Evolution of Host-Plant use in Cymothoe (Nymphalidae) Feeding on Rinorea (Violaceae) Robin van Velzen (791105 866 070) Supervisors: Dr. Freek Bakker Dr. Joop van Loon Biosystematics Group Laboratory of Entomology November 2006 Wageningen University 2 TITLEPAGE. 1. Harma theobene male; 2. Cymothoe beckeri female; 3. Cymothoe indamora female; 4. Cymothoe herminia female RVVB226; 5. Cymothoe caenis male RVV B364; 6. Cymothoe coccinata male RVV B353; 7. Cymothoe oemilius female. 8. Rinorea longisepala young fruit; 9. Rinorea dentata young fruit; 10. Rinorea caudata fruit RVV49; 11. Rinorea subintegrifolia capsule; 12. Cymothoe fumana caterpillar RVV C009 on Rinorea oblongifolia leaf. 3 Preface This is the report of the second Msc thesis for my biology studies at the Wageningen University, supervised by Freek Bakker from the Nationaal Herbarium Nederland – Wageningen Branch & Biosystematics Group and Joop van Loon from the Laboratory of Entomology. This thesis was meant to be a preparative and orientating pilot for the planned PhD project “Evolution of host-specificity in the tropical African butterfly genus Cymothoe (Nymphalidae) feeding on Rinorea (Violaceae)” for which an application has been submitted to the Nederlandse Organisatie voor Wetenschappelijk onderzoek, Aard- en Levenswetenschappen (NWO-ALW). The pilot study aimed at evaluating the feasibility of studying Cymothoe butterflies and their associated host plants in the field and in the laboratory, and my work consisted consequently of field work as well as analytical work. The field work was carried out in the tropical forests of Cameroon, in collaboration with the Herbier National in Yaoundé. The analyticalwork could be carried out thanks to data that was kindly provided by laboratories in Guelph (Canada), Wageningen (Netherlands) and Davis (USA). This report is divided into two chapters, with different objectives. The first chapter is the scientific thesis report, where an introduction to the context of the subject is given and the analytical questions will be adressed. The second chapter is the field report and can be found at page 45. It deals with the work that has been done in Cameroon and gives details about the expedition. The results, in terms of collections and observations are discussed. I thank the reader for the interest and hope that this report will give a satisfactory overview of my work on this subject and a boost to the study of this highly fascinating subject. Robin van Velzen. Wageningen, November 2006 4 CHAPTER 1 Thesis Report 5 Index Abstract............................................................................................................................ 7 1 Introduction ........................................................................................................ 8 1.1 Host plant use in phytophagous insects...................................................................... 8 Phytochemistry............................................................................................................................. 8 Evolutionof host plant use............................................................................................................. 8 1.2 Host specificity ........................................................................................................ 9 Evolutionof host specificity ......................................................................................................... 10 Causes of host specificity............................................................................................................ 10 1.3 The case of Cymothoe (Nymphalidae) feeding on Rinorea (Violaceae)...........................11 Rinorea...................................................................................................................................... 11 Cymothoe and Harma ................................................................................................................. 12 Host plant relationships............................................................................................................... 14 Host plant chemistry................................................................................................................... 15 1.4 DNA barcoding........................................................................................................16 1.5 Research questions.................................................................................................18 Phylogeny estimates................................................................................................................... 18 Host plant associations ............................................................................................................... 19 DNA barcoding ........................................................................................................................... 19 2 Materials & Methods ......................................................................................... 20 2.1 Taxon sampling.......................................................................................................20 2.2 DNA extraction, amplification and sequencing............................................................22 2.3 DNA barcoding........................................................................................................23 2.4 Phylogeny reconstruction.........................................................................................23 3 Results............................................................................................................. 24 3.1 DNA barcoding........................................................................................................24 3.2 Phylogeny reconstruction.........................................................................................25 Cymothoe and Harma ................................................................................................................. 25 The C. egesta complex ............................................................................................................... 27 Rinorea...................................................................................................................................... 28 4 Discussion........................................................................................................ 30 4.1 DNA barcoding........................................................................................................30 4.2 Phylogenetic estimation ...........................................................................................30 Rinorea...................................................................................................................................... 30 Cymothoe and Harma ................................................................................................................. 31 4.3 Cryptic sibling species in Cymothoe..........................................................................32 4.4 Synthesis................................................................................................................34 5 Conclusions...................................................................................................... 37 6 Recommendations ............................................................................................ 37 7 Acknowledgements ........................................................................................... 38 8 References....................................................................................................... 39 6 Abstract Together, living plants and the herbivorous insects that eat them make up half of all extant organisms on earth. Here, I focus on two aspects of host plant association patterns. First, the majority herbivorous insect groups do not feed indiscriminative on any plant, but are usually associated with plants that are somehow related, an observation that has led authors to hypothesise a process of coevolution where plants and herbivorous insects have influenced each others evolution through reciprocal selection pressures, mediated through plant secondary compounds and the butterflies’ adaptations to overcome this anti-herbivore phytochemistry. However, phylogenetic evidence for chemical coevolution is limited and evidence must be sought for at other levels of resolution than most previous studies have achieved. Second, most phytophagous insects are highly host specific, with many species being monophagous. But the proximate and ultimate factors underlying this specificity remain largely unclear and phylogenetic studies of highly specific insect-plant associations are very few. Here, I present a study on the evolution of host plant use and host specificity in the highly specific interaction between Cymothoe butterflies and their Rinorea host plants in the Afrotropics; the first detailed study of a system where the specificity is at the species level and involves many congeneric species of both butterflies and plants and the first such published study in the tropics. The research questions consider the scale and extent of this herbivore plant interaction, the phylogeny of both butterflies and host plants, and the mechanisms involved in host plant recognition, but given the limitations for an Msc thesis, a few number of questions have been selected as a basis of a pilot study aiming at evaluating the feasibility of studying Cymothoe butterflies and their associated host plants in the field and