Population genetic and morphological studies in a hybrid zone between two subspecies of Tephroseris helenitis (L.) B. NORD. (Asteraceae) at the northern fringe of the Alps Masterarbeit Zur Erlangung des Mastergrades an der Naturwissenschaftlichen Fakultät der Paris-Lodron-Universität Salzburg eingereicht von Georg Pflugbeil Gutachter: Univ.-Prof. Dr.rer.nat. Hans-Peter Comes Fachbereich: Organismische Biologie Salzburg, November 2012 ii “Is it all ready? Right. Come on then. Back to creation. We mustn't waste any more time. They'll think I've lost control again and put it all down to evolution.” Supreme Being in “Time Bandits” (1981) iii iv Contents Abstract ......................................................................................................................................... ix Zusammenfassung ........................................................................................................................ xi 1. Introduction .......................................................................................................................... 1 1.1. General introduction ..................................................................................................... 1 1.2. Aims of the study .......................................................................................................... 4 2. Material and methods ........................................................................................................... 6 2.1. Study system ................................................................................................................. 6 2.2. Plant material .............................................................................................................. 13 2.3. Genetic analyses ......................................................................................................... 15 2.3.1. DNA-Extraction .................................................................................................... 15 2.3.2. Generating AFLPs ................................................................................................ 16 2.3.3. Data scoring......................................................................................................... 18 2.3.4. Quantifying the error rate ................................................................................... 18 2.3.5. Pruning the binary matrix ................................................................................... 18 2.4. Genetic data analyses ................................................................................................. 19 2.4.1. Genetic differentiation ........................................................................................ 19 2.4.2. Genetic relationships .......................................................................................... 20 2.4.3. Principal coordinates analysis (PCoA) ................................................................. 20 2.4.4. Analyses of molecular variance (AMOVAs) ......................................................... 21 2.4.5. AMOVA-derived FST values (ΦST) and patterns of isolation by distance (IBD) .... 21 2.4.6. Genetic diversity and rarity ................................................................................. 22 2.4.7. Detection of FST outlier loci and clinal distribution of allele frequencies at single AFLP loci .............................................................................................................. 22 2.5. Morphological analyses .............................................................................................. 23 2.6. Vegetation data analyses ............................................................................................ 24 2.7. Correlations of genetic, morphological and vegetation data ..................................... 25 2.8. Self-incompatibility tests ............................................................................................. 25 3. Results ................................................................................................................................. 28 3.1. AFLP data set and error rate ....................................................................................... 28 3.2. Genetic differentiation ................................................................................................ 28 3.2.1. STRUCTURE assignment tests using independent allele frequencies ................. 28 3.2.2. STRUCTURE assignment tests using correlated allele frequencies ..................... 35 v 3.2.3. BAPS non-spatial clustering ................................................................................. 38 3.2.4. BAPS spatial clustering ........................................................................................ 41 3.3. Genetic relationships ................................................................................................... 43 3.3.1. Individual-based relationships ............................................................................ 43 3.3.2. Population-based relationships ........................................................................... 46 3.4. Principal coordinates analysis (PCoA) ......................................................................... 49 3.5. Analyses of molecular variance (AMOVAs) ................................................................. 52 3.5.1. Non-hierarchical AMOVAs ................................................................................... 52 3.5.2. Hierarchical AMOVAs .......................................................................................... 54 3.6. Genetic diversity and rarity ......................................................................................... 55 3.7. AMOVA-derived FST values (ΦST) and patterns of isolation by distance (IBD) ............. 62 3.8. Detection of FST outlier loci and clinal distribution of allele frequencies at single AFLP loci ............................................................................................................................... 65 3.9. Morphological analyses ............................................................................................... 69 3.10. Vegetation data analyses ........................................................................................ 75 3.11. Association of morphological characters and vegetation data with genetic data .. 79 3.11.1. Mapping of morphological characters onto the genetic data............................. 79 3.11.2. Mapping of vegetation data onto the genetic data ............................................ 83 3.12. Self-incompatibility tests ......................................................................................... 86 4. Discussion ............................................................................................................................ 87 4.1. Taxonomical aspects ................................................................................................... 87 4.1.1. Distribution patterns of Tephroseris helenitis subspp. helenitis and salisburgensis at the northern fringe of the Alps ................................................ 87 4.1.2. Association of achene type with genetic, morphological and vegetation data .. 88 4.1.3. Is the taxonomic rank of “subspecies” for individuals with glabrous achenes justifiable? ........................................................................................................... 90 4.2. Population history of Tephroseris helenitis at the northern fringe of the Alps .......... 91 4.3. Hybrid zone between subspp. helenitis and salisburgensis ........................................ 93 4.3.1. Do the two subspecies form a hybrid zone? ....................................................... 93 4.3.2. Is the hybrid zone a primary or secondary one? ................................................. 95 4.3.3. Origin and maintenance of characters varying along the cline ........................... 97 4.4. Miscellaneous aspects ................................................................................................. 98 4.4.1. Red bract/stem color and anthocyanin synthesis ............................................... 98 4.4.2. Replacement of “glabrous individuals” by “pubescent” ones ............................ 98 4.4.3. Conservation aspects .......................................................................................... 99 4.4.4. Self-incompatibility tests ................................................................................... 100 vi 4.5. Outlook ...................................................................................................................... 100 Conclusions ............................................................................................................................... 101 Acknowledgements ................................................................................................................... 103 References................................................................................................................................. 105 A. Appendix ........................................................................................................................... 114 vii viii Abstract Abstract This study has three major aims. First, the distribution of Tephroseris helenitis subspp. helenitis and salisburgensis at the northern fringe of the Alps was explored according to their achene type as character of subspecies designation.