protection against infectious diseases. A connection with the increasing population density and a sedentary lifestyle is very likely here. i Contents Abstract i Acknowledgement ii 1 Demography and Sample grouping 1 1.1 Introduction . .1 1.2 Archaeological background . .9 1.3 Methods . 14 1.3.1 Sawing and Milling . 14 1.3.2 DNA Extraction . 14 1.3.3 Library preparation . 14 1.3.4 Quantification and Quality control . 14 1.3.5 Nuclear Capture enrichment . 15 1.3.6 Read processing . 15 1.3.7 Estimating contamination . 15 1.3.8 Variant Detection . 16 1.3.9 Mitochondrial haplogroups . 16 1.3.10 Sex determination . 16 1.3.11 Principal Component Analysis and Clustering . 17 1.3.12 Model based clustering . 17 1.4 Results . 18 1.4.1 Coverage . 18 1.4.2 Contamination . 26 1.4.3 Sex determination . 26 1.4.4 Mitochondrial haplogroups . 26 1.4.5 Model-based clustering . 27 1.4.6 Principal component analysis . 29 1.5 Discussion . 31 2 Methods used for the analyses of the functional markers 34 2.1 Introduction . 34 2.2 Capture design . 38 2.3 Reference populations . 38 2.4 Population genetic estimations . 38 2.4.1 Allele frequencies . 38 2.4.2 Mean difference of allele frequencies . 39 2.4.3 Fst .......................................... 39 2.4.4 Pairwise Fst comparisons . 39 2.4.5 Hardy-Weinberg equilibrium . 40 2.4.6 Neutrality tests . 40 iii 2.4.7 Averaged expected heterozygosity or gene diversity De ............. 40 2.5 Phenotypic predictions . 41 2.5.1 Predictions of N-acetyltransferase 2 phenotpyes . 41 2.5.2 Predictions of hair and eye color with the HirisPlex webtool . 41 2.6 Tracking changes in allele frequencies over multiple loci . 41 2.6.1 Genetic risk scores . 41 2.6.2 Pigmentation score . 42 2.7 Beta-binomial forward simulation . 43 3 The history of the Central European pigmentation phenotypes 46 3.1 Introduction . 46 3.2 Hypotheses . 52 3.3 Results . 53 3.3.1 Allele frequencies . 53 3.3.2 Neutrality tests . 61 3.3.3 Average expected heterozygosity De ....................... 67 3.3.4 Pairwise Fst comparison . 71 3.3.5 Pigmentation Score . 75 3.3.6 Phenotypic predictions with the Hirisplex webtool . 78 3.3.7 Beta-binomial simulation . 80 3.4 Discussion . 82 4 The influence of subsistence and climate on the energy metabolism 93 4.1 Introduction . 93 4.2 Hypotheses . 100 4.3 Results . 101 4.3.1 Allele frequencies . 101 4.3.2 Neutrality tests . 114 4.3.3 Average expected heterozygosity De ....................... 122 4.3.4 Pairwise Fst comparisons . 124 4.3.5 Mean difference of risk allele frequencies . 128 4.3.6 Genetic risk score determination . 131 4.3.7 Beta-binomial simulations . 135 4.4 Discussion . 136 5 The influence of subsistence and climate on the immune system 144 5.1 Introduction . 144 5.2 Hypotheses . 148 5.3 Results . 149 5.3.1 Frequencies . 149 5.3.2 Neutrality tests . 155 5.3.3 Average expected heterozygosity De ....................... 158 iv 5.3.4 Pairwise Fst comparisons . 159 5.3.5 NAT2 acetylation-phenotype prediction as described in Sabbagh et al. [2009] 161 5.3.6 Beta-binomial simulations . 161 5.4 Discussion . 164 6 Synthesis discussion and conclusion 174 References 180 CV 207 v 1 Demography and Sample grouping 1.1 Introduction The aim of this study is to investigate the influence of subsistence and eco-region on the genomes of European populations. Studies have shown that genetic variation in European populations is the product of migration and admixture of several ancient populations. There is also evidence suggesting that selection shaped regions of the genome in response to new environments and living conditions. To shed further light on the development of the genomes of Central European populations, a di- achronic data set was used, consisting of genome-wide data of more than 100 ancient samples. This data set spans from the early Mesolithic to the Bronze Age, and covers an area from north-western Turkey, to the Baltic and western Russia. A pre-selected set of markers associated with distinct phe- notypic traits was analyzed, accompanied by genome-wide data from an extensive reference set of modern-day populations. The set of markers was selected to address long-standing questions related to the population history of Europe, and the transition to farming. This first chapter addresses the demographic background of the samples and explains the reasoning for the sample groupings, which provides the foundation of this study. The following chapters will discuss the methods that were used in this study and subsequently the specific questions relating to the selected markers. The first inhabitants of Europe The emergence of the first anatomically modern humans (AMH) is estimated to have happened over 300,000 years ago in Africa [Schlebusch et al., 2017]. This is more than 250,000 years before AMH settled in Europe [Haber et al., 2016] and more than 280,000 years before the migration to the Americas [Slatkin and Racimo, 2016]. The specifics of how and when exactly AMH migrated out of Africa are still part of debate, but it is estimated to have happened between 80,000 - 50,000 years ago [Seguin-Orlando et al., 2014]. Early on, during the migration, AMH met other hominins, such as Neanderthals that already lived outside of Africa for thousands of years. Neanderthal remains were found all over western Eurasia, from France to southern Siberia. The oldest confirmed Nean- derthal bones were found in Croatia and Italy, with an estimated age of 130,000 and 120,000 years respectively [Hublin, 2009]. The interactions between AMH and Neanderthals led to interbreeding, with the result that Neanderthal ancestry still can be traced in the genomes of modern populations [Green et al., 2010, Reich et al., 2010]. The fact that this ancestry can be found in populations from Central Europe as well as South-east-Asia it is assumed that these interactions occurred during a time before the initial split of the populations, which became ancestral to Central Europeans and South-east Asians [Fu et al., 2015]. Approximately 45,000 to 40,000 years ago, modern humans started to arrive in Central Europe. The population density is estimated to have been low and people are assumed to have lived in small bands as hunter-gatherers. However, some form of cultural complexity can be assumed from the cave paintings and figurines that can be found all over western Eurasia. From the depicted scenes.