Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Eva M. Wolf born in: Karlsruhe, Germany Oral examination: 30.08.2017 The evolutionary history of Cochlearia L. Cytogenetics, phylogenomics and metabolomics of a cold relic in a warming world Referees: Prof. Dr. Marcus A. Koch apl. Prof. Dr. Claudia Erbar Table of Contents SUMMARY ........................................................................................................................................ 1 ZUSAMMENFASSUNG ....................................................................................................................... 3 GENERAL INTRODUCTION ................................................................................................................. 5 EVOLUTION IN THE CONTEXT OF A CHANGING ENVIRONMENT ........................................................................... 5 THE STUDY SYSTEM: COCHLEARIA AND TRIBE COCHLEARIEAE ............................................................................ 6 REFERENCES ......................................................................................................................................... 14 CHAPTER 1 – COCHLEARIA CYTOGENETICS ..................................................................................... 21 1.1 ABSTRACT ...................................................................................................................................... 22 1.2 INTRODUCTION ............................................................................................................................... 23 1.2.1 Genome evolution in flowering plants ................................................................................ 23 1.2.2 Genome evolution in the Brassicaceae family ..................................................................... 24 1.2.3 Genome evolution in the genus Cochlearia ......................................................................... 26 1.3 MATERIAL AND METHODS ................................................................................................................. 28 1.3.1 Cytogenetic methods ........................................................................................................... 28 1.3.2 Geographical distribution of chromosome numbers/genome sizes .................................... 31 1.4 RESULTS ........................................................................................................................................ 33 1.4.1 Chromosome counting and flow cytrometric analysis ........................................................ 33 1.4.2 Flow cytometry .................................................................................................................... 42 1.5 DISCUSSION ................................................................................................................................... 46 1.5.1 Two diploid karyotypes and the question of the ancestral base chromosome number ...... 46 1.5.2 Stability, instability and chaos? – Diploidy, polyploidy and aneuploidy in Cochlearia ........ 47 1.5.3 Genome duplication, genome size increase and subsequent parallel genome downsizing 48 1.6 REFERENCES ................................................................................................................................... 50 CHAPTER 2 – COCHLEARIA PHYLOGENOMICS ................................................................................. 57 2.1 ABSTRACT ...................................................................................................................................... 58 2.2 INTRODUCTION ............................................................................................................................... 59 2.2.1 NGS sequencing in phylogenetics and phylogeography ...................................................... 59 2.2.2 Cochlearia phylogenetics..................................................................................................... 60 2.3 MATERIAL AND METHODS ................................................................................................................. 62 2.3.1 Plant material and taxon sampling ..................................................................................... 62 2.3.2 DNA extraction and NGS sequencing .................................................................................. 65 2.3.3 Chloroplast genome data analysis ...................................................................................... 68 2.3.4 Mitochondrial genome data analysis .................................................................................. 72 2.3.5 Nuclear genome data analysis ............................................................................................ 73 2.4 RESULTS ........................................................................................................................................ 77 2.4.1 Illumina sequencing data .................................................................................................... 77 2.4.2 Chloroplast dataset ............................................................................................................. 77 2.4.3 Mitochondrial dataset ......................................................................................................... 85 i 2.4.4 Nuclear dataset................................................................................................................... 88 2.5 DISCUSSION ................................................................................................................................... 97 2.5.1 Congruence and incongruence in organellar phylogenies .................................................. 97 2.5.2 Organellar genome phylogenies, divergence time estimates and nuclear SNP data analyses – rewriting the evolutionary history of the genus Cochlearia ...................................................... 98 2.5.3 New insights into the complex evolutionary histories of polyploid taxa ........................... 101 2.5.4 Repeated adaptation to arctic/alpine habitats ................................................................ 105 2.6 REFERENCES ................................................................................................................................. 107 CHAPTER 3 – COCHLEARIA METABOLOMICS ................................................................................ 115 3.1 ABSTRACT .................................................................................................................................... 116 3.2 INTRODUCTION ............................................................................................................................. 117 3.2.1 New approaches to study plant responses to abiotic stresses and adaptation ................ 117 3.2.2 Edaphic and cold adaptation in arctic/alpine systems and the potential of the Cochlearia study system .............................................................................................................................. 118 3.3 MATERIAL AND METHODS ............................................................................................................... 120 3.3.1 Plant material and taxon sampling ................................................................................... 120 3.3.2 Habitat characterization and ecotype definition .............................................................. 122 3.3.3 Temperature treatment .................................................................................................... 122 3.3.4 Metabolite extraction and analyses ................................................................................. 123 3.3.5 Multivariate statistical analysis of metabolomics and integration of bioclimatic population clusters ....................................................................................................................................... 124 3.4 RESULTS ...................................................................................................................................... 127 3.4.1 Climatic data and habitat characterization ...................................................................... 127 3.4.2 Metabolomics – compound analysis (ANOVA) ................................................................. 129 3.4.3 Metabolomics – DAPC ....................................................................................................... 136 3.5 DISCUSSION AND OUTLOOK ............................................................................................................. 148 3.5.1 Insights from metabolomic compound analysis – investigating the cold metabolome of Cochlearia .................................................................................................................................. 148 3.5.2 DAPC analyses – in search of intrageneric variation in the Cochlearia cold metabolome 149 3.5.3 Outlook ............................................................................................................................. 151 3.5 REFERENCES ................................................................................................................................. 152 AVAILABILITY OF ADDITIONAL MATERIAL .................................................................................... 158 ACKNOWLEDGEMENTS