Evolution of associations between Cymothoe butterflies and their Rinorea host plants in tropical Africa Robin van Velzen Thesis committee Promotor Prof. Dr M.S.M. Sosef Professor of Biosystematics Wageningen University Co-promotor Dr F.T. Bakker Assistant professor, Biosystematics Group Wageningen University Other members Prof. Dr M. Dicke, Wageningen University Prof. Dr S.B.J. Menken, University of Amsterdam Prof. Dr B.J. Zwaan, Wageningen University Dr N. Wahlberg, University of Turku, Finland This research was conducted under the auspices of the Graduate School of Production Ecology & Resource Conservation Evolution of associations between Cymothoe butterflies and their Rinorea host plants in tropical Africa Robin van Velzen Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 11 December 2013 at 11 a.m. in the Aula. Robin van Velzen Evolution of associations between Cymothoe butterflies and their Rinorea host plants in tropical Africa 248 pages PhD thesis, Wageningen University, Wageningen, NL (2013) With references, with summaries in Dutch and English ISBN 978-94-6173-778-6 To Marleen for her love and support To Jean-Louis Amiet for laying the foundation for my research on Cymothoe and Rinorea Contents 1 General Introduction 11 1.1 Evolution of insect{host plant associations . 12 1.2 Cymothoe ................................. 14 1.3 Rinorea .................................. 15 1.4 Cymothoe-Rinorea host plant associations . 16 1.5 Methodologies & Analytics used in this thesis . 17 1.5.1 Field work . 17 1.5.2 Species identification and DNA barcoding . 18 1.5.3 Phylogenetics and divergence time estimation . 19 1.5.4 Statistics . 20 1.5.5 Phytochemistry . 22 1.6 Research Questions . 24 1.7 Outline of this thesis . 25 2 Systematics of Cymothoe butterflies 27 2.1 Introduction . 29 2.1.1 Theoretical framework . 31 2.2 Materials and methods . 33 2.2.1 Taxon sampling . 33 2.2.2 Molecular methods . 33 2.2.3 An integrative taxonomic decision pipeline . 34 2.3 Results . 38 2.3.1 Sequencing and quality control . 38 2.3.2 Confirmation of current classification . 40 2.3.3 Delimitation of candidate species . 45 2.3.4 Corroboration of candidate species and revision of taxonomic classification . 48 2.3.5 Candidates requiring more data . 51 7 Contents 2.3.6 Rejected candidates . 58 2.3.7 Additional taxonomic findings . 63 2.3.8 Identification of immature specimens . 66 2.4 Discussion . 67 2.4.1 Integrative taxonomic decision pipeline . 67 2.4.2 Niger Delta endemism . 68 2.4.3 Technical issues . 68 2.4.4 Incomplete lineage sorting and introgression . 69 2.4.5 Future directions . 71 2.5 Conclusions . 72 3 DNA barcoding of recently diverged species 75 3.1 Background . 77 3.2 Materials and methods . 82 3.2.1 Data simulation . 82 3.2.2 Empirical data sets . 83 3.2.3 Species `barcode gap' and monophyly . 84 3.2.4 Method performance . 84 3.2.5 Statistical tests . 86 3.3 Results . 86 3.3.1 Data simulation . 86 3.3.2 Species `barcode gap' and monophyly . 86 3.3.3 Method performance . 87 3.3.4 Empirical data sets . 92 3.4 Discussion . 93 3.4.1 Method performance . 93 3.5 Conclusions . 97 4 Species diversification in Cymothoe tropical forest butterflies 99 4.1 Background . 101 4.2 Materials and methods . 103 4.2.1 Taxon sampling . 103 4.2.2 Molecular methods . 103 4.2.3 Phylogenetic inference . 108 4.2.4 Timing of divergences . 109 4.2.5 Diversification analyses . 110 8 Contents 4.3 Results . 113 4.3.1 Phylogenetic inference . 113 4.3.2 Timing of divergences . 116 4.3.3 Diversification analyses . 116 4.4 Discussion . 119 4.4.1 Diversification and host association . 120 4.4.2 Diversification and climate change . 122 4.4.3 Speciation versus extinction . 123 4.4.4 Diversification rate versus phylogenetic resolution . 123 4.5 Conclusions . 124 5 Phylogenetics and historical biogeography of Rinorea 125 5.1 Background . 127 5.2 Materials & methods . 129 5.2.1 Phylogenetic analysis . 138 5.2.2 Divergence time estimation . 139 5.3 Results . 141 5.3.1 Phylogenetic results . 142 5.3.2 Divergence timing results . 142 5.4 Discussion . 143 5.4.1 Trans-Atlantic dispersal . 149 5.4.2 Dispersal to Asia . 151 5.4.3 Multiple independent colonizations of Madagascar . 151 5.5 Conclusions . 152 6 Asynchronous divergence of Cymothoe forest butterflies and their Rinorea host plants in tropical Africa 155 6.1 Background . 157 6.1.1 Host shifts . 158 6.1.2 Synchronicity . 159 6.1.3 Species as fundamental units . 160 6.1.4 Cymothoe butterflies and their Rinorea host plants . 160 6.1.5 Objectives . 162 6.2 Materials and methods . 162 6.2.1 Sampling . 162 6.2.2 Phylogeny estimation and divergence timing . 164 9 Contents 6.2.3 Cophylogeny estimation . 167 6.3 Results . 172 6.3.1 Host plant observations . 172 6.3.2 Phylogeny estimation and distribution of associations . 173 6.3.3 Comparative dating . 176 6.3.4 Comparison of pairwise phylogenetic distances . 176 6.3.5 Cophylogenetic analyses . 177 6.4 Discussion . 178 6.4.1 An evolutionary scenario for Cymothoe host plant associations . 181 6.5 Conclusion . 184 7 General discussion 185 7.1 Summarizing results . 187 7.2 A chronology of reconstructed historical events in Cymothoe and Rinorea ..................................190 7.3 Unruliness of rapid radiations . 192 7.4 The end of cospeciation theory? . 194 7.5 Chemical ecology . 196 7.6 Wolbachia and insect speciation . 198 7.7 Herbivorous insects and food security . 198 7.8 A plea for integration of taxonomic data . 200 References 203 Summary 233 Samenvatting 237 Acknowledgements 241 Publications 244 Curriculum Vitae 245 Education statement 246 10 Chapter1 General Introduction 11 1.1. Evolution of insect{host plant associations 1.1 Evolution of insect{host plant associations 1 Insects are by far the most diverse group of multicellular organisms on earth (Øde- gaard 2000, Price 2002). Most insect species are herbivores and plants constitute the vast majority of terrestrial biomass. Understanding the evolution of interactions between herbivorous insects and their host plants is therefore crucial to comprehend- ing global patterns in terrestrial biodiversity (Mitter, Farrell et al. 1988, Farrell and Mitter 1998, Price 2002, Lewinsohn and Roslin 2008, Futuyma and Agrawal 2009, Novotny, Miller et al. 2010). One important observation is that herbivorous insects are generally highly host specific (Futuyma and Moreno 1988). They may restrict feeding to particular clades, species or plant tissues (Weiblen, Webb et al. 2006). This is probably due to the diversity of mechanisms with which plants defend themselves against herbivore attacks (Coley and Barone 1996). In the context of this wide variety of chemical defences, specialization is one way to overcome at least some of these barriers. Phylogenetically, one therefore might expect generalist ! specialist transitions to be more common and specialist clades to be more species rich compared with their non-herbivore sister groups. This has indeed be found in various insect clades (Kelley and Farrell 1998, Janz, Nylin et al. 2006). In addition, host plant-specific toxins may be sequestered for the defence of the insect, reducing mortality from natural enemies (Nishida 2002). Most phylogenetic studies show that related insect species tend to feed on related plant species (Mitter, Farrell et al. 1991, Janz and Nylin 1998, Ronquist and Liljeblad 2001, Braby and Trueman 2006, Lopez-Vaamonde, Wikstr¨om et al. 2006, Wilson, Forister et al. 2012). This pattern can be explained by two alternative processes. First, in accordance with Fahrenholz' rule that parasites follow the speciation events of their hosts (Fahrenholz 1913), if the association between two species is very close, they may speciate in parallel. Such concomitant occurrence of speciation in hosts and their parasites is referred to as `cospeciation' (Page 2003), the associations between pocket gophers and their chewing lice being a well-known example (Hafner and Nadler 1988). Highly specialized insect herbivores can be regarded as parasites and may therefore also speciate in parallel with their host plants (Farrell and Mitter 1990, Farrell and Mitter 1998). The assumption is that when host plant populations become isolated, so will the associated specialist insect herbivores (Janz 2011). A cospeciation process therefore predicts that the divergence times of the insects and associated herbivores are synchronous. I note that this process is sometimes regarded as a special case of `coevolution'. However, although this term has been used to label a wide variety of different processes (Janzen 1980), in a narrow sense it usually assumes reciprocal selection pressures and.