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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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