THE PHYLOGEOGRAPHY AND
MOLECULAR EVOLUTION OF
ITHOMIINE BUTTERFLIES
by
ALAINE JEAN WHINNETT
A thesis submitted for the degree of
Doctor of Philosophy of the University of London
September 2005
Department of Biology
University College London
4 Stephenson Way
London 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
2
a novel region, Tektin, which is shown to have phylogenetic utility at the genus, tribe
and subfamily levels.
3
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.
4
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
5
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
6
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
7
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
8
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
9
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
10
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
11
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
12
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
13
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
14
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
15
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, MingMin 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
Candidate
Professor James Mallet
Supervisor
17