Chromosome differentiation and the radiation of the butterfly subgenus Agrodiaetus (Lepidoptera: Lycaenidae: Polyommatus) – a molecular phylogenetic approach Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Martin Wiemers aus Münster (Westf.) Bonn – September 2003 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Referent: Prof. Dr. Clas M. Naumann 2. Referent: Priv.-Doz. Dr. Bernhard Misof Tag der Promotion: _________________ 2 Contents CONTENTS CHAPTER 1: INTRODUCTION........................................................................................................................ 5 SCOPE AND AIMS................................................................................................................................................. 7 CHAPTER 2: NEW KARYOLOGICAL RESULTS IN AGRODIAETUS...................................................... 9 INTRODUCTION ................................................................................................................................................... 9 MATERIAL AND METHODS................................................................................................................................... 9 RESULTS ........................................................................................................................................................... 10 Karyotype photographs of Agrodiaetus....................................................................................................... 11 Overview of karyotypes in the subgenus Agrodiaetus Hübner, 1822 .......................................................... 15 DISCUSSION ...................................................................................................................................................... 29 CHAPTER 3: A MOLECULAR PHYLOGENY OF AGRODIAETUS ......................................................... 33 MATERIAL AND METHODS................................................................................................................................. 33 Material....................................................................................................................................................... 33 Selection of taxa .......................................................................................................................................... 36 DNA extraction............................................................................................................................................ 37 Selection of genes ........................................................................................................................................ 37 PCR and sequencing.................................................................................................................................... 38 Sequence alignment..................................................................................................................................... 39 Choice of methods for phylogenetic inference............................................................................................. 40 Sequence comparisons and phylogenetic inference..................................................................................... 41 RESULTS ........................................................................................................................................................... 43 Sequences and alignment............................................................................................................................. 43 Base composition and sequence divergence................................................................................................ 43 Bayesian inference of phylogeny ................................................................................................................. 48 Maximum parsimony analysis ..................................................................................................................... 60 Statistical parsimony networks.................................................................................................................... 74 DISCUSSION ...................................................................................................................................................... 87 Comparisons with traditional systematics................................................................................................... 87 Congruence between gene trees and species trees in outgroups................................................................. 90 Comparison with allozyme results............................................................................................................... 94 Hybridization in Agrodiaetus ...................................................................................................................... 94 Radiation of Agrodiaetus and the colonization of Europe........................................................................... 96 CHAPTER 4: SYSTEMATICS OF AGRODIAETUS BASED ON MOLECULAR EVIDENCE ............... 97 CHAPTER 5: EVOLUTION OF MORPHOLOGICAL TRAITS IN AGRODIAETUS............................. 107 INTRODUCTION ............................................................................................................................................... 107 MATERIAL AND METHODS............................................................................................................................... 107 RESULTS ......................................................................................................................................................... 108 DISCUSSION .................................................................................................................................................... 110 SUMMARY....................................................................................................................................................... 112 CHAPTER 6: THE ROLE OF CHROMOSOME EVOLUTION IN THE RADIATION OF AGRODIAETUS ................................................................................................................................................ 113 INTRODUCTION ............................................................................................................................................... 113 MATERIAL AND METHODS............................................................................................................................... 113 RESULTS ......................................................................................................................................................... 114 DISCUSSION .................................................................................................................................................... 116 SUMMARY....................................................................................................................................................... 119 SUMMARY ....................................................................................................................................................... 120 ZUSAMMENFASSUNG .................................................................................................................................. 120 ACKNOWLEDGEMENTS.............................................................................................................................. 121 REFERENCES.................................................................................................................................................. 123 HILFSMITTEL................................................................................................................................................. 143 3 Contents APPENDIX 1. KARYOLOGICAL RESULTS IN AGRODIAETUS....................................................... 145 APPENDIX 2. LIST OF MATERIAL............................................................................................. 149 APPENDIX 3. SEQUENCE DATA ............................................................................................... 165 PLATES. AGRODIAETUS WINGS ............................................................................................... 201 4 Introduction Chapter 1: Introduction The Holarctic butterfly genus Polyommatus Latreille, 1804 consists of approximately 120-180 species (HÄUSER & ECKWEILER 1997), many of which have been placed into subgroups which are considered separate genera according to numerous authors (e.g. the subgenera Meleageria, Cyaniris, Plebicula, Sublysandra, Neolysandra). The subgenus Agrodiaetus Hübner, 1822, which is distributed in the southern Palaearctic, is the most rich in species. ECKWEILER & HÄUSER (1997) recognize between 56 and 84 species whereas BÁLINT & JOHNSON (1997) list 102 species (including 8 species placed under Paragrodiaetus Rose & Schurian, 1977). Since 1997 alone, 23 further species rank taxa have been described from Anatolia and Iran. The distribution of this subgenus ranges from the Iberian Peninsula to northern Pakistan and Central Mongolia, but the majority of species is found in Anatolia, Transcaucasia and Iran. The characteristic morphological feature of this subgenus is the almost complete reduction of the submarginal elements