Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN Biodiversità ed Evoluzione Ciclo XXIV Settore Concorsuale di afferenza: 05/B1 - ZOOLOGIA E ANTROPOLOGIA Settore Scientifico disciplinare: BIO/05 - ZOOLOGIA TITOLO TESI “Study of variability and genetic structure of European populations of Myotis emarginatus and Myotis capaccinii (Chiroptera, Vespertilionidae)" Presentata da: Dott. Andrea Viglino Coordinatore Dottorato Relatore Prof. Barbara Mantovani Prof. Ettore Randi Esame finale anno 2012 INDEX INDEX........................................................................................................................................1 CHAPTER 1...............................................................................................................................4 GENERAL INTRODUCTION ..............................................................................................4 1.1 CONSERVATION GENETICS...................................................................................4 1.2 PHYLOGEOGRAPHY ................................................................................................6 1.3 NON INVASIVE GENETICS .....................................................................................7 1.4 THE ORDER CHIROPTERA......................................................................................8 1.5 GENUS MYOTIS ......................................................................................................14 1.6 PHYLOGENETICS OF MYOTIS .............................................................................15 1.7 LEGISLATION..........................................................................................................17 1.7 BATS IN ITALY........................................................................................................18 1.7.1 Summary Details of Resident Specie ......................................................................18 1.8 STUDIED SPECIES ..................................................................................................21 1.8.1 Myotis emarginatus .................................................................................................21 1.8.2 Myotis capaccinii .....................................................................................................22 1.8.3 Threats on Myotis emarginatus and Myotis capaccinii ..........................................25 1.9 GENETIC APPLICATION ON BATS......................................................................28 1.10 AIMS OF THE THESIS...........................................................................................30 CHAPTER 2.............................................................................................................................31 MATHERIALS AND METHODS ......................................................................................31 2.1 SAMPLING COLLECTION LOCALITIES..............................................................31 2.2 SAMPLE COLLECTION AND PRESERVATION..................................................34 2.2.a TISSUE COLLECTION..........................................................................................34 2.2.1 Capture ....................................................................................................................34 2.2.2 Mist Nets .................................................................................................................35 2.2.3 Harp Traps ...............................................................................................................36 2.2.b NON-INVASIVE SAMPLING...............................................................................38 2.3 DNA MARKERS USED IN THE ANALYSES........................................................38 2.3.1 Nuclear DNA: Microsatellites.................................................................................38 2.3.2 Mitocondrial DNA: CytB and CR...........................................................................39 2.3.3 Nuclear Introns ........................................................................................................41 2.4 ANALYSIS PROCEDURES .....................................................................................42 2.4.1 DNA extraction .......................................................................................................42 2.4.2 Manual extraction....................................................................................................43 2.4.3 Automated extraction ..............................................................................................44 2.4.4 DNA amplification ..................................................................................................44 2.5.1 Microsatellite Amplification....................................................................................45 2.5.3 Analysis of microsatellites in automated capillary sequencers ...............................46 2.6 MICROSATELLITE DATA ELABORATION ........................................................48 2.6.1 Data reliability: RelioType ......................................................................................48 2.6.2 Multilocus genotype comparison: Gimlet ...............................................................48 2.6.3 Species detection: Structure.....................................................................................49 2.6.4 Genetic population study: GeneAlex.......................................................................50 2.6.5 Adegenet package....................................................................................................51 2.7 SEQUENCES DATA ELABORATION....................................................................51 2.7.1 BioEdit.....................................................................................................................52 2.7.2 MEGA .....................................................................................................................52 2.7.3 ARLEQUIN.............................................................................................................52 2.7.4 NETWORK .............................................................................................................54 2.7.5 MODELTEST .........................................................................................................55 2.7.6 PAUP.......................................................................................................................55 2 2.7.7 MRBAYES..............................................................................................................55 2.7.8 GENEIOUS .............................................................................................................56 2.7.9 TREE CONSTRUCTION METHODS...................................................................56 CHAPTER 3.............................................................................................................................60 RESULTS.............................................................................................................................60 3.1 MICROSATELLITES................................................................................................61 3.1.2 NON-INVASIVE ANALYSES ..............................................................................61 3.1.3 Myotis emarginatus .................................................................................................65 3.1.3.1 Genetic structure of Myotis emarginatus ’s populations......................................65 3.1.3.2 Population genetics analysis and microsatellite variability..................................68 3.1.2 Myotis capaccinii .....................................................................................................72 3.1.2.1 Genetic structure of Myotis capaccinii’s populations ..........................................72 3.1.2.2 Population genetics analysis and microsatellite variability..................................75 3.1.3 ASSIGNMENTE TEST ..........................................................................................79 3.2 SEQUENCES .............................................................................................................80 mtDNA CONTROL REGION analyses...........................................................................80 3.2.1 Myotis emarginatus .................................................................................................80 3.2.1.1 HAPLOTYPE ANALYSIS ..................................................................................81 3.2.1.2 COLONIES ..........................................................................................................83 3.2.1.3 GLOBAL DLOOP RESUME ..............................................................................93 3.2.2 Myotis capaccinii .....................................................................................................94 3.2.2.1 HAPLOTYPE ANALYSIS ..................................................................................95 3.2.2.2 COLONIES ..........................................................................................................97 3.2.2.3 GLOBAL DLOOP RESUME ............................................................................103 CYTOCHROME B ........................................................................................................105 3.2.3 Myotis emarginatus ...............................................................................................105