Genetic structure and pollination biology of the FFH-protected Gladiolus palustris GAUDIN (Iridaceae) in Salzburg and adjacent areas Masterarbeit Zur Erlangung des Mastergrades an der Naturwissenschaftlichen Fakultät der Paris-Lodron-Universität Salzburg Eingereicht von Bettina Michaela Leitner Gutachter: Assoz. Prof. Mag. Dr. Andreas Tribsch Department of Biosciences University of Salzburg, Austria Salzburg, April, 2018 The one process now going on that will take millions of years to correct is the loss of genetic and species diversity by the destruction of natural habitats. This is the folly our descendants are least likely to forgive us (Edward O. Wilson, 1984). Contents 1 Introduction ..................................................................................................................................... 1 1.1 General introduction ............................................................................................................... 1 1.2 Study subject Gladiolus palustris GAUDIN .............................................................................. 4 1.2.1 Distribution of Gladiolus palustris ................................................................................... 4 1.2.2 Biological features of Gladiolus palustris ........................................................................ 6 1.2.3 Habitat of Gladiolus palustris .......................................................................................... 8 1.2.4 Conservation status and endangerment ......................................................................... 8 1.3 Aims of the study ................................................................................................................... 10 1.3.1 Genetic structure within and among populations ........................................................ 10 1.3.2 Unique color variation in a single population ............................................................... 10 1.3.3 Pollination biology of Gladiolus palustris ...................................................................... 11 2 Material and Methods ................................................................................................................... 12 2.1 Genetic structure within and among populations in Salzburg and adjacent areas .............. 12 2.1.1 Sampling ........................................................................................................................ 12 2.1.2 Genetic analyses ............................................................................................................ 17 2.1.2.1 DNA-Extraction .............................................................................................................. 17 2.1.2.2 Generating AFLPs........................................................................................................... 18 2.1.2.3 AFLP data scoring .......................................................................................................... 20 2.1.2.4 Quantifying the error rate and pruning the binary matrix ............................................ 21 2.1.3 Genetic data analysis ..................................................................................................... 21 2.1.3.1 Data matrices ................................................................................................................ 21 2.1.3.2 Genetic relationships (Trees) distance-based clustering methods ............................... 22 2.1.3.3 Principal Coordinates Analysis (PCoA) ........................................................................... 23 2.1.3.4 Model-based clustering ................................................................................................. 23 2.1.3.5 Genetic diversity and rarity ........................................................................................... 24 2.1.3.6 Isolation by distance (IBD) ............................................................................................. 25 2.1.3.7 Analyses of molecular variance (AMOVA) ..................................................................... 25 2.2 Investigation of one population with color variation ............................................................ 27 2.2.1 Plant material ................................................................................................................ 27 2.2.2 Data analysis .................................................................................................................. 28 2.3 Pollination biology study ....................................................................................................... 29 2.3.1 Experimental setting ..................................................................................................... 29 2.3.2 Evaluation of the data ................................................................................................... 30 3 Results ........................................................................................................................................... 31 3.1 Results of the investigation of the genetic structure within and among populations in Salzburg and adjacent areas .............................................................................................................. 31 3.1.1 Results of the AFLP data ................................................................................................ 31 3.1.2 Identification of identical genotypes ............................................................................. 31 3.1.3 Results of the data analysis ........................................................................................... 33 3.1.3.1 Genetic relationships ..................................................................................................... 33 3.1.3.2 Results of the Principal Coordinates Analysis (PCoA) .................................................... 35 3.1.3.3 Results of the Bayesian clustering ................................................................................. 37 3.1.3.4 Results of the genetic diversity analysis ........................................................................ 46 3.1.3.5 Results of the AMOVA ................................................................................................... 50 3.1.3.6 Isolation by distance ...................................................................................................... 56 3.2 Results of the investigation of one population with color variation ..................................... 59 3.2.1 Results of the AFLP data ................................................................................................ 59 3.2.2 Results of the data analysis ........................................................................................... 59 3.3 Results of the pollination biology study ................................................................................ 61 4 Discussion ...................................................................................................................................... 64 4.1 Genetic structure of Gladiolus palustris in Salzburg and adjacent areas .............................. 64 4.1.1 Genetic variation in Gladiolus palustris ......................................................................... 64 4.1.2 Genetic structure of Gladiolus palustris in Salzburg and adjacent areas ...................... 65 4.1.3 Gene diversity and genetic rarity .................................................................................. 66 4.1.4 Differentiation (FST) of Gladiolus palustris and isolation by distance (IBD) ................... 69 4.2 Discussion of the color variation ........................................................................................... 72 4.3 Discussion of the pollination biology .................................................................................... 74 4.4 Habitat fragmentation, potential consequences and conservation ..................................... 76 4.4.1 Habitat fragmentation and decline of Gladiolus palustris in Salzburg .......................... 76 4.4.2 Potential consequences of habitat loss for Gladiolus palustris ..................................... 79 4.4.3 Implications for conservation of Gladiolus palustris ..................................................... 82 5 Acknowledgements ....................................................................................................................... 84 6 References ..................................................................................................................................... 85 7 Appendix ........................................................................................................................................ 93 Abstract Due to the loss of potential habitats because of anthropogenic fragmentation many plant populations became small and isolated. Small populations are often facing negative genetic consequences like reduced genetic variation, genetic drift, founder effects, inbreeding depression and accumulation of deleterious mutations. These factors may lead on to a weaker future adaption potential to environmental changes, reduced plant fitness and consequential higher risk of becoming extinct. In this particular field, conservation genetics becomes enormously applicable. It targets the comprehension of dynamics of genes in populations via genetic methods to develop management plans for persistence and protection of the population. A