THE PHYLOGEOGRAPHY AND MOLECULAR EVOLUTION OF

ITHOMIINE BUTTERFLIES

byALAINE JEAN WHINNETT

A thesis submitted for the degree ofDoctor of Philosophy of the University of London

September 2005

Department of BiologyUniversity College London

4 Stephenson WayLondon NW1 2HE

ABSTRACT

This thesis uses molecular techniques to investigate aspects of the evolution of

ithomiine butterflies (Lepidoptera: Nymphalidae: Ithomiinae).

1) This thesis takes a comparative phylogeographic approach to investigate the

diversification of ithomiines collected across an Amazonian suture zone in N. E. Peru.

I recovered a high variability of mitochondrial DNA (mtDNA) and triosephosphate

isomerase (Tpi) sequence divergences which, i) suggested that diversification of the

ithomiines studied here was inconsistent with predictions of the Pleistocene forest

refugia theory, one of the leading hypotheses used to explain the record richness of

Amazonian biodiversity, and ii) challenged the categorisation of taxa based purely on

DNA divergence thresholds, as proposed by DNA barcoding.

2) This thesis also investigates the contribution of ecological adaptation verses

allopatric differentiation in explaining the distribution patterns of 4 subspecies

belonging to the ithomiine species Hyposcada anchiala. The mtDNA sequence data

revealed that the most recent radiations were consistent with allopatric divergence

during the Pleistocene.

3) In addition, this thesis generates gene genealogies for the ithomiine tribe Oleriini,

based on regions of mtDNA, wingless and elongation factor 1-. In nearly all cases

individuals were clustered by species into the four recognised genera, however, the

relationships between the genera remains undetermined. This data contributes to a

complete Oleriini phylogeny, which will be used to examine aspects of the evolution

of this tribe.

4) Finally, this thesis contributes to the development of nuclear loci for PCR in

Lepidoptera. Tpi had previously been used for phylogenetics, but here was further

developed so that a longer region could be amplified. Primers were also developed for

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a novel region, Tektin, which is shown to have phylogenetic utility at the genus, tribe

and subfamily levels.

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ACKNOWLEDGEMENTS

I would like to extend thanks to the many people, in many countries, who so

generously contributed to the work presented in this thesis.

Special mention goes to my enthusiastic supervisor, Jim Mallet. My PhD has been an

amazing experience and I thank Jim wholeheartedly, not only for his tremendous

academic support, but also for giving me so many wonderful opportunities. Not many

PhDs involve a helicopter ride with the Ecuadorian Army into the rainforest, or

travelling through Mongolia to get to a conference in Siberia!

Similar, profound gratitude goes to Andy Brower, who has been a truly dedicated

mentor. I am particularly indebted to Andy for his constant faith in my lab work, and

for his support when so generously hosting me in Oregon. I have very fond memories

of my time there.

I am also hugely appreciative to Keith Willmott, especially for sharing his taxonomic

expertise so willingly, and for being so dedicated to his role as my secondary

supervisor.

Special mention goes to Marga Beltrán, Russ Naisbit, Marie Zimmermann, Vanessa

Bull, Ming-Min Lee, Ian Evans, Jacques Gianino, Chris Jiggins, Gerardo Lamas and

Fraser Simpson, for going far beyond the call of duty. To Nick Mundy, for

encouraging me to embark on the molecular biology path, and for providing me with a

fantastic lab training. And to Vernon Reynolds, Peter Davies and Mr Sumner, for

nurturing my enthusiasm for biology.

Finally, but by no means least, thanks go to mum, dad and Steve for almost

unbelievable support. They are the most important people in my world and I dedicate

this thesis to them.

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TABLE OF CONTENTS

Title Page 1

Abstract 2

Acknowledgements 3

Table of Contents 4

List of Figures 10

List of Tables 13

Declaration 16

Chapter One. Introduction 18

Ithomiine butterflies 18

Ithomiine butterflies and mimicry 19

Ithomiine butterflies and pyrrolizidine alkaloids 22

Ithomiine butterflies and Neotropical diversification theories 23

Vicariance hypotheses 24

Ecological or gradient hypotheses 26

Scientific rationale and thesis outline 27

References 30

Chapter Two. Strikingly variable divergence times inferred across an Amazonian

butterfly ‘suture zone’ 38

Abstract 38

Introduction 39

Materials and methods 42

Results 45

Discussion 50

References 55

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Chapter Three. Divergences at triosephosphate isomerase (Tpi) and mitochondrial

loci in Amazonian butterflies: further insights into Neotropical diversification and

comments on the molecular evolution of Tpi 60

Abstract 60

Introduction 62

Materials and methods 64

Sampling method 64

Primer development and PCR protocols 64

Sequence analysis 67

Results 69

Sequence results and phylogenetic analysis 69

Tpi exon and Tpi intron 69

Pairwise divergences and dN/dS ratios 71

Discussion 75

Phylogenetic relationships 75

MtDNA and Tpi divergences 75

Hybridisation 76

The molecular evolution of Tpi 77

References 80

Chapter Four. The Phylogenetic Utility of Tektin, a Novel Region for Inferring

Systematic Relationships Amongst Lepidoptera 84

Abstract 84

Introduction 85

Materials and methods 91

DNA extraction 91

Primer development, PCR and sequencing. 91

Taxonomic sampling 92

Data analyses 93

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Results 95

Discussion 100

References 103

Chapter Five. Mitochondrial DNA provides an insight into the mechanisms driving

diversification in the ithomiine butterfly Hyposcada anchiala (Lepidoptera:

Nymphalidae, Ithomiinae). 115

Abstract 115

Introduction 116

Materials and methods 120

Results 125

Discussion 128

References 132

Chapter Six. A molecular phylogeny of the Neotropical butterfly tribe Oleriini

(Lepidoptera: Nymphalidae: Ithomiinae) 135

Abstract 135

Introduction 136

Materials and methods 138

Taxonomic sampling and DNA extraction 138

Primer development, PCR and sequencing 138

Data analyses 139

Results and Discussion 152

Molecular results 152

Generic level phylogenetics 152

Species level phylogenetics 154

Phylogenetic construction method 158

Conclusion 160

References 161

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Chapter Seven. Conclusions. 170

Phylogeography 170

1) The use of nuclear DNA 170

2) An increase in the integration of molecular data with information of

profound relevance to phylogeographic patterns. 171

3) The application of ‘comparative phylogeography on a regional

scale, using multiple co-distributed species’ 172

Finally 172

References 174

Appendix One. Molecular systematics of the butterfly genus Ithomia (Lepidotera:

Ithomiinae): a composite phylogenetic hypothesis based on seven genes. 175

Abstract 176

Introduction 176

Materials and methods 177

Loci analysed 177

Samples, DNA extraction, amplification, and sequencing 177

Molecular characterization and phylogenetic analysis 180

Molecular clock analysis 180

Results 180

MtDNA 180

Nuclear DNA 180

Ef1 181

Tektin 181

Wingless 185

Rpl5 intron 185

Relative rates of molecular evolution 185

Monophyly of Ithomia 185

Comparison of the mtDNA and nuclear DNA trees 185

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The iphianassa/salapia relationship 189

Combined evidence phylogeny 189

Discussion 190

Topological discordance 190

Taxonomic implications 193

Acknowledgements 193

References 194

Appendix Two. Phylogenetic relationships among the Ithomiini (Lepidoptera:

Nymphalidae) inferred from one mitochondrial and two nuclear gene regions 196

Abstract 196

Introduction 197

Materials and methods 204

Taxon sampling 204

DNA extraction, PCR and sequencing 204

Phylogenetic Analysis 211

Results 213

Discussion 219

Implications of this analysis for the phylogeny and classification of

Ithomiinae 219

Evolution of larval host plant affinities 223

References 224

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LIST OF FIGURES

Chapter Two

Figure 1. Map of the study area 41

Figure 2. Phylogenetic hypothesis based on mitochondrial nucleotide data 46

Figure 3. Histogram of fitted GTR +I +G distances across nodes between

subspecies and species in the rate-smoothed tree 47

Chapter Three

Figure 1. Phylogenetic hypothesis based on Tpi exon data 70

Figure 2. Within genera JC corrected Tpi pairwise distances, plotted as a

function of corresponding JC corrected coding mitochondrial pairwise

distances 74

Figure 3. dS values for Tpi exon and coding mitochondrial regions, plotted as a

function of corresponding JC corrected Tpi intron pairwise distances 74

Chapter Four

Figure 1. Base compositions of the 807 bp aligned Tektin region, averaged over

all specimens 96

Figure 2. Proportion of transitions or transversions against proportion total

pairwise divergence for; Tektin, Ef1, wg and mitochondrial data 97

Figure 3. Phylogenetic hypothesis based on Tektin nucleotide data 107

Figure 4. Phylogenetic hypothesis based on wg nucleotide data 108

Figure 5. Phylogenetic hypothesis based on Ef1 nucleotide data 109

Figure 6. Phylogenetic hypothesis based on mitochondrial nucleotide data

110

Figure 7. Total evidence phylogenetic hypothesis, inferred using Bayesian

methods 111

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Figure 8. Total evidence phylogenetic hypothesis, inferred with maximum

parsimony 112

Figure 9. Phylogenetic hypothesis based on Tektin amino acid data 113

Figure 10. Most parsimonious tree inferred from combined morphological and

ecological data 114

Chapter Five

Figure 1. Map showing the butterfly collection localities for H. a. mendax, H.

a. fallax, H. a. interrupta, H. a richardsi and H. a. ecuadorina 119

Figure 2. Phylogenetic hypothesis inferred with maximum parsimony 127

Figure 3. Phylogenetic hypothesis inferred with Bayesian methods 127

Chapter Six

Figure 1. Phylogenetic hypothesis based on ‘complete’ data, inferred with

maximum parsimony 163

Figure 2. Phylogenetic hypothesis based on wg data 164

Figure 3. Phylogenetic hypothesis based on Ef1 data 165

Figure 4. Phylogenetic hypothesis based on mitochondrial data 166

Figure 5. Phylogenetic hypothesis based on ‘complete’ data, inferred with

neighbour joining 167

Figure 6. Phylogenetic hypothesis based on ‘total’ data 168

Figure 7. Phylogenetic hypothesis based on ‘complete’ data, inferred with

Bayesian methods 169

Appendix One

Figure 1. Phylogenetic hypothesis for Ithomia based on mtDNA 182

Figure 2. Phylogenetic hypothesis for Ithomia based on the Ef1 gene 183

Figure 3. Phylogenetic hypothesis for Ithomia based on the tektin gene 184

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Figure 4. Phylogenetic hypothesis for Ithomia based on the wg gene 186

Figure 5. Phylogenetic hypothesis for Ithomia based on RpL5 187

Figure 6. Plot of relative divergence rates of different Ithomia genes 188

Figure 7. Phylogenetic hypothesis for Ithomia based on sequences of the Co1,

leucine tRNA, Co2, Ef1, tektin, and wg genes 191

Figure 8. Maximum parsimony tree for Ithomia based on sequences of the Co1,

leucine tRNA, Co2, Ef1, tektin, and wg genes 192

Appendix Two

Figure 1. Phylogenetic hypotheses for Ithomiini redrawn from (A) Brown &

Freitas (1994) and (B) Motta (2003) 203

Figure 2. Single most parsimonious tree 215

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LIST OF TABLES

Chapter Two

Table 1. Study site details 42

Table 2. Table showing fitted between-species pairwise % divergences within

genera, assuming a GTR+I+G model 48

Table 3. Table showing fitted between-subspecies pairwise % divergences

within species, assuming a GTR+I+G model 49

Chapter Three

Table 1. Primer details 66

Table 2. PCR parameters 66

Table 3. Sequenced specimens 68

Table 4. Nucleotide compositions 71

Table 5. Mean Jukes Cantor corrected % pairwise divergences 73

Table 6. Mean within genera JC corrected % pairwise divergences and dN:dS

statistics

Chapter Four

Table 1. Specimen information and Genbank Accession codes 88

Table 2. Primers used to amplify Tektin 92

Table 3. The number of Bayesian inferred branches with strong Bayesian,

Parsimony, or Bayesian and Parsimony support 97

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Chapter Five

Table 1. Specimen information and Genbank accession numbers 122

Table 2. Absolute pairwise distances for all H. anchiala individuals, mean

HKY85 subspecies pairwise distances, and mean estimated time since

divergence of subspecies. 123

Chapter Six

Table 1. Specimen information and Genbank accession codes 140

Table 2. Primer details 151

Table 3. Oleria species groups based on morphological characters 155

Appendix One

Table 1. Specimens of Ithomia and related genera included in the present study

178

Table 2. Amplification primers and conditions used to amplify the Ithomia

genes used in this study 179

Table 3. Parameter estimates of sequence evolution for the Ithomia genes used

in this study 181

Table 4. Results of SH tests comparing the ‘best fit’ Ithomia tree topologies

inferred from different gene regions 188

Table 5. SH tests of specific topological hypotheses for Ithomia species

inferred from different gene regions 188

Table 6. Partitioned Bremer support analysis for the combined data set 189

Appendix Two

Table 1. Representative classifications of ithomiine genera 199

Table 2. Ithomiini and outgroup taxa examined in this study 205

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Table 3. New PCR and sequencing primers employed in this study 210

Table 4. Parameters of the data for individual gene regions and the entire

matrix 213

Table 5. Support indices for the branches in Figure 2 216

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DECLARATION

The work presented in Chapters 1, 3, 6 and 7 is entirely my own.

I am the principal author of Chapter 2. It is co-authored with Marie Zimmermann,

Keith Willmott, Nimiadina Herrera, Ricardo Mallarino, Fraser Simpson, Mathieu

Joron, Gerardo Lamas and Jim Mallet, who contributed to discussion. I performed all

of the lab work, except for individuals of the genera Melinaea and Hypothryis which

were sequenced by Marie Zimmermann. The data contained in the paper were

compiled by myself and I performed all initial analyses.

I am the first author of Chapter 4, which is co-authored with Andrew Brower, Ming-

Min Lee, Keith Willmott and Jim Mallet. I carried out all of the Tektin primer

development, generated all of the Tektin sequences and performed all of the analyses.

Much of the comparative data was already available from other projects, as detailed in

the chapter.

I am the first author of Chapter 5, which is co-authored with Keith Willmott, Andrew

Brower, Fraser Simpson, Marie Zimmermann, Gerardo Lamas and Jim Mallet. I

performed all of the lab work in this chapter, except for one individual which was

sequenced by Fraser Simpson.

Appendix one was written by Ricardo Mallarino, who also performed the lab work. I

helped by providing the Tektin primers that I had designed (prior to their publication),

by providing specimens, and contributing to discussion.

Appendix two was written by Andrew Brower, who also led this research program. I

contributed by providing specimens and generating nuclear sequence data.

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In addition, the co-authors of chapters 2, 4 and 5, and my supervisor Jim Mallet,

contributed by proof reading the text. Keith Willmott and Gerardo Lamas helped with

butterfly identification, and a number of collaborators helped by supplying specimens.

Signed:

Alaine Whinnett Professor James MalletCandidate Supervisor

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