The Aquatic Leaf Beetle Macroplea mutica (Coleoptera: Chrysomelidae): Population Structure in Europe and the Signature of Zoochorous Dispersal Dissertation with the aim of achieving a doctoral degree at the Faculty of Mathematics, Informatics and Natural Sciences Department of Biology of Universität Hamburg submitted by Jan-Jakob Laux Hamburg, 2014 ii ABSTRACT .............................................................................................................. VII ZUSAMMENFASSUNG ............................................................................................ IX LIST OF FIGURES .................................................................................................... XI LIST OF TABLES .................................................................................................... XII 1- INTRODUCTION .................................................................................................... 1 1.1 - Dispersal ........................................................................................................................................................ 1 1.1.1 - Passive Dispersal of Freshwater Organisms ........................................................................................... 2 1.1.2 - Dispersal of Aquatic Organisms by Waterbirds ...................................................................................... 3 1.2 - Population Genetic Analyses in Dispersal Ecology .................................................................................... 5 1.2.1 - Detecting Genetic Evidence of Vector-Mediated Dispersal .................................................................... 6 1.2.2 - Population Genetic Markers .................................................................................................................... 8 1.2.2.1 - Microsatellites ................................................................................................................................. 8 1.2.2.2 - Mitochondrial DNA ........................................................................................................................ 9 1.2.3 - Population Genetic Software Tools....................................................................................................... 10 1.3 - Fully Aquatic Reed Beetles - The Genus Macroplea ................................................................................ 11 1.3.1 - Macroplea mutica (FABRICIUS 1792) .................................................................................................... 14 1.3.1.1 - Life Cycle ...................................................................................................................................... 14 1.3.1.2 - Respiration and Mobility ............................................................................................................... 15 1.3.1.3 - Population Genetic Structure of M. mutica ................................................................................... 16 1.3.1.4 - Passive Dispersal of M. mutica: Water and Waterbirds as Potential Vectors ................................ 17 1.3.1.5 - The Mute Swan Cygnus olor - a Potential Vector Species ............................................................ 18 1.4 - Objectives .................................................................................................................................................... 20 2 - MATERIAL AND METHODS .............................................................................. 21 2.1 - Origin of Samples........................................................................................................................................ 21 2.2 - Molecular Biology ....................................................................................................................................... 22 2.2.1 - DNA Extraction .................................................................................................................................... 22 2.2.2 - Development and Characterization of Microsatellite Markers for Macroplea mutica .......................... 22 2.2.2.1 - Construction of a Genomic DNA Library Enriched for Microsatellites ........................................ 22 2.2.2.2 - Microsatellite Primer Design ......................................................................................................... 23 2.2.2.3 - Microsatellite Primer Testing and Locus Screening ...................................................................... 23 2.2.2.4 - PCR Conditions ............................................................................................................................. 24 2.2.2.5 - Genotyping / Scoring..................................................................................................................... 24 2.2.3 - Amplification and Sequencing of Mitochondrial DNA ........................................................................ 25 2.3 - Population Genetic Analyses ...................................................................................................................... 25 2.3.1 - Characterization of Developed Microsatellite Markers ........................................................................ 25 2.3.1.1 - Tests for Null Alleles, Stuttering, Hardy-Weinberg Equilibrium and Linkage Disequilibrium .... 25 2.3.1.2 - Validation of Markers - Test for Congruence with AFLP Data .................................................... 26 2.3.2 - Population Genetic Differentiation and Isolation by Distance .............................................................. 26 2.3.2.1 - Assessing Statistical Power of the Used Set of Microsatellites ..................................................... 26 2.3.2.2 - Population Genetic Differentiation ................................................................................................ 27 2.3.2.3 - Testing the Relative Performance of FST and RST Estimates ......................................................... 27 2.3.2.4 - Subdivision of Macroplea mutica Sampling Sites ......................................................................... 28 2.3.2.5 - Testing for Isolation by Distance .................................................................................................. 29 2.3.2.6 - Calculating Residual RST-Values................................................................................................... 30 2.3.2.7 - Construction of a Neighbor-Joining Tree ...................................................................................... 30 iii 2.3.2.8 - Principal Coordinate Analysis ....................................................................................................... 30 2.3.3 - Inferring the Number of Genetically Distinct Clusters ......................................................................... 30 2.3.3.1 - Inferring the Uppermost Level of Population Structure ................................................................ 31 2.3.3.2 - BAPS 5.2 ....................................................................................................................................... 32 2.3.3.3 - GENELAND ................................................................................................................................. 32 2.3.4 - Bayesian Inference of Migration Rates with MIGRATE-N .................................................................. 33 2.3.5 - Analysis of Mitochondrial DNA ........................................................................................................... 33 2.4 – GIS-Based Analyses ................................................................................................................................... 34 2.4.1 - Mapping and Visualizing of Swan Sightings and Movements.............................................................. 34 2.4.2 - Inferring the Direction of Swan Movements ......................................................................................... 34 2.4.3 - Local Swan Abundances and Swan Traffic between Sites.................................................................... 36 2.4.4 - Geographic Distances between Macroplea mutica Sampling Sites ...................................................... 38 2.4.5 - Mapping Beetle Sampling Locations against Landscape Features ....................................................... 38 2.4.6 - Testing for Correlation of Swan Movements and Abundances with Population Genetic Structure in Macroplea mutica ............................................................................................................................................ 38 2.4.6.1 - Pairwise Genetic Differentiation in M. mutica and Swan Sighting Data ...................................... 38 2.4.6.2 - Breakdown of Isolation by Distance and Swan Abundances ........................................................ 39 2.5 - Feeding Trials and Simulation of Gut Passage......................................................................................... 40 2.5.1 - Origin and Keeping Conditions of Plant Material and Beetles ............................................................. 40 2.5.2 - Simulation of Gut Passage .................................................................................................................... 40 2.5.2.1 - Gizzard Phase ................................................................................................................................ 40 2.5.2.2 - Intestinal Phase .............................................................................................................................