Comparative phylogeography of five swallowtail butterfly species (Lepidoptera: Papilionidae) in South Africa: ecological and taxonomic implications A thesis submitted in fulfilment of the requirements of the degree of MASTER OF SCIENCE of RHODES UNIVERSITY By Götz-Georg Neef March 2014 i Abstract With current biota under constant threat of extinction, it is important to ascertain where and how biological diversity is generated and partitioned. Phylogeographic studies can assist in the identification of places and processes that indicate the origin and maintenance of biodiversity. Forest fragmentation has a big effect on local extinction and loss of genetic diversity of forest-restricted taxa, along with divergence and speciation of forest biota. This study aims to understand the effects of these processes on a number of forest-dwelling butterflies using a comparative phylogeographic approach. Mitochondrial DNA of five different Papilio species with different degrees of forest specificity was analysed using phylogenetic methods. In addition, the subspecific taxonomy of P. ophidicephalus was investigated using morphometrics of discal spots on the wings and nuclear DNA analysis along with mitochondrial DNA analysis. The results show that the forest-restricted species (P. ophidicephalus and P. echerioides) have more genetic structure and less genetic diversity than the more generalist species (P. dardanus, P. demodocus and P. nireus). This could be due to inbreeding depression and bottlenecks caused by forest fragmentation. As forest patches become smaller, the population size is affected and that causes a loss in genetic diversity, and increasing habitat fragmentation disrupts gene flow. The intraspecific taxonomy of P. ophidicephalus is far from revealed. However, this study shows there is evidence for the different subspecies when comparing morphological results and genetic results. From the evidence provided here it is suggested that P. ophidicephalus should be divided into two separate species rather than five subspecies. vii Table of Contents Comparative phylogeography of five swallowtail butterfly species (Lepidoptera: Papilionidae) in South Africa: ecological and taxonomic implications ................................ i Abstract .................................................................................................................... vii List of Tables ............................................................................................................ xi List of Figures .......................................................................................................... xii List of Abbreviations ............................................................................................... xv Acknowledgements ................................................................................................. xvi Disclaimer .............................................................................................................. xvii Declaration ........................................................................................................... xviii General Introduction ................................................................................................... 1 Forests in South Africa .............................................................................................. 1 African Forest Biodiversity........................................................................................ 1 Role of phylogeography in elucidating forest histories ............................................. 2 Aim ............................................................................................................................ 3 Papilio (swallowtail butterflies) ................................................................................ 4 Thesis structure .......................................................................................................... 5 Afromontane forest history as indicated by the phylogeography of Papilio species in South Africa ......................................................................................................................... 6 Introduction ................................................................................................................ 6 Aims and Hypotheses ................................................................................................ 8 Species selected to test these hypotheses ................................................................... 8 Materials and Methods ............................................................................................. 10 Sampling .............................................................................................................. 10 Molecular techniques ........................................................................................... 10 Phylogenetic analyses .......................................................................................... 12 viii Molecular Dating ................................................................................................. 12 Network-building Methods .................................................................................. 13 Isolation By Distance ........................................................................................... 13 Genetic Diversity ................................................................................................. 13 Results ...................................................................................................................... 14 Data characteristics .............................................................................................. 14 Genetic structure of forest-restricted vs widespread species ............................... 14 Haplotype networks ............................................................................................. 18 Molecular dating .................................................................................................. 23 Isolation By Distance ........................................................................................... 25 Genetic diversity indices ...................................................................................... 26 Discussion ................................................................................................................ 30 Do forest-restricted butterfly species have more genetic structure than more widespread species? ......................................................................................................... 30 Molecular dating and forest fragmentation .......................................................... 31 Is isolation by distance stronger in forest-restricted species? .............................. 32 Does fragmentation decrease genetic diversity in forests? .................................. 32 Conclusions .............................................................................................................. 33 A molecular and morphological investigation of the intraspecific taxonomy of P. ophidicephalus ........................................................................................................................ 35 Introduction .............................................................................................................. 35 Subspecies concept .............................................................................................. 35 Aims ..................................................................................................................... 36 Materials and Methods ............................................................................................. 36 Phylogenetic analysis ........................................................................................... 36 Morphometric analysis of wing spots .................................................................. 37 Statistical Analyses .............................................................................................. 38 Results ...................................................................................................................... 38 ix ITS........................................................................................................................ 38 Principal Component Analysis ............................................................................ 41 Discriminant Function Analysis .......................................................................... 42 Discussion ................................................................................................................ 44 Conclusions .............................................................................................................. 44 General Discussion ..................................................................................................... 46 Summary of findings................................................................................................ 46 Correlation with Forest types ................................................................................... 46 Genetic and morphological investigation of P. ophidicephalus .............................. 46 The Lydenburg anomaly .......................................................................................... 47 References ................................................................................................................... 49 Appendix 1 .................................................................................................................