Evolution of the wild tomato species Solanum chilense: demography and natural selection Dissertation an der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Katharina Barbara Böndel aus Rinteln München, April 2014 Dekan: Prof. Dr. Heinrich Leonhardt 1. Gutachter: Prof. Dr. Wolfgang Stephan 2. Gutachter: Prof. Dr. Wolfgang Enard Tag der Abgabe: 15. April 2014 Tag der mündlichen Prüfung: 10. Juli 2014 Erklärung: Diese Dissertation wurde im Sinne von §12 der Promotionsordnung von Prof. Dr. Stephan betreut. Ich erkläre hiermit, dass die Dissertation nicht ganz oder in wesentlichen Teilen einer anderen Prüfungskommission vorgelegt worden ist und dass ich mich nicht anderweitig einer Doktorprüfung ohne Erfolg unterzogen habe. Eidesstattliche Erklärung: Ich versichere hiermit an Eides statt, dass die vorgelegte Dissertation von mir selbstständig und ohne unerlaubte Hilfe angefertigt ist. München, den 15.04.2014 Katharina Böndel Note In this thesis I present the results of my doctoral research which I conducted from November 2010 until April 2014 in the area of evolutionary biology. This research comprises predominantly genetic analyses of the wild tomato species Solanum chilense, but also a phenotypic experiment, and was done in collaboration with several other scientists. The project for the genetic part was designed by Mamadou Mboup, Aurélien Tellier, Wolfgang Stephan and me. The salt stress experiment was designed by Tetyana Nosenko and me. All of the experimental and analytical work has been done by myself except for the following: Hilde Lainer did about half of the DNA and PCR product preparation for the S. chilense sequencing, the S. chilense sequencing itself (including library preparation) was done by the GATC Biotech AG in Konstanz, Hilde Lainer and Gaby Kumpfmüller did most of the outgroup sequencing, Pablo Duchén provided the PERL script to extract SNP information from the pileup file, Armin Scheben analysed the synonymous and nonsynonymous polymorphism and divergence and did the McDonald-Kreitman tests for half of the candidate genes as his IRT1 (‘individual research training’) project, Paula Brücher performed the population genetic analyses of the consensus sequence data set as part of her bachelor thesis project, Tetyana Nosenko did the phylogenetic analysis of the consensus sequence data set, and the salt stress treatment and documentation was done together with Tetyana Nosenko. Contents Contents 1 Summary 3 Zusammenfassung 5 Abbreviations 7 List of figures 9 List of tables 11 CHAPTER 1: INTRODUCTION 15 1.1 Adaptation in plants 15 1.2 Wild tomatoes as a model system to study evolution 16 1.2.1 Natural habitat and adaptations 16 1.2.2 Genomic features, mating system, and genetic variation 17 1.2.3 The cultivated tomato 17 1.3 Abiotic stress response in plants 18 1.4 Molecular signatures of local adaptation to abiotic stresses in wild tomato species 20 1.5 The aim of this study 22 CHAPTER 2: MATERIAL AND METHODS 25 2.1 Sequence evolution in Solanum chilense 25 2.1.1 Plant material and plant growing 25 2.1.2 Choice of genes and primer design 28 2.1.3 Sequencing approach for Solanum chilense 30 2.1.4 Sequencing of the outgroup species S. ochranthum and S. lycopersicoides 32 2.1.5 Sequence data analyses 32 2.1.5.1 Data assembly 32 2.1.5.2 Summary statistics of the whole data set 34 2.1.5.3 Regressions and correlations 35 2.1.5.4 Population differentiation and isolation by distance 35 2.1.5.5 Detecting selection in Solanum chilense 36 2.1.5.6 Analysis of the consensus sequence data set 37 2.2 Salt stress experiment 39 CHAPTER 3: RESULTS 41 3.1 Sequence data 41 1 3.2 Demographic history of Solanum chilense 42 3.2.1 Within population levels of variation and population averages of Tajima’s D 42 3.2.2 Population differentiation and isolation by distance 45 3.2.3 Genetic differentiation and divergence from other Solanaceae species 49 3.3 Genes under selection in Solanum chilense 52 3.3.1 Candidate genes vs. reference genes 52 3.3.2 Regulatory genes vs. functional genes 54 3.3.3 Detection of local adaptation 57 3.3.4 Single gene evolutionary histories 66 3.4 Analysis of the consensus sequence data set 75 3.4.1 Gene evolution on the species level 75 3.4.2 Phylogenetic analyses 78 3.4.3 Footprints of selection 80 3.5 Differential behaviour after application of salt 82 CHAPTER 4: DISCUSSION 83 4.1 Evaluation of the data generation 83 4.2 Demographic history of Solanum chilense 85 4.2.1 North-south cline indicates migration from north to south 85 4.2.2 Isolation by distance and genetic differentiation define population groups 87 4.3 Local adaptation in Solanum chilense 90 4.3.1 Gene evolution on the species level 90 4.3.2 Different methods to detect outliers in the data set 92 4.3.3 Signatures of selection in the coastal populations 95 4.3.4 Adaptation in high altitude populations 99 4.3.5 Signatures of balancing selection in abiotic stress genes 100 4.4 The utility of consensus sequences in molecular evolution 103 4.5 Conclusion and outlook 105 APPENDIX A: Material and methods 107 APPENDIX B: Results 115 Bibliography 165 Acknowledgements 175 2 Summary Demography and adaptation are important factors determining the evolution of plant species. Many plant species are substructured into populations or demes connected by migration (metapopulations). The spatial distribution of populations and migration patterns depend on the means of dispersal. Since plants are sessile organisms, they also have to cope with both biotic and abiotic stresses. Therefore adaptations to local environmental conditions are essential to ensure survival and duration of the species. Wild tomato species (Solanum section Lycopersicon) are native to western South America. They occur in diverse and often extreme habitats including rain forests, coastal regions, high altitude habitats in the Andean Mountains and also hyperarid deserts in the Atacama Desert. Therefore, wild tomatoes are a good model system to study plant evolution and genomic bases for plant adaptation. This study focuses on the wild tomato species Solanum chilense, which exhibits a metapopulation structure with populations distributed from southern Peru to northern Chile. In its native range, S. chilense is confronted with different abiotic stresses including drought, cold and salinity. I sequenced 30 unlinked nuclear genes from 23 populations using next generation sequencing. 16 genes are involved in the abiotic stress response and serve as candidates for selection and adaptation. The remaining 14 genes are used as references to study the genomic average and species past demography. In the first part of this study, I investigated the demographic history of the wild tomato species Solanum chilense. Genetic data analyses revealed a north-south cline. This cline includes 1) a decrease of genetic variation from north to south, 2) an increase in the strength of population expansion along the cline, and 3) an increase in genetic differentiation from other wild tomato species towards the south of the range. Results further revealed that the populations form four groups: a central group and three peripheral groups. Altogether the results suggest that S. chilense originated in the northern part of its current distribution and migrated to the south, via two routes, along the coast and higher up in the Andes. During this north-south colonization, at least three bottlenecks occurred. In the second part of this study, I investigated natural selection and local adaptation in S. chilense. Signatures of selection and local adaptation were detected in the abiotic stress- related genes, for example signatures of positive selection in high altitude populations were found possibly indicating adaptation to low temperatures. Interestingly, signatures of balancing selection were detected as well in high altitude populations reflecting probable 3 adaptation to different types of abiotic stresses. The coastal populations showed a distinct pattern. Several genes involved in the salt stress response exhibited signatures of local adaptation. Performing a salt stress experiment, I revealed that low altitude populations cope better with such stress than populations from intermediate or high altitudes. The coastal populations also showed an accumulation of nonsynonymous and possibly deleterious genetic variation, which can be explained by extreme bottlenecks and potential occurrence of selfing in some populations. Signatures of selection and local adaptation in S. chilense were mainly detected in populations from the peripheral groups and not in the central region, in agreement with the hypothesis that local adaptation is associated with the colonization of new territories. In summary, this study showed that demography plays an important role in the evolutionary history of S. chilense and that local adaptation for key abiotic stresses occurs more frequently in the marginal ranges of the species distribution. 4 Zusammenfassung Demographie und Anpassung spielen eine wichtige Rolle in der Evolution von Pflanzen. Viele Pflanzenarten sind unterteilt in Populationen die durch Migration miteinander verbunden sind (Metapopulationsstruktur). Ihre räumliche Ausbreitung und die Migrationsraten sind abhängig von den Verbreitungsmechanismen. Da es sessile Lebewesen sind, können sie biotischen und abiotischen Stressfaktoren nicht ausweichen. Daher sind Anpassungsmechanismen an die lokalen Umweltbedingungen essentiell um das Überleben und Fortbestehen der Art zu gewährleisten. Wildtomaten (Solanum sect. Lycopersicon) sind im Westen Südamerikas beheimatet. Sie kommen in unterschiedlichen und teilweise extremen Habitaten vor. Diese reichen von Regenwäldern, Küstenregionen, Bergregionen in den Anden zu den hyperariden Wüstenregionen der Atacama. Daher sind Wildtomaten geeignet um Evolution und die genomischen Grundlagen für die Anpassung an verschiedene Umweltbedingungen zu untersuchen. Diese Studie beschäftigt sich mit der Wildtomatenart Solanum chilense. Diese Art hat eine Metapopulationsstruktur und die einzelnen Populationen sind vom südlichen Peru bis ins nördliche Chile verteilt. In ihrem natürlichen Verbreitungsgebiet ist S. chilense mit verschiedenen abiotischen Stressfaktoren konfrontiert, u.a.