Mitochondrial Genomes and Their Utility For The Recovery of Phylo

University of Wollongong
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2014
Mitochondrial genomes and their utility for the

recovery of phylogeny in the Hymenoptera
Meng Mao
Recommended Citation
Mao, Meng, Mitochondrial genomes and their utility for the recovery of phylogeny in the Hymenoptera, Doctor of Philosophy thesis,
School of Biological Sciences, University of Wollongong, 2014. http://ro.uow.edu.au/theses/4206
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Mitochondrial genomes and their utility for the recovery of
phylogeny in the Hymenoptera
A thesis submitted in fulfillment of the requirements for the award of the degree
Doctor of Philosophy
from
by
Meng Mao
(BSc, MAg)
School of Biological Sciences
2014
Thesis Certification
I, Meng Mao, declare that this thesis, submitted in fulfillment of the requirements for the award of
Doctor of Philosophy, in the School of Biological Sciences, University of Wollongong, is wholly my
own work unless otherwise referenced or acknowledged. The document has not been submitted for
qualifications at any other academic institution.
Meng Mao
18th August 2014
ii
Table of Contents
Thesis Certification ............................................................................................................................ ii
List of Tables .................................................................................................................................... viii
List of Figures .................................................................................................................................... ix
List of Abbreviations ......................................................................................................................... xi
Abstract ............................................................................................................................................ xiv
Acknowledgements.......................................................................................................................... xvi
List of publications ......................................................................................................................... xvii
Statement of Contribution ............................................................................................................ xviii
CHAPTER 1: General Introduction................................................................................................. 1
1.1 Preamble ............................................................................................................................. 1

1.2 The mitochondrial genome and phylogenetics................................................................. 2
1.3 Mitochondrial gene rearrangements ................................................................................ 4
1.3.1 Mechanisms of gene rearrangement .................................................................................... 5
1.3.2 Gene rearrangements as phylogenetic markers ................................................................... 8
1.4 Higher-level phylogenetic relationships within the Hymenoptera ................................. 9
1.4.1 Suborder Symphyta ............................................................................................................. 9
1.4.2 Suborder Apocrita ............................................................................................................. 10
1.5 Conclusions ....................................................................................................................... 13

1.6 Aims ................................................................................................................................... 14
CHAPTER 2: The first mitochondrial genome for the wasp superfamily Platygastroidea: the
egg parasitoid Trissolcus basalis ...................................................................................................... 17
2.1 Introduction ...................................................................................................................... 17

2.2 Materials and methods..................................................................................................... 18
DNA extraction, PCR amplification, and sequencing ................................................................ 19
Sequence analyses ...................................................................................................................... 20
Sequence alignment ................................................................................................................... 21
Phylogenetic analysis ................................................................................................................. 22
2.3 Results and discussion...................................................................................................... 23
Genome size and gene content ................................................................................................... 23

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Nucleotide content ..................................................................................................................... 25
Codon usage and amino acid composition ................................................................................. 25
Translation initiation and termination codons ............................................................................ 26
Transfer RNA genes ................................................................................................................... 27
Ribosomal RNA genes ............................................................................................................... 27
Non-coding regions .................................................................................................................... 28
Genome organization ................................................................................................................. 29
Phylogenetic analysis and considerations .................................................................................. 31
2.4 Conclusions ....................................................................................................................... 34
CHAPTER 3: Complete mitochondrial genomes of Ceratobaeus sp. and Idris sp. (Hymenoptera:
Scelionidae): shared gene rearrangements as potential phylogenetic markers at the tribal level
............................................................................................................................................................ 35
3.1 Introduction ...................................................................................................................... 35

DNA extraction .......................................................................................................................... 36
Amplification, sequencing, and annotation of entire mt genomes ............................................. 37
Sequence alignment ................................................................................................................... 38
Genetic divergence and phylogenetic analysis ........................................................................... 39
3.3 Results and discussion...................................................................................................... 41
Genome size and gene content ................................................................................................... 41
Protein-coding genes .................................................................................................................. 42
Transfer RNA and Ribosomal RNA ........................................................................................... 43
Noncoding regions ..................................................................................................................... 44
Genome organizations ................................................................................................................ 46
Ka/Ks ratios among the three Scelionidae taxa .......................................................................... 48
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CHAPTER 4: Coexistence of minicircular and a highly rearranged mtDNA molecule suggests

that recombination shapes mitochondrial genome organization ................................................. 52
4.1 Introduction ...................................................................................................................... 52

DNA extraction .......................................................................................................................... 55
Amplification, sequencing, and annotation of the entire mt genome ......................................... 55
Amplification and sequencing of minicircles and the maxicircle .............................................. 56
4.3 Results ............................................................................................................................... 57
The mt genome of Conostigmus sp. ........................................................................................... 57
Initial characterization of the putative maxicircle and minicircles of Conostigmus sp. ............. 58
Are the minicircles amplification artifacts? ............................................................................... 62
Elimination of the possibility of numts ...................................................................................... 64
4.4 Discussion .......................................................................................................................... 65
Importance of control reactions.................................................................................................. 65
Possible explanations for low copy number of the maxicircle ................................................... 66
Comparison with minicircles from other Metazoa ..................................................................... 66
Possible mechanism yielding the minicircle and maxicircle ...................................................... 68
Direct link between intra-mitochondrial recombination and mt gene rearrangement ................ 69
CHAPTER 5: Evolutionary dynamics of the mitochondrial genome in the Evaniomorpha

(Hymenoptera) – a group with an intermediate rate of gene rearrangement............................. 71
5.1 Introduction ...................................................................................................................... 71

5.2 Materials and Methods .................................................................................................... 74
DNA extraction .......................................................................................................................... 74
Mt genome amplification, sequencing, and annotation .............................................................. 75
Nucleotide composition, gene rearrangement, and repeat analyses ........................................... 76
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Sequence alignment and phylogenetic analysis ......................................................................... 77
Rogue taxon identification ......................................................................................................... 78
5.3 Results and Discussion ..................................................................................................... 78
General features of mt genomes ................................................................................................. 78
Genome organizations ................................................................................................................ 80
The mt genome of Gasteruption sp. ........................................................................................... 83
The mt genomes of Megalyra sp. and O. pulchella ................................................................... 85
Evolutionary dynamics of evaniomorph mt genomes ................................................................ 87
The utility of gene rearrangements for assessing phylogeny in the Evaniomorpha ................... 89
Rates of gene rearrangement in the Evaniomorpha .................................................................... 90
CHAPTER 6: Higher-level phylogeny of the Hymenoptera inferred from mitochondrial

genomes ............................................................................................................................................. 97
6.1 Introduction ...................................................................................................................... 97

6.2 Materials and Methods .................................................................................................. 100
DNA extraction ........................................................................................................................ 100
Mt genome amplification, sequencing, annotation, and bioinformatic analysis ...................... 101
Sequence alignment and phylogenetic analysis ....................................................................... 102
6.3 Results and Discussion ................................................................................................... 103
General features of mt genomes ............................................................................................... 103
Genome organizations .............................................................................................................. 105
Phylogenetic analysis ............................................................................................................... 107
6.4 Conclusions ......................................................................................................................113
CHAPTER 7: General Conclusions...............................................................................................115
7.1 Main outcomes .................................................................................................................115

7.2 Recommendations and future research .........................................................................115
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7.2.1 Increasing taxon sampling of the Apocrita ................................................................115
7.2.2 Incorporating genome-based collection of markers ..................................................117
7.3 Conclusions ......................................................................................................................118
References ....................................................................................................................................... 120
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List of Tables
Table 1.1 Mt genomes (complete/near-complete) for the Hymenoptera available in GenBank
and unpublished in this study ..................................................................................................... 14
Table 2.1 List of taxa used in phylogenetic analyses. ................................................................ 22
Table 2.2 Locations and nucleotide sequence lengths of genes in the Trissolcus basalis mt
genome, the lengths of predicted amino acid sequences of protein-coding genes, and their
initiation and termination codons ............................................................................................... 24
Table 2.3 Nucleotide composition (%) of the Trissolcus basalis mt genome ............................ 25
Table 2.4 Codon usage for protein-coding genes of the Trissolcus basalis mt genome ............ 26
Table 3.1 Datasets used for phylogenetic analysis..................................................................... 39
Table 3.2 Estimation of Bayes Factors of different partition schemes. ..................................... 39
Table 3.3 Comparison of genetic divergences for all protein-coding genes among scelionds. . 48
Table 5.1 Species sequenced in this study, collection data and GeneBank accession numbers . 75
Table 5.2 The number of repeats (including direct and inverted repeats) within the A+T-rich
region and gene rearrangements identified for seven evaniomorph taxa ................................... 88
Table 5.3 Breakpoint numbers of the evaniomorph, hemipteroid and nematoceran mt genomes
relative to the ancestral mt genome organization. ...................................................................... 89
Table 6.1 Species sequenced in this study, collection data, and GeneBank accession numbers
.................................................................................................................................................. 101
Table 6.2 Characteristics of the three mitochondrial genomes sequenced in this study. ......... 104
Table 6.3 Summary of the major clades recovered by different dataset and analytical approach
.................................................................................................................................................. 107
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List of Figures
Figure 1.1 Map of the typical mitochondrial genome of animals ................................................ 2
Figure 1.2 Simple diagram illustrating the duplication/random loss model ................................ 6
Figure 1.3 The duplication/nonrandom loss model ..................................................................... 7
Figure 1.4 Model for the generation of mitochondrial gene inversions by recombination ......... 8
Figure 1.5 Major hymenopteran relationships proposed by Rasnitsyn (1988) .......................... 10
Figure 2.1 Mitochondrial genome organizations of four Proctotrupomorpha taxa ................... 30
Figure 2.2 Bayesian analysis of mitogenomic sequences based on the Muscle alignment dataset
.................................................................................................................................................... 31
Figure 3.1 Mitochondrial genome organization of three sceliond taxa, compared with the
ancestral pancrustacean mt genome organization ...................................................................... 42
Figure 3.2 Inferred secondary structures of tRNA genes .......................................................... 43
Figure 4.1 Organizations of the entire mt genome, maxicircle and minicircles that were initially
characterized by amplification ................................................................................................... 57
Figure 4.2 Mitochondrial genome organization of Conostigmus sp., compared with the
ancestral pancrustacean mt genome organization ...................................................................... 58
Figure 4.3 Size separations of amplicons on 1% agarose gels .................................................. 62
Figure 4.4 Model for the generation of minicircle and maxicircle mtDNA by recombination
during repair of DSBs. ............................................................................................................... 69
Figure 5.1 Simplified interpretation of the phylogenetic relationships among the major lineages
within the Apocrita from Rasnitsyn (1998) ................................................................................ 73
Figure 5.2 Structure of the A+T-rich region in the Ceraphron sp., Gasteruption sp., Megalyra
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sp. and Orthogonalys pulchella mt genomes ............................................................................. 81
Figure 5.3 Mitochondrial genome organization of two taxa from the Ceraphronoidea,
Ceraphron sp. (present study) and Conostigmus sp. (Mao et al. 2014) ..................................... 83
Figure 5.4 Mitochondrial genome organization of three taxa from the Evanioidea: Gasteruption
sp. (present study), Evania appendigaster (Wei et al., 2010b) and Pristaulacus compressus (Wei
et al., 2010b)............................................................................................................................... 85
Figure 5.5 Mitochondrial genome organization of Megalyra sp. (Megalyroidea) (present study)
and Orthogonalys pulchella (Trigonalyoidea) (present study) ................................................... 87
Figure 5.6 Bayesian analysis of the initial taxon dataset (without rogue taxon deletion) based
on mitogenomic sequences......................................................................................................... 93
Figure 5.7 Bayesian analysis of the reduced taxon dataset (with rogue taxon deletion) based on
mitogenomic sequences.............................................................................................................. 94
Figure 6.1 Mitochondrial genome organization of Ibalia leucospoides, Pelecinus polyturator,
and Monomachis antipodalis, compared with the ancestral pancrustacean/hymenopteran mt
genome organization ................................................................................................................ 105
Figure 6.2 Bayesian analysis of the Muscle alignment dataset ............................................... 109
x
List of Abbreviations
°C degrees Celsius
μl micro-litre
μm micro-metre
A adenine
a6 ATPase subunit 6
a8 ATPase subunit 8
BLAST Basic Local Alignment Search Tool
bp base pairs
C cytosine
cox1 cytochrome oxidase subunit I
cox2 cytochrome oxidase subunit II
cox3 cytochrome oxidase subunit III
cob cytochrome oxidase subunit b
DNA deoxyribonucleic acid
EDTA ethylendiaminetetraacetic acid
EST expressed sequenced tag
G guanine
GTR + G + I general time reversible model with rates invgamma
kb kilo base pair
Mb Mega base pairs
xi
MEGA Molecular Evolutionary Genetics Analysis
mg micro-gram
min minute
ml milli-litre
mM milli-molar
mt mitochondrial
nad1 NADH dehydrogenase subunit 1
nad4L NADH dehydrogenase subunit 4L
numts nuclear copies of mitochondrial DNA
ORF Open reading frame
PCR polymerase chain reaction
Poly T polythymidine
RNA ribonucleic acid
rRNA ribosomal RNA
rrnL gene for ribosomal RNA large-subunit
rrnS gene for ribosomal RNA small-subunit
s second
xii
T thymidine
TE Tris-EDTA
Tris tris (hydroxymethyl) aminomethane
tRNA transfer RNA
trnA transfer RNA gene for Alanine
trnC transfer RNA gene for cytosine
trnD transfer RNA gene for aspartic acid
trnE transfer RNA gene for glutamic acid
trnF transfer RNA gene for phenylalanine
trnH transfer RNA gene for histidine
trnK transfer RNA gene for lysine
trnL transfer RNA gene for leucine
trnM transfer RNA gene for metionine
trnN transfer RNA gene for asparagine
trnP transfer RNA gene for proline
trnR transfer RNA gene for arginine
trnS transfer RNA gene for serine
trnT transfer RNA gene for threonine
trnV transfer RNA gene for valine
trnW transfer RNA gene for triptophan
trnY transfer RNA gene for tyrosine
UOW University of Wollongong, Wollongong, NSW, Australia
xiii
Abstract
The Hymenoptera, comprising of sawflies, wasps, bees and ants, is the third largest order of insects.
It contains more than 155,000 described species, placed into two traditional suborders (Symphyta
and Apocrita). Many hymenopteran species play important roles in biological control, ecosystems
and production of commercial products. Unfortunately, higher-level relationships of the
Hymenoptera, particularly within the highly diverse parasitic lineages, remain unresolved in both
morphological and molecular studies.
The mitochondrial (mt) genome sequence is becoming a powerful tool for reconstructing
phylogenetic relationships. In addition, mt gene rearrangements also provide useful phylogenetic
information. However, deficient representation of a broad range of lineages restricts the evolutionary
utility of the mt genome in the Hymenoptera. For the four infraorders of the Apocrita, the Aculeata
and Ichneumonomorpha are reasonably sampled. By contrast, the Evaniomorpha and
Proctotrupomorpha are poorly represented. Therefore, this PhD research was focussed on increasing
our understanding of mt genomes of the Hymenoptera and their utility for the recovery of the
higher-level phylogeny by extending the taxonomic range of available mt genome sequences.
Eleven new mt genomes for representatives of four evaniomorph and four proctotrupomorph
superfamilies were determined. Several gene rearrangements were detected in each of the eleven mt
genomes when compared with the ancestral positions. In particular, protein-coding or rRNA gene
rearrangements were identified in six mt genomes. Mt genome organizations were also compared
within the Evaniomorpha and Proctotrupomorpha. The results showed that there were no shared,
derived gene rearrangements at the family level, indicating that the frequency of gene
xiv
rearrangements of these groups is too high to be useful for assessing relationships between families.
By contrast, comparison of mt genome organizations within the subfamily Scelioninae suggested
that gene rearrangements have much potential as phylogenetic markers at the subfamily level. The
large number of gene rearrangements identified in this study also facilitated the investigation of gene
rearrangement mechanisms. Direct and indirect evidence that supports the notion that recombination
is an important aspect of the gene rearrangement mechanism was identified. Most importantly, the
end products of recombination were detected (minicircles), in a megaspilid wasp Conostigmus sp.,
which provided support for the link between recombination and mt gene rearrangement. Furthermore,
a model of recombination which is important for our understanding of mtDNA evolution was
developed.
The first mt genome phylogeny of the Apocrita with a complete representation of superfamilies
(excluding Mymmaromatoidea) was reconstructed. The influence of inclusion/exclusion of 3rd codon
positions, alignment approaches, partition schemes and phylogenetic approaches on topology and
nodal support within the Hymenoptera was assessed. The results showed that the topologies were
sensitive to the variation of dataset and analytical approach. However, some robust and highly
supported relationships were recovered: the Ichneumonomorpha was monophyletic; the
Trigonalyoidea + Megalyroidea and the Diaprioidea + Chalcidoidea were consistently recovered; the
Cynipoidea was generally recovered as the sister group to the Diaprioidea + Chalcidoidea. In
addition, the monophyletic Aculeata and Proctotrupomorpha were recovered in some analyses.
Further phylogenetic analysis of the Hymenoptera will rely on increased sampling of both taxa and
sequence data.
xv
Acknowledgements
First of all, I would like to express my deepest appreciation to my principal supervisor, Professor
Mark Dowton. I have appreciated your guidance, great support, and encouragement throughout my
degree. Thank you for sharing your knowledge and laboratory techniques with me. I am also grateful
for your time and patience spent improving my writing skills in English as a second language.
Without your help, I would not have reached my dream of obtaining my PhD. I would also like to
thank my co-supervisor, Associate Professor James Wallman for his continual support and
encouragement. I feel so lucky and honoured to have had these two great supervisors. Gratitude also
goes to all the members of the Dowton lab, particularly Tracey Gibson, Angelique Hoolahan and
Kelly Meiklejohn, for their support, guidance and friendship.
I would like to express my sincere thanks to Andy Austin, Gary Gibson, Dave Smith, and John
Jennings for providing the biological specimens. A huge thank you also goes to Andy Austin,
Norman Johnson and Alejandro Valerio for providing the sequence data of Trissolcus basalis, and
Tracey Gibson for providing the sequence data of Ibalia leucospoides and Orthogonalys pulchella.
I would like to acknowledge the financial support from the Centre for Medical and Molecular
Bioscience, University of Wollongong.
Thanks to Xiushuai for his love, patience and support. It would not have been easy for me to
complete this thesis without his computing assistance. Finally, I would like to thank my wonderful
family, my mum, dad and younger sister. Thank you for your immense support and encouragement. I
appreciate everything you have done to help me to achieve my dream.

xvi
List of publications
1. Mao M, Valerio A, Austin AD, Dowton M, Johnson NF (2012) The first mitochondrial genome
for the wasp superfamily Platygastroidea: the egg parasitoid Trissolcus basalis. Genome
55:194-204.
2. Mao M, Austin AD, Johnson NF, Dowton M (2014) Coexistence of minicircular and a highly
rearranged mtDNA molecule suggests that recombination shapes mitochondrial genome
organization. Molecular Biology and Evolution 31:636-644.
3. Mao M, Dowton M (2014) Complete mitochondrial genomes of Ceratobaeus sp. and Idris sp.
(Hymenoptera: Scelionidae): shared gene rearrangements as potential phylogenetic markers at
the tribal level. Molecular Biology Reports doi: 10.1007/s11033-014-3522-x.
4. Mao M, Gibson T, Dowton M (2014) Evolutionary dynamics of the mitochondrial genome in
the Evaniomorpha (Hymenoptera) — a group with an intermediate rate of gene rearrangement.
Genome Biology and Evolution 6, 1862-1874.
5. Mao M, Gibson T, Dowton M Higher-level phylogeny of the Hymenoptera inferred from
mitochondrial genomes. Molecular Phylogenetics and Evolution (Under review).
xvii
xvi
ii
CHAPTER 1: General Introduction
1.1 Preamble
The Hymenoptera, comprising sawflies, ants, bees and wasps, is considered to be the third largest
order of insects, behind the Coleoptera and the Lepidoptera. There are more than 155,000 described
species in the Hymenoptera (Aguiar et al., 2013). The first fossil record of the Hymenoptera
appeared in the late Triassic, about 230 million years ago (Rasnitsyn, 2010). However, according to
recent divergence time estimation based on morphological and molecular data, the origin of the
Hymenoptera might date back to the Carboniferous, about 310 million years ago (Ronquist et al.,
2012b). Many hymenopterans play important ecological roles in the biological control of crop and
forest pests, the pollination of flowers and the production of commercial products, such as honey and
wax (LaSalle and Gauld, 1993).
Since most of this thesis has been published or submitted for publication, each chapter has been
written as a journal article. The chapters written as journal articles have been left in the general
submission format, except for minor changes for consistency. Consequently there is some repetition
between the General Introduction with the introduction of each chapter. To reduce the level of
repetition, the General Introduction chapter is not written as a comprehensive literature review, but
as a general introduction to the topics contained within the thesis. The literature is more intensely
reviewed in the introduction to each thesis chapter. Similarly, the General Conclusions chapter is not
written as a comprehensive discussion of the thesis chapters, as this would duplicate much of the
content in each chapter. Instead, the General Conclusions chapter outlines the main areas of future
research that the results of the thesis suggest. There is also some repetition among chapters of the
1
methodology used. The title page of each chapter details publication information. All references have
been compiled within a single list at the end of the thesis.
1.2 The mitochondrial genome and phylogenetics
The mitochondrion is an essential organelle present in most eukaryotic cells. It plays a central role in
energy metabolism, apoptosis, disease and aging (Boore, 1999). The mitochondrion contains its own
independent genome, known as the mitochondrial (mt) genome. In animals, the mt genome is a
double-stranded circular molecule (~16 kb in size), and usually consists of two ribosomal RNA
(rRNA) genes, 22 transfer RNA (tRNA) genes, along with 13 protein coding genes which code for
subunits of the enzymes required in electron transport or ATP synthesis (Moritz et al., 1987). There
is also generally a single large non-coding region (the A+T-rich region or control region) which is
known to contain regulatory elements for replication and transcription (Zhang and Hewitt 1997). The
37 genes distribute asymmetrically between the two strands, allowing the identification of a major (J)
and a minor (N) strand according to the number of encoded genes (Gissi et al., 2008) (Fig. 1.1).
Figure 1.1 Map of the typical mitochondrial genome of animals. The gene content on the major and minor DNA
strands are indicated, with the gene labels either external or internal to the molecule, respectively. tRNA genes are
indicated by single-letter amino acid codes, L1, L2, S1 and S2 denote trnLCUN, trnLUUR, trnSAGN and trnSUCN,
respectively. Adapted from Cook (2005).
2
Several features promote the mt genome as a good model for evolutionary genomic studies: (1) it is
easy to amplify due to the relatively small size and high copy number in the cell; (2) it is maternally
inherited in most species (Birky, 2001); (3) the nucleotide substitution rate of mtDNA is much higher
when compared with nuclear DNA (Lin and Danforth, 2004; Oliveira et al., 2008); (4) it lacks
recombination, so the whole molecule can be assumed to have the same genealogical history (Moritz
et al., 1987; Rokas et al., 2003). (5) In addition to the sequence data, some other mt genome
characters, such as gene rearrangements (Boore and Brown, 1998) and tRNA secondary structure
(Chen et al., 2014) also provide useful phylogenetic information.
However, the utility of mtDNA as a molecular marker is not consistently supported. Some
researchers have expressed their concerns about the usefulness of mtDNA. For example, mtDNA has
been suggested to be less informative than nuclear DNA to resolve deep relationships due to AT bias
and saturation (Burger et al., 2003). Furthermore, Galtier et al. (2009) argued that mtDNA is not
always clonal, that it does not evolve in a neutral manner and that it has a highly variable mutation
rate among lineages. These evolutionary properties of mtDNA might mislead phylogenetic
reconstructions (Galtier et al., 2009). Nevertheless, with the development of appropriate analytical
strategies and the rapid accumulation of sequenced mt genomes, most of these concerns can be
resolved. One such successful example of using mt genomes from a large taxon sampling for
phylogenetic reconstruction was the study of deuterostome phylogeny (Perseke et al., 2013). In this
study, more than 300 complete mt genomes were employed to deduce internal relationships within
the Deuterostomia. The results robustly recovered three chordata subgroups, Echinodermata and
their five subgroups, Enteropneusta and Ambulacraria. Similarly, a more recent phylogenetic study
of higher-level relationships within the Lepidoptera based on 106 mt genomes recovered topologies
3
that were highly congruent with nuclear and/or morphological studies (Timmermans et al., 2014). In
addition, several strategies have been developed to increase the performance of mitochondrial
phylogenetic analyses. For example, exclusion of 3rd codon positions or recoding them as purines
and pyrimidines (RY-coding) in the analysis can reduce the effect of saturation, and therefore recover
a more accurate topology (Cameron et al., 2012; Dowton et al., 2009a; Harrison et al., 2004).
Furthermore, appropriate data partitioning schemes and substitution models that account for
heterogeneity in evolutionary processes among sites are important to avoid artifactual relationships.
Recently, a program (named PartitionFinder) for simultaneously choosing partitioning schemes and
substitution models has been developed (Lanfear et al., 2012). This program can reduce systematic
error for large dataset as it allows optimal partitioning schemes to be efficiently and accurately
selected from predefined character sets (Lemmon and Lemmon, 2013). An example of using this
type of analytical strategy to improve the mitochondrial phylogeny was recently reported by Gibson
et al. (Gibson et al., 2005). In this study, Gibson et al. developed a customized three-state DNA
substitution model to reduce base composition bias in mammalian mt genomes. Phylogenetic trees
obtained using the new model showed increased congruence with studies of large sets of nuclear
genes. However, the performance of any particular analytical strategy can vary among different
lineages. For example, exclusion of 3rd codon positions has been found to improve the accuracy of
termite phylogenetic reconstructions (Cameron et al., 2012), while it did not appear to have any
effect on the phylogeny of the Orthoptera (Fenn et al., 2008). Therefore, extensive tests of the
effectiveness of different analytical strategies is crucial for accuracy when constructing a
mitochondrial phylogeny (Cameron, 2014).
1.3 Mitochondrial gene rearrangements

4
Mitochondrial gene rearrangements were generally considered to be rare evolutionary events in
animals (Boore, 1999). However, with more mt genomes from different groups available, the
diversity of mt genome organization has been shown to be much higher than expected. For example,
in insects, 13 orders have been found to possess gene rearrangements and the rate of gene
rearrangements are extremely high in the Hymenoptera (Dowton and Austin, 1999) and the
hemipteroid orders (Shao et al., 2001). The increasing number of gene rearrangements found in
different groups facilitates our understanding of molecular evolutionary processes and mechanisms
of gene rearrangement.
Gene rearrangements can be classified into four types: long-range translocation (across
protein-coding or rRNA genes), local inversion, remote inversion and local translocation (within a
tRNA gene cluster) (Dowton et al., 2003). Long-range translocation and inversion are rare in
vertebrates (but see Amer and Kumazawa, 2007; Inoue et al., 2001), while they are more common in
invertebrates (Dowton et al., 2003).
1.3.1 Mechanisms of gene rearrangement
Considerable effort has been devoted to explore the mechanisms of gene rearrangement. To date,
three main models have been proposed: the duplication/random loss model (Boore, 2000; Moritz et
al., 1987), the duplication/nonrandom loss model (Lavrov et al., 2002) and recombination (Dowton
and Campbell, 2001; Lunt and Hyman, 1997).
(1) The duplication/random loss model. During replication, a tandem duplication of part of the mt
genome may occur due to slipped-strand mispairing or imprecise termination. One copy of each
duplicated gene is likely to be rapidly eliminated or become nonfunctional due to the
accumulation of mutations. Therefore, the original gene arrangement is either restored or altered
5
depending on which copy of the genes is lost (Fig. 1.2). This model is supported by the
existence of pseudogenes and the position of intergenic spacers observed in some rearranged mt
genome organizations. It has long been considered as the principal mechanism of gene
rearrangement in vertebrates (San Mauro et al., 2006). Many translocations observed in
invertebrates are also consistent with this model. For example the rearrangement trnW-trnC →
trnC-trnW, which occurred in neuropteran mt genomes, can be well explained by this model
(Beckenbach and Stewart, 2009). However, this model cannot explain gene inversions and
long-range translocations.
Figure 1.2 Simple diagram illustrating the duplication/random loss model. A and B represent two adjacent genes.
(2) The duplication/nonrandom loss model. Duplication of the entire mt genome can result in a
dimeric molecule with the two monomers covalently linked head to tail. Such a dimeric
molecule would contain two copies of transcriptional promoters. Mutation to one copy of the
promoters would result in the genes under the control of the disabled promoter becoming
pseudogenes, and the sequences containing them would be free to be eliminated from the
genome. Therefore, the loss of genes would be predetermined by their transcriptional polarity.
After rearrangement, genes with identical transcriptional direction will be clustered in the mt
genome (Fig. 1.3). A typical example in insects that is consistent with this mechanism is the mt
genome of a winter crane fly, Paracladura trichoptera (Diptera) (Beckenbach, 2012). In this mt
genome, the genes are highly rearranged, and include protein-coding, rRNA and tRNA genes.
6
All these rearrangements fall into two main groups. Within each group, both the ancestral gene
order and transcriptional direction are maintained. The duplication/nonrandom loss model can
account for almost all gene rearrangements in Paracladura. However, as is the case with the
duplication/random loss model, this model cannot explain gene inversions and long-range
translocations.
Figure 1.3 The duplication/nonrandom loss model proposed by Lavrov et al. (2002) explains the gene
rearrangements (excepting trnC and trnT) observed in two millipedes. The proposed position for transcription
initiation and termination are marked by TI and TT. Genes are transcribed from left to right except when
underlined; underlining indicates the opposite transcriptional polarity. The copies of the genes that became
pseudogenes in a hypothetical intermediate arrangement are indicated by blue boxes. Adapted from Lavrov et al.
(2002).
(3) Recombination. A defining feature of this mechanism is the breakage and rejoining of mtDNA
strands (Fig. 1.4). Recombination had been considered to be absent in the animal mt genome
(Moritz et al., 1987; Satoh and Kuroiwa, 1991). However, with more effort devoted to mt
genome research in recent years, both direct and indirect evidence of animal mtDNA
recombination has been provided (Gantenbein et al., 2005; Kraytsberg et al., 2004; Ladoukakis
and Zouros, 2001; Lunt and Hyman, 1997; Tsaousis et al., 2005). Recombination can provide a
good explanation to the gene inversions and long-range translocations commonly observed in
7
invertebrate mt genomes. It is also supported by the non-tandem duplications present in some
vertebrate mt genomes. For example, the duplicated control regions in the mantellid frog mt
genomes were hypothesized to be produced by recombination (Kurabayashi et al., 2008).
Figure 1.4 Model for the generation of mitochondrial gene inversions by recombination. After double-strand
breakage at two neighbouring loci, rejoining of the broken ends can produce a genome with a short, inverted segment.
The linear fragment represents the minifragment that is deleted from the entire mt genome; the circle represents the
main fragment of the entire mt genome (excluding the minifragment). Adapted from Dowton et al. (1999).
1.3.2 Gene rearrangements as phylogenetic markers
Comparison of mitochondrial gene arrangements has been argued to be a powerful tool for deducing
evolutionary relationships mainly because (1) the enormous number of potential arrangements
makes convergence unlikely; (2) gene arrangements are selectively neutral (Boore and Brown, 1998).
It has been shown to be useful in resolving both shallow and deep evolutionary relationships across a
broad range of animal groups (Boore, 2006). For example, gene arrangement analysis of Sipuncula
taxa provided additional evidence for their close relationship with Annelida rather than Mollusca
(Boore and Staton, 2002; Mwinyi et al., 2009). Several analytical methods of gene rearrangement
have been developed in recent years, such as Crex (Bernt et al., 2007), TreeREx (Bernt et al., 2008),
BADGER (Larget et al., 2002) and UniMoG (Hilker et al., 2012). These methods facilitate the use of
gene rearrangements to infer phylogenetic relationships. However, gene rearrangement analysis is
8
still at a premature stage. Each of the available analytical methods has its limitations and has not
been extensively applied (Bernt et al., 2013). For example, gene duplications are commonly found in
rearranged mt genomes (Akasaki et al., 2005; Castro et al., 2006). However, most of the analytical
methods, such as Crex and TreeREx, cannot encode them. Furthermore, cases of convergent
evolution of mt gene organization have been occasionally reported (Dowton et al., 2009b; Flook et
al., 1995; Kilpert et al., 2012). Therefore, more taxa with gene rearrangements and more
sophisticated analytical methods are needed for detecting the potential of mt gene arrangements as
phylogenetic markers, as well as their performance and levels of homoplasy (Grande et al., 2008).
1.4 Higher-level phylogenetic relationships within the Hymenoptera
The Hymenoptera are traditionally divided into the Symphyta (without discernible waist) and
Apocrita (with wasp-waist) (Sharkey, 2007). The Symphyta are the most primitive members and
comprise about 7% of the Hymenoptera (Huber, 2009). Except for the parasitic Orussidae, all
symphytan families are herbivorous, with a fairly unspecialized, caterpillar-like lifestyle in larval
stage (Vilhelmsen, 2001). The Apocrita comprise about 93% of the Hymenoptera and are subdivided
into the Aculeata (including bees, ants and stinging wasps) and the Parastica (most of which are
parasitoids of insects and spiders) (Huber, 2009).
1.4.1 Suborder Symphyta
It has long been recognized that the Symphyta are paraphyletic with respect to the Apocrita (Sharkey,
2007) (Fig. 1.5). Both morphological and molecular evidence supports that the Orussoidea is sister
group to the Apocrita (Heraty et al., 2011; Rasnitsyn, 1988; Schulmeister, 2003b; Sharkey et al.,
2011; Vilhelmsen, 2001; Vilhelmsen et al., 2010). Several comprehensive phylogenetic studies based
9
on morphological and/or molecular characters have recovered many well-supported relationships:
the Xyelidae is sister group to all the remaining Hymenoptera; the clade Xiphydrioidea + Orussoidea
+ Apocrita is frequently recovered; and the Unicalcarida (all Hymenoptera except Xyelidae,
Tenthredinoidea and Pamphilioidea) is well supported (Ronquist et al., 1999; Schulmeister, 2003a, b;
Sharkey et al., 2011; Vilhelmsen, 2001).
Figure 1.5 Major hymenopteran relationships proposed by Rasnitsyn (1988). Adapted from Sharkey et al. (2007).
1.4.2 Suborder Apocrita
The Apocrita has long been recognized as a natural group (Sharkey, 2007). However, the
phylogenetic relationships within the Apocrita remain elusive. Neither morphological nor molecular
data have provided a convincingly resolved phylogeny. In 1988, Rasnitsyn presented the first
comprehensive, fully resolved phylogenetic hypothesis for the Apocrita. He suggested four major
infraorders based on morphological and fossil evidence: the Ichneumonomorpha, the Aculeata, the
Proctotrupomorpha, and the Evaniomorpha, with the Ichneumonomorpha sister to the Aculeata and
the Proctotrupomorpha sister to the Evaniomorpha (Rasnitsyn, 1988) (Fig 1.5). Although his analysis
was not performed in a strictly cladistic context, it sets a stage for subsequent hymenopteran
phylogenetic studies. Johnson’s (1988) analysis based on the midcoxal articulation characters
10
recovered the Stephanoidea as the most basal apocritan lineage and supported the remaining
Evaniomorpha as monophyletic (Johnson, 1988). The basal position of the Stephanoidea was
supported by most subsequent studies (Heraty et al., 2011; Klopfstein et al., 2013; Sharkey et al.,
2011; Vilhelmsen et al., 2010; Whitfield, 1992). Ronquist et al. (1999) recoded Rasnitsyn’s (1988)
data into a numerical cladistics matrix and conducted a parsimony analysis. However, only the
Ichneumonomorpha and Aculeata were recovered as monophyletic groups; the Evaniomorpha
appeared as a paraphyletic grade of basal apocritan lineages; the Proctotrupomorpha was also
paraphyletic due to the position of the Ceraphronoidea (Ronquist et al., 1999). A subsequent
reanalysis with a revised set of wing characters produced an even less resolved tree (Sharkey and
Roy, 2002). Recently, Vilhelmsen et al. (2010) conducted a comprehensive analysis based on 273
mesosomal characters scored for 89 taxa. The results recovered the Ichneumonomorpha and the
Aculeata as monophyletic and supported their sister relationship; the Evaniomorpha (excluding the
Stephanoidea) was also retrieved but placed as the basal apocritan lineage; the Proctotrupomorpha
failed to be recovered (Vilhelmsen et al., 2010). In a later study, Vilhelmsen analyzed the head
capsule characters and mapped these characters onto a phylogeny from Vilhelmsen et al. (2010).
Most of the characters provided further support at the superfamily or family level (Vilhelmsen,
2011).
Dowton and Austin (1994) provided the first comprehensive molecular phylogenetic analysis of the
Hymenoptera based on 16S rRNA data. The results supported the clade Ichneumonomorpha +
Aculeata and a monophyletic Proctotrupomorpha. A subsequent study with expanded taxon sampling
based on the same sequence data supported the above relationships and recovered a monophyletic
Evaniomorpha (excluding the Stephanoidea) (Dowton et al., 1997). Dowton and Austin (2001)
11
performed multiple analyses based on three genes (16S, COI and 28S) and morphological characters.
However, the relationships were sensitive to the analytical approaches and the inclusion of
morphological data (Dowton and Austin, 2001). More recently, Castro and Dowton (2006) added
18S rRNA sequences to the Dowton and Austin (2001) dataset and conducted Bayesian and
parsimony analyses. The results supported the Evaniomorpha as paraphyletic, with the Aculeata
nested within the Evaniomorpha and recovered a monophyletic Proctotrupomorpha (Castro and
Dowton, 2006). Two studies based on molecular data and one study based on molecular data and
morphological characters have been published recently (Heraty et al., 2011; Klopfstein et al., 2013;
Sharkey et al., 2011). These three studies consistently recovered the Aculeata nested within a
paraphyletic Evaniomorpha and generally supported a sister relationship between the
Ichneumonomorpha and the Proctotrupomorpha.
Several phylogenetic studies using mt genomes from the Hymenoptera have been published. In 2007,
Castro and Dowton performed a range of analyses to assess the ability of mt genome data to recover
a well-accepted phylogeny of five hymenopteran taxa. The results indicated that the outgroup
composition was important to recover the test phylogeny (Castro and Dowton, 2007). Dowton et al.
(2009) performed phylogenetic analyses of mt genomes from a much broader range of the
Hymenoptera using mutiple phylogenetic approaches. The analyses indicated that partitioned
Bayesian analysis of nucleotide data (excluding 3rd codon positions) recovered more
uncontroversial relationships. The results suggested that analysis of mt genomes holds promise for
the resolution of hymenopteran higher-level relationships (Dowton et al., 2009a). Wei and co-authors
published two papers to reconstruct the phylogeny of the Hymenoptera using mt genomes. The
monophyly of the Aculeata, Ichneumonomorpha and Proctotrupomorpha were robustly confirmed.
12
(Wei et al., 2014; Wei et al., 2010a). Some other studies also attempted to assess the utility of the mt
genome for phylogenetic analysis of the Hymenoptera (de Melo Rodovalho et al., 2014; Kaltenpoth
et al., 2012). However, a common drawback of the above studies is the limited taxon sampling,
especially the poorly represented Evaniomorpha and Proctotrupomorpha.
1.5 Conclusions
There has been much effort in generating a good taxon sampling and diversity of markers for
phylogenetic analysis of the Hymenoptera (Castro and Dowton, 2006; Heraty et al., 2011; Sharkey et
al., 2011). But with respect to the mitochondrial phylogeny, previous studies have not sampled the
diversity of the Hymenoptera well. So far, there are 38 mt genomes (this number excludes
congenerics) of the Hymenoptera available in GenBank. As shown in Table 1.1, the Aculeata (in blue
font) and Ichneumonomorpha (in green font) are reasonably sampled. By contrast, the
Evaniomorpha (in purple font – excludes highlighted taxa, which were sequenced in the present
study) and Proctotrupomorpha (red font) are poorly represented. To resolve higher-level
relationships within the Apocrita using mt genomes, there is a pressing need to sample entire mt
genomes from each apocritan superfamily. In addition, with an extended taxon sampling from the
Evaniomorpha and Proctotrupomorpha, more gene rearrangements are likely to be identified. This
will facilitate the investigation of the mechanisms of gene rearrangement and assessing the utility of
shared, derived gene rearrangements as phylogenetic markers in these two lineages. By
understanding the gene rearrangement mechanism, this will further lead to more realistic analyses of
gene rearrangement characters.
13
Table 1.1 Mt genomes (complete/near-complete) for the Hymenoptera available in GenBank and unpublished in this
study. The superfamilies from the same infraorder are shown in the same color. The mt genomes sequenced in this
study are highlighted by yellow.
Cephoidea Cephus
Orussoidea Orussus
Pamphilioidea
Siricoidea
Tenthredinoidea Monocellicampa Perga
Xiphydrioidea
Xyeloidea
Apoidea Apis Bombus Melipona philanthus
Chrysidoidea Cephalonomia Primeuchroeus
Vespoidea Atta Abispa Camponotus Leptomyrmex Polistes
Pristomyrmex Radoszkowskius Solenopsis Wallacidia Pristomyrmex
Ichneumonoidea Aphidius Cotesia Diachasmimorpha Diadegma Diadromus Enicospilus
Macrocentrus Meteorus Phanerotoma Spathius Venturia Macrocentrus
Ceraphronoidea Ceraphron Conostigmus
Evanioidea Evania Gasteruption Pristaulacus
Megalyroidea Megalyra
Stephanoidea Schlettererius
Trigonalyoidea Orthogonalys Taeniogonalys
Chalcidoidea Ceratosolen Nasonia Philotrypesis
Cynipoidea Ibalia
Diaprioidea Monomachis
Mymarommatoidea
Platygastroidea Ceratobaeus Idris Trissolcus
Proctotrupoidea Pelecinus Vanhornia
1.6 Aims
1. To more completely sample mt genomes within the Platygastroidea, and to
investigate mt gene rearrangements and mechanisms within this group (Chapters 2
and 3).
Mt gene positions were highly rearranged in the available Proctotrupomorpha mt genomes (Castro et
al., 2006; Oliveira et al., 2008; Xiao et al., 2011). To further investigate the degree of gene
rearrangements at different taxonomic levels and possible mechanism for these gene rearrangements,
14
I sequenced three sceliond taxa: Trissolcus basalis, Ceratobaeus sp. and Idris sp. These mt genomes
facilitated both closer (within subfamily) and more distant (between superfamilies) comparisons of
gene rearrangements.
2. To more completely sample mt genomes within the Evaniomorpha, and to
investigate phylogeny and mt gene rearrangements and mechanisms within this
group (Chapters 4 and 5).
Evolutionary dynamics of mt gene rearrangements are poorly studied mainly due to a lack of
suitable model systems. To investigate whether the Evaniomorpha might provide such a model
system, I sequenced five new mt genomes for representatives of four evaniomorph superfamilies,
Megalyra sp. (Megalyroidea), Orthogonalys pulchella (Trigonalyoidea), Ceraphron sp.
(Ceraphronoidea), Conostigmus sp. (Ceraphronoidea) and Gasteruption sp. (Evanioidea). A mt
genome phylogeny was also reconstructed. In addition, recombination has been considered as an
important mechanism for invertebrate mt gene rearrangements. The Conostigmus sp. mt genome was
examined for evidence of recombination.
3. To further sample Proctotrupomorpha mt genomes, in order to obtain
representative mt genomes from every apocritan superfamily (excluding
Mymmaromatoidea), and to conduct the first mt genome phylogeny of the
Apocrita with a complete representation of superfamilies (Chapter 6)
The analysis of mt genomes holds promise to resolve higher-level relationships within the Apocrita.
However, a comprehensive mt genome phylogeny of the Apocrita has not been previously reported
due to limited taxon sampling. In an attempt to increase our understanding of the apocritan
phylogeny, I further sequenced three Proctotrupomorpha mt genomes: Ibalia leucospoides
15
(Cynipoidea), Monomachis antipodalis (Diaprioidea) and Pelecinus polyturator (Proctotrupoidea).
With these new genomes, the taxon dataset represented every apocritan superfamily (excluding
Mymmaromattoidea). I analyzed this dataset using a range of phylogenetic analytical approaches.
16
CHAPTER 2: The first mitochondrial genome for the
wasp superfamily Platygastroidea: the egg parasitoid
Trissolcus basalis
This chapter is slightly modified from the paper:
Mao M, Valerio A, Austin AD, Dowton M, Johnson NF (2012) The first mitochondrial genome for
the wasp superfamily Platygastroidea: the egg parasitoid Trissolcus basalis. Genome 55:194-204.
doi: 10.1139/g2012-005.
2.1 Introduction
Mitochondrial (mt) genomes have been widely used for the reconstruction of phylogenetic
relationships at different taxonomic levels (Cameron et al., 2007b; Kim et al., 2009; Li et al., 2011;
Miya et al., 2003). In insects, mtDNA is typically a double-stranded circular molecule of about 16 kb
in size. It contains 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes, and 22 transfer RNA
(tRNA) genes (Boore, 1999). Additionally, there is a major non-coding region, known as the
A+T-rich region or control region, which plays a role in initiation of transcription and replication
(Zhang and Hewitt, 1997).
The Hymenoptera is one of the most species-rich and biologically diverse insect orders (LaSalle and
Gauld, 1993). Although much progress has been made to investigate the phylogeny of the
Hymenoptera (Castro and Dowton, 2006; Dowton and Austin, 1994, 2001; Heraty et al., 2011;
Ronquist et al., 1999; Sharkey et al., 2011), there are also many relationships remaining unresolved.
For example, the monophyly of the Proctotrupomorpha (sensu Rasnitsyn 1988) is well supported by
recent studies, but the relationships among the proctotrupomorph families are still controversial
17
(Castro and Dowton, 2006; Dowton and Austin, 2001; Heraty et al., 2011; Rasnitsyn and Zhang,
2010). Several analyses have demonstrated that the whole mt genome is a useful tool to resolve
hymenopteran evolutionary relationships (Cameron et al., 2008; Castro and Dowton, 2007; Dowton
et al., 2009a; Wei et al., 2010a). To date, 18 complete mt genomes and 15 nearly complete mt
genomes have been successfully sequenced in the Hymenoptera, with 18 taxa added in the last 3
years. This large amount of data holds promise to improve phylogenetic analysis. Notwithstanding,
there remain shortcomings with this dataset, with only 11 superfamilies represented. For
approximately half of the hymenopteran superfamilies, there is no mt genome data available.
In this paper, we provide the mt genomic sequence of Trissolcus basalis (Wollaston) from the
superfamily Platygastroidea, the third largest of the parasitic superfamilies after the Ichneumonoidea
and Chalcidoidea (Austin et al., 2005). No mt genome sequences for the Platygastroidea have
previously been published. The characteristics of this mt genome, with respect to genome
composition, nucleotide content, and codon usage, were compared with other hymenopteran mt
genomes. We conducted phylogenetic analyses of mt genomes from 29 hymenopteran taxa and 7
other orders of holometabolous insects. Bayesian analyses were performed using nucleotide
sequences (including and excluding the third codon position). The performance of two alignment
methods (Clustal W and Muscle) was also compared. Another goal of this research was to further
explore the evolution of mt gene rearrangement. The hymenopteran mt genome has been reported to
rearrange more frequently than those from other insect orders (Crozier and Crozier, 1993; Dowton
and Austin, 1999; Dowton et al., 2009b). Here we compared the genome structures of four
Proctotrupomorpha taxa.
2.2 Materials and methods

18
DNA extraction, PCR amplification, and sequencing
454 sequencing. Genomic DNA was extracted from whole bodies of 25 adult male specimens of T.
basalis from a laboratory culture maintained by F. Bin at the Universitàdi Perugia. The extract was
sent to the DNA Sequencing Facility at the University of Pennsylvania Perelman School of Medicine.
Five full plates were run on a Roche/454 GS FLX sequencer (454 Life Sciences, Branfort, Conn.,
USA) using Titanium chemistry. This resulted in 5,080,113 reads of a total of 1,535,920,544 bases.
These reads were assembled using gsAssembler 2.5.3 with a minimum overlap of 30 bases and the
complex genome assembly option.
Illumina sequencing. To address the issue of possible homopolymer errors in 454 sequencing,
genomic DNA was extracted from an additional five female specimens from the same culture. The
extract was processed using the Nextera DNA sample preparation kit from Epicentre
Biotechnologies (Madison, Wis., USA) following the manufacturer’s instructions. The sample was
submitted to the Nucleic Acid Shared Resource of the College of Medicine at The Ohio State
University. A single lane of 51 base reads was run on an Illumina Genome Analyzer IIx (Illumina,
San Diego, Calif., USA) resulting in 29,780,645 reads and a total of 1,518,812,895 bases. These
reads were assembled using VCAKE 1.5 into repetitive and non-repetive contigs, and short contigs
(< 70 base) were then removed. The remaining contigs were then assembled together with the final
454 assembly using gsAssembler 2.5.3. Nesoni 0.42 software was used to correct errors in the hybrid
assembly.
Contigs of mt origin from the entire assembly were identified by a BLAST search using known
hymenopteran mt proteins. Two contigs emerged from the search, with lengths of 8,067 bases and
19
7,719 bases.
Sequence analyses
The two contigs were imported into Bioedit version 7.0.5 (Hall, 1999). Visual inspection revealed
that the same dinucleotide repeat, (TA)n was present at one end of each of the contigs. When the
contigs were oriented in the ancestral orientation (i.e. with protein-coding genes oriented on the
same strand and direction as the ancestral pancrustacean), the shorter contig had a (TA)13 repeat at
the 3’ end, while the longer contig had a (TA)9 repeat at the 5’ end. We interpret this as indicating
that a dinucleotide repeat that varies in length is situated between these two contigs. Joining these,
the ancestral orientation of all protein-coding and rRNA genes is maintained. The dinucleotide repeat
is situated in the intergenic space, between the trnH and nad4 genes.
Nineteen tRNA genes were identified by tRNA-Scan SE version 1.21
(lowelab.ucsc.edu/tRNAscan-SE/) (Lowe and Eddy, 1997), specifying mitochondrial/chloroplast
DNA as the source and choosing the invertebrate mitochondrial genetic code for tRNA isotype
prediction. The cove cutoff score was set to 5 in order to avoid missing tRNA genes with lower
cutoff scores. Two tRNA genes (trnN and trnS1) failed to be detected by tRNA-Scan SE, but they
were identified by visually comparing unassigned regions of the genome with previously determined
tRNA counterparts from the Hymenoptera. Thirteen protein-coding genes were identified using an
open reading frame (ORF) finder (www.ncbi.nlm.nih.gov/gorf/orfig.cgi), specifying the invertebrate
mt genetic code. The initiation and termination codons of some genes were identified according to
the boundaries of tRNA genes and comparison with other insect mt sequences. The precise
boundaries of rRNA genes are difficult to define. They were assumed to be bounded by the
20
neighboring tRNA genes (Beckenbach, 2011; Beckenbach and Stewart, 2009; Dowton et al., 2009a).
However, the boundary between rrnL and rrnS was difficult to define in T. basalis, as the trnV that is
usually between the two rRNA genes is not present here. Instead, we determined this boundary by
aligning the rRNA regions from T. basalis with other closely related insect rrnL and rrnS genes.
Sequence alignment
Mitogenomic sequences from 29 hymenopteran taxa and 7 taxa from other holometabolous insects
were obtained from GenBank (Table 2.1). Nucleotide sequences for each of the 13 protein-coding
genes and the 2 rRNA genes were imported into separate files using MEGA version 5.05 (Tamura et
al., 2011) and aligned using Clustal W (Thompson et al., 1994) or Muscle (Edgar, 2004) as
implemented within MEGA 5. For the protein-coding genes (excluding the stop codons), the
nucleotide sequences were translated into the amino acid sequences using the invertebrate mt genetic
code, and the amino acid sequences were aligned using Clustal W or Muscle. MEGA 5 then creates a
nucleotide alignment using the amino acid sequences as a guide. The alignment parameters were the
default settings for all genes. The Clustal W alignment parameters were as specified in Cameron et al.
(2008). The Muscle alignment parameters for the rRNA genes were gap open penalty = -400, gap
extend penalty = 0; and for the protein-coding genes were gap open penalty = -2.9, gap extend
penalty = 0. For both types of gene, the maximum memory allocated was 2,047 MB, with a
maximum of eight iterations. The clustering method used for all iterations was UPGMB, with the
length of the minimum diagonal set to 24. Following alignment, individual genes were concatenated
prior to phylogenetic analysis.
21
Table 2.1 List of taxa used in phylogenetic analyses.
Order Family Genus GenBank Accession Referance

Coleoptera Tenebrionidae Tribolium AJ312413 Friedrich and Muqim 2003
Diptera Ceratopogonidae Culicoides AB361004 Matsumoto et al. 2009
Mecoptera Bittacidae Bittacus HQ696578 Beckenbach 2011
Megaloptera Sialidae Sialis FJ859905 Cameron et al. 2009
Neuroptera Ascalaphidae Ascaloptynx FJ171324 Beckenbach and Stewart 2009
Lepidoptera Tortricidae Adoxophyes NC_008141 Lee et al. 2006
Raphidioptera Raphidiidae Mongoloraphidia FJ859902 Cameron et al. 2009
Hymenoptera
Suborder
Symphyta
Cephoidea Cephidae Cephus NC_012688 Dowton et al. 2009a, b
Orussoidea Orussidae Orussus NC_012689 Dowton et al. 2009a, b
Tenthredinoidea Pergidae Perga AY787816 Castro and Dowton 2005
Apocrita
Apoidea Apidae Apis NC_001566 Crozier and Crozier 1993
Apidae Bombus DQ870926 Cha et al. 2007
Apidae Melipona NC_004529 Silvestre et al. 2008
Chalcidoidea Agaonidae Ceratosolen AH016005 Chen et al. unpublished
Pteromalidae Nasonia NW_001815691 Oliveira et al. 2008
Chrysidoidea Bethylidae Cephalonomia FJ823227 Wei et al. unpublished
Chrysididae Primeuchroeus AH015389 Castro et al. 2006
Evanioidea Evaniidae Evania NC_013238 Wei et al. 2010b
Ichneumonoidea Braconidae Aphidius GU097658 Wei et al. 2010a
Braconidae Cotesia NC_014272 Wei et al. 2010a
Braconidae Diachasmimorpha GU097655 Wei et al. 2010a
Braconidae Macrocentrus GU097656 Wei et al. 2010a
Braconidae Meteorus GU097657 Wei et al. 2010a
Braconidae Phanerotoma GU097654 Wei et al. 2010a
Braconidae Spathius NC_014278 Wei et al. 2010a
Ichneumonidae Diadegma NC_012708 Wei et al. 2009
Ichneumonidae Enicospilus FJ478177 Dowton et al. 2009a, b
Ichneumonidae Venturia FJ478176 Dowton et al. 2009a, b
Platygastroidea Scelionidae Trissolcus JN903532 Present study
Proctotrupoidea Vanhorniidae Vanhornia NC_008323 Castro et al. 2006
Stephanoidea Stephanidae Schlettererius FJ478175 Dowton et al. 2009a, b
Vespoidea Formicidae Pristomyrmex NC_015075 Hasegawa et al. 2011
Formicidae Solenopsis NC_014669 Gotzek et al. 2010
Mutillidae Radoszkowskius NC_014485 Wei and Chen unpublished
Vespidae Abispa NC_011520 Cameron et al. 2008
Vespidae Polistes EU024653 Cameron et al. 2008
Phylogenetic analysis
Previous phylogenetic studies using hymenopteran mt genome sequences indicated that nucleotide
data analyses were superior to amino acid data analyses, and that Bayesian analyses were superior to
22
parsimony analyses (Castro and Dowton, 2007; Dowton et al., 2009a). For this reason, we employed
Bayesian analyses in this study. The analyses were conducted using MrBayes version 3.1.2
(Ronquist and Huelsenbeck, 2003) at the freely available Bioportal server (www.bioportal.uio.no).
The two datasets (one aligned using Clustal W, the other aligned using Muscle) were divided into
four partitions: the first, second, and third codon positions and rRNA genes. The GTR+G+I model
(nst = 6, rate = invgamma) was chosen as the best-fit model for all of the partitions (MrModelTest
2.3, Nylander et al., 2004). Two analyses (all codon positions included or the third codon positions
excluded) were performed for the two alignment datasets. The Markov chain Monte Carlo (MCMC)
process was set so that four chains (three heated and one cold) ran simultaneously. For each analysis,
we conducted four independent runs for 1,000,000 generations, with trees being sampled every 100
generations. Burn-in was discarded according to the plot of generation against the likelihood scores
and the sump command in MrBayes. The percentage of trees recovering a particular clade was used
as a measure of that clade’s posterior probability (Huelsenbeck and Ronquist, 2001).
2.3 Results and discussion
Genome size and gene content
The almost complete mt genome sequence of T. basalis is 15,768 bp in size (Fig. 2.1). We were not
able to obtain the sequence of part of the non-coding region. Although the sequencing strategy
should have sequenced the non-coding region just as frequently as the coding regions, the inability to
identify fragments that could be added to our mt contigs is possibly due to the presence of repeats.
We did attempt to amplify the noncoding region by conventional PCR, but we were unable to obtain
amplicons, as has been reported for a number of hymenopteran mt genomes (Castro and Dowton,
23
2005; Castro et al., 2006; Oliveira et al., 2008). We were able to identify 36 of the 37 genes usually
found in animal mt genomes (Table 2.2); 13 protein coding genes, 21 of the 22 tRNA genes, 2 rRNA
genes, and part of the A+T-rich region. Several short non-coding regions were identified. The longest
of these is 60 bp long, and it is located between the trnS1 and nad5 genes. There are five genes that
overlap: two between protein-coding genes (a8 and a6, and nad4 and nad4L) and three between
tRNA genes (trnD and trnK, trnN and trnE, and trnT and trnP).
Table 2.2 Locations and nucleotide sequence lengths of genes in the Trissolcus basalis mt genome, the lengths of
predicted amino acid sequences of protein-coding genes, and their initiation and termination codons.
Gene/region Nucleotide position Size Codon

No. of nucleotides No. of amino acids Initiation Termination
trnC 2-62 61
trnY 82-147 66
trnQ 178-249 73
trnI 253-318 66
trnA 322-387 66
nad2 447-1425 979 327 ATA T
trnW 1426-1489 64
cox1 1490-3025 1536 512 ATA TAA
trnL2 3040-3109 70
cox2 3110-3787 678 226 ATT TAA
trnD 3790-3858 69
trnK 3856-3927 72
a8 3928-4095 168 56 ATT TAA
a6 4079-4737 659 220 ATG TA
cox3 4738-5530 793 265 ATG T
trnG 5531-5598 68
nad3 5605-5949 345 115 ATA TAA
trnN 5952-6019 68
trnE 6017-6080 64
trnF 6084-6150 67
trnS1 6154-6212 59
nad5 6275-7984 1710 570 ATT TAA
trnH 7985-8049 65
nad4 8080-9429 1350 450 ATG TAA
nad4L 9423-9695 273 91 ATA TAG
trnT 9709-9774 66
trnP 9774-9837 64
nad6 9839-10428 590 197 ATA TA
cob 10429-11565 1137 379 ATG TAA
trnS2 11581-11650 70
nad1 11655-12566 912 304 ATA TAA
trnL1 12573-12638 66
rrnL 12639-13908 1270
rrnS 13909-14660 752
trnM 14661-14727 67
trnV 14729-14793 65
24
Nucleotide content
As has been widely published in other insect mt genomes (Simon et al., 1994), the nucleotide
content of T. basalis is heavily biased towards adenine and thymine (Table 2.3). The entire sequence
has 43.7% A, 9.8% C, 40.5% T, and 6.0% G. The total A+T content is 84.2%, which is typical of
other hymenopteran mt genomes. For example, it is almost as high as that of Apis mellifera (84.9%:
Crozier and Crozier, 1993) but higher than that of Perga condei (78%: Castro and Dowton, 2005)
and Vanhornia eucnemidarum (80.1%: Castro et al., 2006).
For the mt protein-coding genes of T. basalis, the A+T content is a little lower than that of the entire
mt genome sequence. However, the first and second codon positions have a lower A+T content
(78.9% and 75.4%, respectively), while the third codon position shows a much higher A+T content
(92.3%). Conversely, both the rRNA and tRNA genes have a higher A+T content (87.9% and 88.7%,
respectively) when compared with the average for the genome. The nucleotide content bias is
inferred to result from mutational pressure (Asakawa et al., 1991; Foster et al., 1997).
Table 2.3 Nucleotide composition (%) of the Trissolcus basalis mt genome.
Nucleotide Length (bp) A C T G A+T

Entire sequence 15768 43.7 9.8 40.5 6.0 84.2
Protein-coding sequence 11130 36.5 9.1 45.7 8.6 82.2
J-strand 6885 38.5 11.4 42.8 7.4 81.3
N-strand 4245 33.4 5.5 50.5 10.6 83.9
Codon position
1st 3710 40.9 8.7 38.0 12.4 78.9
2nd 3710 23.6 14.0 51.8 10.6 75.4
3rd 3710 45.0 4.8 47.3 2.9 92.3
Ribosomal RNA gene sequences 2022 42.7 4.0 45.1 8.1 87.9
Transfer RNA gene sequences 1396 46.4 4.7 42.3 6.5 88.7
A+T-rich
J-strand region 974
1002 42.8
46.6 5.1
5.3 47.0
42.1 5.0
5.9 89.8
88.7
N-strand 394 45.9 3.3 42.6 7.9 88.5
Codon usage and amino acid composition
25
The codon usage in the T. basalis mt genome is highly skewed towards codons that are high in A+T
content (Table 2.4). Codons such as CTG (Leu) and CGC (Arg), encoded by both C- and G-rich
codons, are rarely used. Conversely, the AT-rich codons ATT (Ile), TTA (Leu), TTT (Phe), and ATA
(Met) are the four most frequently used codons (465, 460, 407, and 341 times, respectively). Within
a particular synonymous codon family, AT-rich codons are predominantly used. For example, leucine
can be coded by six alternative codons, but TTA is used much more frequently than any other. The
relative synonymous codon usage (RSCU: Nei and Kumar, 2000) for TTA is 5.23. The RSCU
measures the relative proportion that a codon is used within a particular synonymous codon family –
the maximum RSCU value for the leucine codon family is 6. For each codon family, the sum of the
A- and T-rich codons is close to the maximum possible RSCU value. Similar codon usage bias has
been observed in other hymenopteran mt genomes (Castro et al., 2006; Cha et al., 2007; Wei et al.,
2009).
Table 2.4 Codon usage for protein-coding genes of the Trissolcus basalis mt genome.
codon aa nc RSCU codon aa nc RSCU codon aa nc RSCU codon aa nc RSCU

TTT Phe 407 1.92 TCT Ser 92 2.45 TAT Tyr 161 1.69 TGT Cys 25 1.85
TTC Phe 18 0.08 TCC Ser 9 0.24 TAC Tyr 30 0.31 TGC Cys 2 0.15
TTA Leu 460 5.23 TCA Ser 102 2.71 TAA * 12 1.85 TGA Trp 79 1.93
TTG Leu 15 0.17 TCG Ser 4 0.11 TAG * 1 0.15 TGG Trp 3 0.07
CTT Leu 28 0.32 CCT Pro 41 1.45 CAT His 50 1.64 CGT Arg 8 0.84
CTC Leu 1 0.01 CCC Pro 17 0.6 CAC His 11 0.36 CGC Arg 1 0.11
CTA Leu 23 0.26 CCA Pro 55 1.95 CAA Gln 54 1.93 CGA Arg 26 2.74
CTG Leu 1 0.01 CCG Pro 0 0.0 CAG Gln 2 0.07 CGG Arg 3 0.32
ATT Ile 465 1.88 ACT Thr 48 1.52 AAT Asn 252 1.82 AGT Ser 20 0.53
ATC Ile 30 0.12 ACC Thr 11 0.35 AAC Asn 25 0.18 AGC Ser 0 0.0
ATA Met 341 1.92 ACA Thr 66 2.1 AAA Lys 155 1.9 AGA Ser 70 1.86
ATG Met 15 0.08 ACG Thr 1 0.03 AAG Lys 8 0.1 AGG Ser 4 0.11
GTT Val 55 1.93 GCT Ala 34 1.92 GAT Asp 46 1.74 GGT Gly 17 0.45
GTC Val 4 0.14 GCC Ala 7 0.39 GAC Asp 7 0.26 GGC Gly 4 0.11
GTA Val 48 1.68 GCA Ala 27 1.52 GAA Glu 61 1.74 GGA Gly 99 2.61
GTG Val 7 0.25 GCG Ala 3 0.17 GAG Glu 9 0.26 GGG Gly 32 0.84
Note: aa, amino acid; nc, number of codons; RSCU, relative synonymous codon usage; *, stop codon.
Translation initiation and termination codons
26
Thirteen protein-coding genes were identified using an ORF finder. Table 2.2 shows the position of
these genes in the mt genome of T. basalis, their lengths, and translation initiation and termination
codons. Conventional ATA, ATT, or ATG initiation codons are assigned to all of the 13
protein-coding genes. Nine genes (a8, cob, cox1, cox2, nad1, nad3, nad4, nad4L, and nad5) are
predicted to use the complete termination codons TAA or TAG. Four other genes have incomplete
termination codons: TA for a6 and nad6, and T for nad2 and cox3. Incomplete termination codons
have been commonly reported for other insect species (Clary and Wolstenholme, 1985; Crozier and
Crozier, 1993). It has been proposed that the complete termination codon (TAA) is created by
polyadenylation after transcription (Ojala et al., 1981).
Transfer RNA genes
Twenty-one tRNA genes (59-73 bp in size) were identified in the T. basalis mt genome. Fourteen
tRNA genes are encoded on the J-strand (the strand encoding the majority of genes, as defined by
Simon et al 1994), with 7 encoded on the N-strand (the minority strand). Their predicted secondary
structures are typical cloverleaf structures, except for trnS1 (Supplementary Fig. 1,
http://nrcresearchpress.com/doi/suppl/10.1139/g2012-005). The D-stem in the dihydrouridine (DHU)
arm is absent in the trnS1 gene, which has been reported in other hymenopteran species (Castro et al.,
2006; Wei et al., 2010b). In addition, mismatched base pairs appear in the DHU stem and acceptor
stem, which is common in the insect tRNA genes (Shao and Barker, 2003; Zhou et al., 2009). The
anticodons are identical to their counterparts in most other published hymenopteran mt genomes.
Ribosomal RNA genes
The small and large ribosomal RNA genes (rrnS and rrnL) are located on the N-strand. Unlike other
27
hymenopteran mt genomes, they are directly adjacent to each other, without the trnV gene between
them. The rrnS gene is 752 bp in size with an A+T content of 86.2%. The rrnL gene is 1270 bp in
size with an A+T content of 88.8%. These sizes are similar when compared with other Hymenoptera.
Based on the range of hymenopteran taxa included in Table 2.1, rrnS ranges from 600 to 811
nucleotides, while rrnL ranges from 868 to 1541 nucleotides. The A+T content of rrnS is similar
when compared with other hymnopteran taxa (Table 2.1), in which the A+T content ranges from
74.1% to 91.2%. The A+T content of rrnL is at the higher end of the range; it is only lower than that
of Cotesia (89.72%) and Macrocentrus (89.55%).
Non-coding regions
The longest non-coding region (partial A+T-rich region) of T. basalis is 974 bp in size, which is
located between the rrnS and trnC genes (Fig. 2.1). The A+T content is 89.8%, which is not
markedly higher compared with the remainder of the genome. There are several other short
non-coding regions ranging in size from 2 to 60 bp, which are located throughout the genome.
Interestingly, the 60 bp non-coding region has the anticodon of trnR (the tRNA gene that could not
be found) and the length is similar to the tRNA genes, but it could not be folded into a clover-leaf
structure unless overlapping more than 30 bp with nad5. If this section is ascribed to the trnR gene,
the slightly shorter nad5 gene lacks a stop codon (either complete or incomplete). Recently, some
truncated tRNA genes were reported in the mt genomes of velvet worms, with tRNA editing
converting the primary transcripts to conventional tRNA structures (Segovia et al., 2011). It is
conceivable that the missing trnR gene is present in this non-coding region, and it is completed by a
similar mechanism. We also investigated whether trnR was present in other non-coding regions,
including the trnA-nad2 junction, but we could find no regions that could be folded into a trnR gene.
28
Genome organization
The mitochondrial genome organization of T. basalis is shown in Fig. 2.1. All of the protein-coding
and rRNA genes have identical positions and transcriptional directions when compared with the
organization of the ancestral pancrustacean (Cook, 2005; Crease, 1999) (Fig. 2.1), which (as far as is
known) is the same organization as the ancestral hymenopteran (Dowton et al., 2009b). However, the
positions of a number of tRNA genes differ from the ancestral organization. For example, the
ancestral positions of trnC, trnY, and trnW are between the nad2 and cox1 genes, while in T. basalis,
trnC and trnY are between nad2 and the non-sequenced region (presumably the non-coding region).
Similarly, trnA, trnD, trnI, trnM, trnQ, trnR, and trnS1 are not in the ancestral positions.
We then compared the organization of the T. basalis mt genome with three other hymenopteran taxa
(all from the Proctotrupomorpha), to investigate whether any of the tRNA gene rearrangements were
shared with close allies. The Proctotrupomorpha have been consistently recovered as a monophyletic
group in molecular phylogenetic studies (Castro and Dowton, 2006; Dowton and Austin, 2001;
Heraty et al., 2011). Shared gene rearrangements are considered reliable indicators of shared
ancestry (Boore et al., 1995), and they have been used to infer phylogeny in the Hymenoptera
(Dowton, 1999), although examples of homoplastic gene rearrangements have been reported
(Dowton and Austin, 1999; Flook et al., 1995). The mt genome organizations of these three
additional taxa from the Proctotrupomorpha are shown in Fig. 2.1 to facilitate comparisons. All of
the protein-coding and rRNA genes are positioned identically in T. basalis and Vanhornia. In the
available mitogenomic sequences of Ceratosolen and Nasonia, Ceratosolen has an inversion of at
least five protein-coding genes (Xiao et al., 2011), while Nasonia has an inversion of six
protein-coding genes and a translocation of one protein-coding gene (Oliveira et al., 2008). With
29
respect to the position of the larger protein-coding and rRNA genes, there are three blocks of genes
that are present in all four taxa: cox1-cox2-a8-a6-cox3, nad4-nad4L-nad6-cob, and
nad1-rrnL-rrnS. However, despite the conservation of the organization of the larger genes in these
blocks, the tRNA genes are not conserved, with only three tRNA genes remaining in their ancestral
locations in all four taxa (trnH, trnT, and trnP). The trnS2 gene is present in its ancestral locations in
three Proctotrupomorpha taxa, with its positions unknown in Nasonia. All remaining tRNA genes
have rearranged in one or more taxa. Interestingly, the trnA gene is in the same derived position
(next to the rrnS gene) in both Ceratosolen and Nasonia. These are the only two included
representatives from the Chalcidoidea – this gene rearrangement may thus be a synapomorphy for a
subset of the Chalcidoidea.
Figure 2.1 Mitochondrial genome organizations of four Proctotrupomorpha taxa, compared with the ancestral
pancrutacean mt genome organization (Crease 1999, Cook 2005). tRNA genes are indicated by single-letter amino
acid codes L1, L2, S1, and S2, and denote trnLCUU, trnLUUR, trnSAGN, and trnSUCN, respectively. Genes are transcribed
from left to right except those indicated by underlining. Gene movements, relative to the ancestral organization, are
indicated with arrows.
30
Phylogenetic analysis and considerations
Phylogenetic analyses were performed using 36 complete or partial mt genomes, with 29 of these
taxa from the Hymenoptera. This is more than double the number of hymenopteran taxa compared
with previous studies (14 taxa: Dowton et al., 2009a). The most comprehensive analyses of
holometabolan relationships place the Hymenoptera at the base of the Holometabola (Savard et al.,
2006; Wiegmann et al., 2009), which make it difficult to choose an outgroup. For this reason, we
included a range of homometabolous taxa in the analysis. MrBayes allows only a single taxon to be
the outgroup, so we chose Tribolium (Coleoptera) as the outgroup, but any of the other
holometabolous insects would have been appropriate. Two Bayesian analyses (all codon positions
included or the third codon positions excluded) were performed for each of the alignment datasets
(Clustal W and Muscle).
Figure 2.2 Bayesian analysis of mitogenomic sequences based on the Muscle alignment dataset, including first and
second codon positions from protein-coding genes, and the two rRNA genes. Tribolium (Coleoptera) was specified as
the outgroup. Posterior probabilities are shown at each node.
The intraordinal relationships within the Hymenoptera were broadly congruent across the four
31
analyses, although there were some differences in the topologies. The major difference occurred
between analyses that included all codon positons and those that excluded the third codon positions.
In the 29 hymenopteran taxa, there are 10 taxa from the Aculeata, which contains the superfamilies
Apoidea, Chrysidoidea and Vespoidea. The analyses with the third codon positions excluded
recovered the Aculeata as a monophyletic group, while the analyses with all codon positions
recovered a controversial clade, with Evania (a member of Evaniomorpha) disrupting the Aculeata.
This contrasts with previous studies (Dowton et al., 2009a), in which the Aculeata were recovered as
a clade in both analyses (i.e. including or excluding third codon positions). This suggests that the
exclusion of third codon positions improves phylogenetic accuracy when analyzing hymenopteran
relationships using the whole mt genome, which is consistent with previous analyses (Castro and
Dowton, 2007; Dowton et al., 2009a). By contrast, exclusion of third codon positions has not had a
positive effect on phylogenetic reconstruction in some insect orders (Diptera: Cameron et al., 2007b;
Orthoptera: Fenn et al., 2008).
Dataset alignment strategies may also affect the recovered topology (Cameron et al., 2004; Cameron
et al., 2009). Comparison of the topologies recovered from the datasets aligned by Clustal W and
Muscle (both with third codon positions excluded) indicated that the topology produced from the
Muscle alignment was more congruent with expected relationships than the Clustal W alignment.
Analysis of both datasets recovered a range of monophyletic superfamilies, such as Apoidea,
Chalcidoidea and Ichneumonoidea, but the relationships within the superfamilies varied between the
two strategies. For example, within the Braconidae, the Muscle analysis recovered relationships
entirely congruent with the most recent analysis of Sharanowski et al. (2011); the monophyly of the
noncyclostomes (Cotesia + Phanerotoma + Macrocentrus + Meteorus), the monophyly of the

32
cyclostomes (Spathius + Diachasmimorpha), the monophyly of the microgastroids (Cotesia +
Phanerotoma), and a sister relationship between the cyclostomes and the Aphidiinae (Aphidius). By
contrast, the Clustal W topology failed to support these expected relationships. This suggests that the
phylogenetic relationships at lower taxonomic levels are more sensitive to the alignment strategy.
For the reasons outlined above, we present the tree of the dataset aligned by Muscle, excluding the
third codon positons (Fig. 2.2). Relationships among the Symphyta are recovered as expected – there
are only three symphytans included, and they form a paraphyletic grade at the base of the
Hymenoptera; Tenthredinoidea + (Cephoidea + (Orussoidea + Apocrita)). As expected, a
monophyletic Aculeata is recovered, as is a monophyletic Apoidea. Evania + Aculeata form a clade,
consistent with the resolution of the Aculeata inside the Evaniomorpha in more taxon-rich analyses
(Castro and Dowton, 2006; Heraty et al., 2011; Sharkey et al., 2011). However, support for a range
of relationships within the Aculeata is low, with posterior probabilities below 0.95. Terminal branch
lengths are very long compared with internode lengths. Further, compared with previous analyses,
internal relationships within the Aculeata are less congruent with expectation, despite the increase in
taxonomic sampling. For example, the Chrysidoidea are not recovered as monophyletic; although
Heraty et al. (2011) also failed to recover a monophyletic Chrysidoidea. Similarly, the Vespoidea are
polyphyletic, but this is in agreement with recent, broader analyses of hymenopteran relationships
(Heraty et al., 2011; Sharkey et al., 2011). Alternatively, Pilgrim et al. (2008) recovered a
paraphyletic Vespoidea. Together, these observations suggest that the uncontroversial relationships
recovered by Dowton et al. (2009a) may have been due to the narrow taxon sampling – for example,
the two vespoids are closely related (both from the Vespidae), biasing the analysis toward a finding
of monophyly. It suggests that a higher taxonomic sampling is necessary to reliably recover
33
relationships within the aculeates.
With respect to the Proctotrupomorpha, four taxa from three superfamilies (Proctotrupoidea,
Platygastroidea, and Chalcidoidea) are included in our analysis. The four taxa failed to form a clade
as Vanhornia (Proctotrupoidea) was misplaced, while the Chalcidoidea and Platygastroidea were
recovered as a grade above the Ichneumonoidea. Within the Ichneumonoidea, the well-established
sister group relationship between the Ichneumonidae and Braconidae are well supported (Dowton et
al., 1997; Rasnitsyn, 1988). In addition, the two groups (cyclostomes and non-cyclostomes) of
Braconidae were well recovered.
2.4 Conclusions
We obtained the nearly complete mt genome of T. basalis, which is the first record of a mt genome
of the superfamily Platygastroidea. Comparison of the genome organization of T. basalis with other
Proctotrupomorpha taxa identifies a number of gene rearrangements, which might provide
informative characters for the resolution of relationships among the Proctotrupomorpha.
Phylogenetic analyses using the entire mt genome sequences indicate that, despite the large number
of characters provided by the mt genome, increased taxonomic sampling is likely necessary to
reliably recover evolutionary relationships at the family and superfamily levels in the Hymenoptera.
34
CHAPTER 3: Complete mitochondrial genomes of
Ceratobaeus sp. and Idris sp. (Hymenoptera:
Scelionidae): shared gene rearrangements as potential
phylogenetic markers at the tribal level
Mao M, Dowton M (2014) Complete mitochondrial genomes of Ceratobaeus sp. and Idris sp.
(Hymenoptera: Scelionidae): shared gene rearrangements as potential phylogenetic markers at the
tribal level. Mol Biol Rep. doi: 10.1007/s11033-014-3522-x.
3.1 Introduction
The Scelionidae (Apocrita: Platygastroidea) is a diverse and speciose family of parasitic
Hymenoptera, encompassing 3308 valid species worldwide (Johnson, 2013). They are generally
small (0.5-12 mm), with most species being morphologically simple compared with other parasitic
wasps (Austin et al., 2005; Masner, 1976). Scelionds are egg parasitoids of grasshoppers, locusts,
crickets, bugs and spiders, so they have great significance in the biological control of agricultural
pests (Galloway and Austin, 1984). The Scelionidae are considered a paraphyletic group based on
morphological and molecular data (Austin and Field, 1997; Murphy et al., 2007). The evolutionary
relationships within the family are poorly resolved, especially at the tribal level. According to the
most recent molecular phylogenetic analysis, none of the tribes was found to be monophyletic except
for the Scelionini, which is probably due to the problems of current classification (Murphy et al.,
2007). A solid phylogenetic scheme for the group is essential to systematic and biological
researchers.
35
Mitochondrial (mt) genome sequences have provided large and diverse datasets useful in improving
phylogenetic relationships (Cameron et al., 2007b; Fenn et al., 2008). Analysis of mt genome
organization is also particularly useful for retracing ancient evolutionary relationships (Boore and
Brown, 1998). In insects, mt genomes are typically double-stranded circular molecules of about 16
kb that contain 13 protein-coding genes, 2 rRNA genes and 22 tRNA genes (Boore, 1999). The mt
genome also contains a large non-coding region, known as the A+T-rich region (in invertebrates) or
control region (in vertebrates), which regulates transcription and replication (Zhang and Hewitt,
1997). Prior to the present study, only a single mt genome from the Scelionidae (Trissolcus basalis)
had been sequenced (Mao et al., 2012). When this sequence was included in a comparison of gene
organizations among the sequenced Proctotrupomorpha taxa, mt gene positions were highly
rearranged at the superfamily level. Thus it would be intriguing to explore mt genome organization
among more closely related taxa, in order to better understand the history of gene rearrangement. In
this paper, we present two complete mt genomes of two other Scelionidae taxa, Ceratobaeus sp. and
Idris sp. Both of these wasps are from the tribe Baeini (Scelionidae: Scelioninae). This provides the
capacity to make both closer (within subfamily) and more distant comparisons (between
superfamilies). We analyze their organizations together with Trissolcus and perform phylogenetic
analyses based on the three mt genome sequences and the Nasonia mt genome. We find that gene
organization is much more conserved at the tribal level and that the mt genome sequences recover
the expected topology with high confidence. These results indicate that the mt genome may provide
a very useful source of data for resolution of the phylogeny of the Scelionidae.
DNA extraction
36
The specimens of Ceratobaeus sp. (UOW wasp alcohol library reference number M453) and Idris sp.
(UOW wasp alcohol library reference number M454) were kindly provided by Andy Austin, were
collected from Wistow, South Australia and preserved in 100% ethanol. Genomic DNA was
extracted as described (Aljanabi and Martinez, 1997). The DNA was resuspended in 100 μl of fresh
TE solution (1 mM Tris–HCl, 0.1 mM EDTA [pH 8]) and stored at 4°C.
Amplification, sequencing, and annotation of entire mt genomes
Internal gene fragments were amplified and sequenced using either published universal animal mt
primers (Simon et al., 1994; Simon et al., 2006), or using primers designed from consensus
hymenopteran mt sequences. Using the sequence information obtained, specific primers were
designed for long amplification of multigenic regions. Short PCRs were performed using
BIOTAQ™ DNA Polymerase (Bioline, Australia) with the following cycling conditions: 94oC for 2
min, 35 cycles of 94oC for 30 sec, 45oC-65oC for 1 min, 72oC for 2 min and a final elongation for 10
min at 72oC. Long PCRs were performed using Takara LA Taq (Takara Biomedical, Japan) with the
following conditions: 94oC for 2 min, 30 cycles of 94oC for 1 min, 45oC-65oC for 30 sec, 68oC for
12 min and a final elongation for 10 min at 68oC. The A+T-rich region of Ceratobaeus sp. was
amplified by nested PCR. All PCR products were visualised by agarose gel electrophoresis and
purified with ExoSAP-IT® (GE Healthcare, Bucks, UK). The primer-walking method was employed
for direct sequencing, using the ABIPRISM®BigDye™ Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems, Australia). Amplicons that were difficult to sequence directly were ligated into
the pGEM-T Easy Vector System (Promega, Madison, Wisconsin) and cloned into E. coli JM109
cells (Promega, Madison, Wisconsin). The sequences of both strands were determined for each of
the entire mt genomes.

37
The sequences were assembled into contigs in ChromasPro Ver 1.33 (Technelysium Ltd., Tewantin,
Australia). ORFinder (www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to identify protein-coding
genes, specifying the invertebrate mt genetic code. The tRNA-scan SE 1.21
(lowelab.ucsc.edu/tRNAscan-SE/) (Lowe and Eddy, 1997) search server was used to annotate tRNA
genes using the following settings: source, Mito/Chloropast; genetic code for tRNA isotype
prediction, Invertebrate Mito; and cove score cutoff, 5. Ribosomal RNA genes were identified both
through sequence comparison with published hymenopteran mt ribosomal RNA sequences and
through comparison of the conserved secondary structure of the 3’ end of rrnS. The sequence data
have been deposited in GenBank, under accession numbers KF696669 and KF696670.
Sequence alignment
Two mt genomes (Ceratobaeus sp. and Idris sp.) sequenced in this study together with one mt
genome (T. basalis) sequenced in a previous study (Mao et al., 2012) were aligned, together with
Nasonia (which would form the outgroup for later phylogenetic analyses). Nucleotide sequences for
each of the 13 protein-coding genes and the 2 rRNA genes were imported into separate files using
MEGA5 (Tamura et al., 2011) and aligned using Clustal W (Thompson et al., 1994) or Muscle
(Edgar, 2004) as implemented within MEGA5. For the protein-coding genes (excluding the stop
codons), an amino acid alignment was generated first for each gene and a nucleotide alignment
inferred from the amino acid alignment. The alignment parameters were the default settings for all
genes, which have been specified in previous studies (Cameron et al., 2008; Mao et al., 2012).
Nucleotide alignments were concatenated prior to phylogenetic analysis.
38
Table 3.1 Datasets used for phylogenetic analysis.
Dataset Alignment method Partitions Number of partitions

All-DNA123 + rRNA Clustal W 1st, 2nd, 3rd codons, rrnL, rrnS 5
Muscle 1st, 2nd, 3rd codons, rrnL, rrnS 5
st st st
Clustal W (rrnS, rrnL) (a6_1 codon, cob_1 codon, cox3_1 10
codon, cox2_1st codon, cox1_1st codon) (a6_2nd
codon, cox3_2nd codon, cox2_2nd codon, nad1_2nd
codon, nad4l_2nd codon, nad4_2nd codon, nad5_2nd
codon) (a6_3rd codon, cob_3rd codon, cox3_3rd
codon, cox2_3rd codon, cox1_3rd codon, nad3_3rd
codon) (a8_1st codon, nad2_1st codon, nad3_1st
codon, nad6_1st codon) (a8_2nd codon, nad2_2nd
codon, nad3_2nd codon, nad6_2nd codon) (a8_3rd
codon, nad2_3rd codon, nad6_3rd codon) (cob_2nd
codon, cox1_2nd codon) (nad1_1st codon, nad4l_1st
codon, nad4_1st codon, nad5_1st codon) (nad1_3rd
codon, nad4l_3rd codon, nad4_3rd codon, nad5_3rd
codon)
Muscle (rrnS, rrnL) (a6_1st codon, cob_1st codon, cox3_1st 10
codon, cox2_1st codon, cox1_1st codon) (a6_2nd
codon, cob_2nd codon, cox3_2nd codon, cox2_2nd
codon, cox1_2nd codon, nad3_2nd codon) (a6_3rd
codon, cob_3rd codon, cox3_3rd codon, cox2_3rd
codon, cox1_3rd codon, nad3_3rd codon) (a8_1st
codon, nad2_1st codon, nad3_1st codon, nad6_1st
codon) (a8_2nd codon, nad2_2nd codon, nad6_2nd
codon) (a8_3rd codon, nad2_3rd codon, nad6_3rd
codon) (nad1_1st codon, nad4l_1st codon, nad4_1st
codon, nad5_1st codon) (nad1_2nd codon,
nad4l_2nd codon, nad4_2nd codon, nad5_2nd codon)
(nad1_3rd codon, nad4l_3rd codon, nad4_3rd codon,
nad5_3rd codon);
All-DNA12 + rRNA Clustal W 1st, 2nd codons, rrnL, rrnS 4
st nd
Muscle 1 , 2 codons, rrnL, rrnS 4
Table 3.2 Estimation of Bayes Factors of different partition schemes.
Partition* Bayes Factor

P4-Muscle/P4-ClustalW 343
P10-Muscle/P5-Muscle 1538
P10-ClustalW/P5-ClustalW 1045
* Partition schemes are outlined in Table 3.1. P4, P5, P10 correspond to the number of partitions given in Table 3.1
Genetic divergence and phylogenetic analysis
The ratios of Ka (the nonsynonymous substitution rate) and Ks (the synonymous substitution rate)
39
for protein coding genes between the Scelionidae species were calculated using the software Ka/Ks
calculator (v. 1.2) based on the model-averaging method (MA) (Zhang et al., 2006). For the
phylogenetic analyses, two partition schemes were used for the two datasets (one aligned using
Clustal W, the other aligned using Muscle). Previous investigations of partitioning approaches using
hymenopteran and termite mt genome sequences indicated that nucleotide data excluding third
codon positions better recovered traditional relationships (Cameron et al., 2012; Dowton et al.,
2009a; Mao et al., 2012). However, inclusion of third codon positions has been found to improve the
accuracy of phylogenetic reconstructions in other studies (Cameron et al., 2007b; Fenn et al., 2008;
Nelson et al., 2012). In addition, a more objective framework for choosing among competing
partition schemes was recently developed, PartitionFinder (Lanfear et al., 2012). For these reasons, a
range of partition schemes were compared (Tables 3.1, 3.2). A total of 41 data blocks were defined
(3 codon positions of 13 protein-coding genes + 2 rRNA genes). PartitionFinder distinguished
between competing partition schemes using the Bayesian Information Criterion (BIC) and a heuristic
search algorithm. In all cases, MrModelTest 2.3 (Nylander et al., 2004) was employed to define
models of nucleotide evolution for each partition.
Phylogenetic analysis was performed with MrBayes v. 3.1.2 (Ronquist and Huelsenbeck, 2003). The
Markov chain Monte Carlo (MCMC) process was set so that four chains (three heated and one cold)
ran simultaneously. For each analysis, we conducted four independent runs for 1,000,000
generations with sampling every 100 generations. Burn-in was discarded according to the plot of
generation against the likelihood scores. Stationarity was assessed by importing the parameter file
into Tracer v. 1.5 (Rambaut and Drummond, 2009), and assessing whether the ESS of all parameter
values were >100. The percentage of trees recovering a particular clade was used as a measure of
40
that clade’s posterior probability (Huelsenbeck and Ronquist, 2001).
3.3 Results and discussion
Genome size and gene content
Two complete mt genomes (Ceratobaeus sp. and Idris sp.) were sequenced. Each genome contained
13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and an A+T-rich region similar to most
other insects (Fig. 3.1). The Ceratobaeus sp. mt genome is 15,851 bp long. This size is well within
the range found in other completely sequenced hymenopteran insects (15,425 bp in Spathius agrili to
19,339 bp in Cephus cinctus) (Dowton et al., 2009a; Wei et al., 2010a). The A+T content of the
entire genome is 77.59% (A=40.87%; T=36.71%; C=14.03%; G=8.38%), which is similar to Perga
condei (78%) (Castro and Dowton, 2005). We detected five overlapping regions between genes
(trnD-trnK, a8-a6, trnN-trnF, trnF-trnS1, and trnE-trnR) for a total of 21 bp (supplementary Table 1,
http://dx.doi.org/10.1007/s11033-014-3522-x). The longest one is 8 bp, involving the trnE and trnR
genes. Twelve short noncoding regions ranging from 2 to 72 bp were identified.
At 15,137 bp, the mt genome of Idris sp. is the shortest hymenopteran complete mt genome to date.
The A+T content of the entire mt genome is 81.22%, which is a little higher than Ceratobaeus sp.
Six overlapping regions were detected. Four of these overlaps are in the same location as found in
Ceratobaeus sp. (trnD-trnK, a8-a6, trnN-trnF, and trnE-trnR), but there are another two located in
the trnQ-trnC and trnT-trnP regions (supplementary Table 1,
http://dx.doi.org/10.1007/s11033-014-3522-x). There are 14 short noncoding regions in Idris sp.,
ranging from 1 to 72 bp, which together total 155 bp.
41
Figure 3.1 Mitochondrial genome organization of three sceliond taxa, compared with the ancestral pancrustacean mt
genome organization (Cook 2005). tRNA genes are indicated by single-letter amino acid codes, L1, L2, S1, and S2
denote tRNA-Leu (CUN), tRNA-Leu (UUR), tRNA-Ser (AGN), and tRNA-Ser (UCN), respectively. Genes are
transcribed from left to right except those indicated by underlining. Gene movements, relative to the ancestral
organization, are indicated with arrows.
Protein-coding genes
The 13 protein-coding genes typical for insect mt genomes were identified using ORF finder. Two of
the conventional start codons ATA or ATG could be assigned to most of the protein-coding genes.
However, nad2, a8, and cox2 use ATC, ATC, and ATT in Ceratobaeus sp., respectively, and cox1
and nad5 use ATT in Idris sp. Nine genes end with complete termination codons (TAA) in
Ceratobaeus sp., while only six genes have complete termination codons in Idris sp. The remaining
genes appear to use incomplete termination codons (TA or T) that are presumably completed by
polyadenylation after transcription (Ojala et al., 1981).
42
The relative synonymous codon usage (RSCU) shows high base compositional biases for AT in the
mt genomes of Ceratobaeus sp. and Idris sp. (supplementary Table 2, 3,
http://dx.doi.org/10.1007/s11033-014-3522-x). Within a particular synonymous codon family,
codons with A or T in the third codon position are generally strongly overrepresented compared to
codons with G or C in the third position. However, the RSCU values of GCC (Ala), AGG (Ser) and
GGG (Gly) are higher than GCA (Ala), AGT (Ser) and GGT (Gly) in Ceratobaeus sp., respectively.
GCC (Ala) and AGG (Ser) have slightly higher RSCU values than GCA (Ala) and AGT (Ser),
respectively, while the RSCU value of GGG (Gly) is much higher than GGT (Gly) (1.31 to 0.35).
Anticodon analysis showed that both trnS-TCT and trnG-TCC contain wobble nucleotides and they
can decode AGG and GGG, respectively. This might be the reason for the higher RSCU value of
AGG (Ser) and GGG (Gly). However, it cannot explain the higher RSCU value of GCC (trnA-TGC).
Figure 3.2 Inferred secondary structures of tRNA genes: A-B. trnS1 identified in the mt genomes of Ceratobaeus sp.
and Idris sp., respectively. C. Pseudo-trnK identified in the mt genome of Idris sp. D-G, trnR identified in the three
sceliond mt genomes (D. Ceratobaeus sp. E. Idris sp. and F. Trissolcus basalis), compared with trnR reported in the
mt genome of Ceratosolen solmsi (Chalcidoidea). Watson-Crick pairs are indicated by lines and noncanonical pairs
are indicated by asterisks.
Transfer RNA and Ribosomal RNA
43
The length of tRNA genes ranges from 58 bp to 74 bp in the two species. Most tRNA genes have a
typical cloverleaf structure except for trnS1 and trnR, in which the D-stem pairings in the
dihydrouridine (DHU)-arm are absent (Fig. 3.2). The missing D-stem has been commonly noted for
the trnS1 gene in insects (Castro et al., 2006; Sheffield et al., 2008; Yang et al., 2013). It is
hypothesized that the atypical trnS1 occurred early in, or before the evolution of Metazoa (Garey and
Wolstenholme, 1989). Although the loss of the DHU arm of trnR has been reported in other
non-insect organisms (e.g. Taenia asiatica) (Jeon et al., 2005), the missing D-stem in trnR, to our
knowledge, has not been previously reported in insect mt genomes. This particular tRNA gene was
the most difficult to locate in all three sceliond mt genomes (Ceratobaeus sp., Idris sp. and T.
basalis); it overlaps with trnE in both Ceratobaeus sp. and Idris sp. In our previous study on the mt
genome of T. basalis, we were unable to find the trnR gene (Mao et al., 2012). However, using the
location and structure of the trnR gene in Ceratobaeus sp. and Idris sp. as a guide, we are now able
to locate a putative trnR gene in T. basalis; all three have similar secondary structures (Fig. 3.2). We
suggest that the atypical trnR might be a shared derived character for the Scelionidae.
Since the rrnL and rrnS genes are directly adjacent to each other in both species, we identified the
gene boundaries by comparing the nucleotide sequences with other hymenopteran mt genomes. The
rrnL and rrnS genes of Ceratobaeus sp. are 1,280 and 754 bp long, which are shorter in size by 9
and 21 bp than those genes of Idris sp.
Noncoding regions
The two mt genomes contain a large noncoding region (A+T-rich region) between trnM and trnQ. It
is 1,098 bp long in Ceratobaeus sp. In contrast to the A+T-rich region in most insect mt genomes,
44
the A+T content (75.16%) of this region is lower than that of the entire mt genome (77.59%). It
contains an array of tandem repeats with a total length of 1,066 bp present in eight copies (the last
one is a partial copy). The first, second, third, fifth, sixth and seventh copies are almost identical in
sequence, with only one to three site differences, while the fourth copy is 24 or 25 bp shorter than
other copies and with six to nine site differences. The A+T-rich region has been difficult to amplify
in other hymenopteran mt genomes (Castro et al., 2006; Oliveira et al., 2008). We could only
amplify this region in Ceratobaeus sp. by nested PCR. It suggests that the long tandem repeats might
cause amplification problems. In addition to the long tandem repeats, a microsatellite (AT)10 was
identified, which is a typical characteristic reported in other insect mitochondrial A+T-rich regions.
Several short non-coding regions with a total of 199 bp are scattered throughout the Ceratobaeus sp.
mt genome. The longest one (72 bp) is located between the nad4L and trnP genes, where the trnT
gene is missing compared with the ancestral organization (Fig. 3.1). Sequence analysis shows that
this noncoding region possesses the anticodon (TTG) of trnT but cannot be folded into a clover-leaf
structure. The 59.7% sequence identity compared with trnT indicates that it might be a remnant of an
historical intermediate generated by the tandem duplication/random loss model (Moritz et al., 1987)
(see below).
The A+T-rich region of Idris sp. is 414 bp with an A+T content of 84.3%, which is relatively higher
than that of the entire mt genome (81.22%). An 8 bp tandem repeat (TTTTACTA) is present in three
copies, as well as a microsatellite (AT)9. The second largest noncoding region of Idris sp. is 72 bp,
between the nad1 and trnL1 genes. This region contains a sequence that can be folded into a
secondary structure resembling an additional trnK with TTT as the anticodon, differing from the
designated trnK using CTT as the anticodon (Fig. 3.2). The cove score of trnKTTT (19.9) is relatively
45
lower than that for trnKCTT (28.8), as determined by tRNAscan-SE. The abnormal anticodon TTT is
more commonly used in the trnK gene of hymenopteran mt genomes (Castro et al., 2006; Gotzek et
al., 2010; Kaltenpoth et al., 2012; Wei et al., 2010a). However, trnK (CTT) is clearly the ancestral
form of this gene, as it is the form that is present in a range of holometabolous and other insects.
Although trnK (TTT) may also function as a bona fide trnK gene, the ancestral nature of the trnK
(CTT) gene, together with the higher cove score leads us to postulate that the trnK (CTT) gene is the
functional version of the trnK. The presence of tRNA-like sequences or extra tRNA genes has been
commonly reported in other insects (Beckenbach, 2011; Castro et al., 2006; Cha et al., 2007; Shao
and Barker, 2003).
Genome organizations
The two mt genome organizations are shown in Fig. 3.1. The protein-coding and rRNA genes are
conserved in positions and orientations relative to the organization of the ancestral pancrustacean
(Cook, 2005). However, the relative positions of tRNA genes are highly variable. As shown in Fig.
3.1, a total of 11 tRNA genes have rearranged in both Ceratobaeus sp. and Idris sp. when compared
with the ancestral positions. The rearrangement events mainly occur at three junctions (nad2-cox1,
cox2-a8 and nad3-nad5) and around the A+T-rich region. The cox2-a8 and nad3-nad5 junctions
have been reported as “hot spots” for gene rearrangements in the Hymenoptera (Dowton and Austin,
1999; Dowton et al., 2003). In addition, trnT and trnP have swapped positions in Ceratobaeus sp.
We then compared the genome organizations among the three Scelionidae taxa. The gene order of
Ceratobaeus sp. is nearly identical to that of Idris sp. Only two tRNA genes (trnT-trnP→trnP-trnT)
are arranged differently between the two mt genomes. The presence of a short noncoding region
46
between nad4L and trnP in Ceratobaeus sp. is highly consistent with the tandem duplication/random
loss model, which is the most plausible mechanism for local gene rearrangements in animal mt
genomes (Boore, 2000). The retention of the ancestral organization (trnT-trnP) in both Idris sp. and
T. basalis indicates the gene rearrangement occurred after the divergence of Ceratobaeus within the
Baeini. There are some derived gene arrangements shared among the three taxa: trnK and trnD have
swapped positions; trnM has inverted and moved into the junction between rrnS and the A+T-rich
region; trnC and trnY have moved out of the nad2-cox1 junction and trnC has moved to the junction
between nad2 and the A+T-rich region in all 3 scelionid taxa; trnV has moved into the junction
between rrnS and the A+T-rich region and trnY is inverted. Some of the rearrangements were also
characterized in other hymenopteran linages. For example, the derived position of trnM reported
here was identical with 7 known braconid (Ichneumonoidea) mt genomes (Wei et al., 2010a). But
these two superfamilies (Platygastroidea and Ichneumonoidea) are not closely related groups,
indicating this arrangement has likely evolved on two occasions independently. Furthermore, trnV
has also moved out of the rrnL-rrnS junction in three chalcidoid taxa (Oliveira et al., 2008; Xiao et
al., 2011). Although its derived position is unknown in these taxa, we speculate that the
rearrangement of trnV might be a shared derived character for Platygastroidea and Chalcidoidea,
which were recovered as sister groups in some phylogenetic analysis (Dowton and Austin, 2001;
Ronquist et al., 1999).
Shared gene rearrangements are considered valuable sources for deducing phylogenetic relationships
(Dowton et al., 2002). The two mt genomes reported here share almost all of the derived gene
positions. Both of them belong to the tribe Baeini, which was not resolved as monophyletic by
molecular data (Carey et al., 2006; Murphy et al., 2007). The morphological phylogeny for the
47
Baeini is also problematic as it is dominated by reductional synapomorphies (Iqbal and Austin,
2000). Idris Foerster and Ceratobaeus Ashmead are the two most speciose genera in Baeini and they
were found to be polyphyletic in previous studies (Carey et al., 2006; Murphy et al., 2007). Although
our data is not yet sufficient to comprehensively address these issues, the evidence of shared gene
positions between closely related groups suggests that these gene rearrangements have much
potential as phylogenetic markers at the tribal level in the subfamily Scelioninae. Further, the three
Scelionidae taxa also shared some derived gene arrangements. However, we consider that we require
more genome representatives before we can conclude that these shared rearrangements are derived
characters for the Scelionidae.
Table 3.3 Comparison of genetic divergences for all protein-coding genes among scelionds.
Comparison Ka Ks Ka/Ks
Ceratobaeus/Idris 0.160421 6.48094 0.0247527
Ceratobaeus/Trissolcus 0.267965 9.04276 0.0296331
Idris/Trissolcus 0.265943 11.7398 0.0226532
Ka/Ks ratios among the three Scelionidae taxa
The calculation of nonsynonymous (Ka) and synonymous (Ks) substitution rate is informative for
understanding the evolutionary dynamics of coding regions across closely related species (Fay and
Wu, 2003). In this study, we measured the Ka and Ks of protein coding genes and determined the
Ka/Ks ratios between the three Scelionidae taxa, respectively (Table 3.3). The results show that Ks
(range 6.48-11.74) is about 34-44 times higher than Ka (range 0.16-0.27) in each comparison, which
indicates these protein coding genes are evolving under strong purifying selection (Roques et al.,
2006). The results also indicated that there was little difference between the Ka/Ks ratio when all
combinations of the various Scelionidae were compared. This indicates that there has not been a
48
substantial rate change during the evolution of the Scelionidae, in contrast to the rate change that is
evident when symphytan wasps are compared with apocritan wasps (Dowton et al., 2009a).
Phylogenetic analyses were performed using four mt genomes, with three of them from the
Scelionidae. Nasonia (Chalcidoidea: Pteromalidae) was chosen as the outgroup. Bayesian analyses
were performed for each dataset, in which the alignment and partitioning approach were varied; the
analyses are outlined in Table 3.1. Each analysis recovered the same uncontroversial relationships
(supplementary Fig. 1, http://dx.doi.org/10.1007/s11033-014-3522-x). The two Baeini taxa
(Ceratobaeus sp. and Idris sp.) group together with strong support (pp = 1). In contrast to previous
studies using hymenopteran mt geomes, inclusion of third codon positions had no effect on topology
and nodal support in the current analysis, indicating that inclusion of third codon positions does not
appear to be problematic when constructing the phylogeny of closely related taxa, which is
consistent with a recent study in which intrafamily relationships within a family of Diptera were
examined (Nelson et al., 2012)
In order to test the effects of different alignment methods and partition schemes, we employed Bayes
Factors following the approach in a previous study (Dowton et al., 2009a). As shown in Table 3.2,
alignment of sequences using Muscle resulted in trees with improved likelihood scores when
compared with those aligned with Clustal W. This indicates that alignment with Muscle is preferable
to those with Clustal W in phylogenetic reconstruction of hymenopteran taxa, which is consistent
with our previous study (Mao et al., 2012). We then examined the effect of different partition
schemes. Choosing an appropriate partitioning scheme is important for accuracy of phylogenetic
49
reconstruction using mt genomes (Dowton et al., 2009a; Fenn et al., 2008). For the All-DNA123 +
rRNA dataset, we first divided the data into four partitions (1st, 2nd, 3rd codon positions and rRNA, as
reported in previous studies). In the second scheme, we employed PartitionFinder, which divided the
data into ten partitions in both the All-DNA123 + rRNA-Clustal W and the All-DNA123 +
rRNA-Muscle datasets.
The results of our PartitionFinder analyses suggest that more complex partitioning schemes can
result in vastly improved likelihood scores. The likelihood score of the tree found using the model
established by PartitionFinder was 769 log units higher than that found using our previous approach
(1st, 2nd, 3rd codon positions, and rRNA partitions). This increase in likelihood is not likely to simply
reflect an increased fit of the data due to the increased number of parameters in the model. This is
because PartitionFinder uses the BIC to distinguish between competing partition schemes, and this
approach penalizes over-parameterization (Sullivan and Joyce, 2005), particularly when the number
of sites is high (as is the case with mt genome based phylogenies). It is also of interest to examine
the details of the partitions found by PartitionFinder, as it may yield clues to the underlying
evolutionary processes operating on the hymenopteran mt genome. Genes or codon positions that are
placed into the same partition may be evolving under the same underlying influences. For example,
the third codon positions of protein coding genes were partitioned into three partitions: (3rd codon
positions of a6, cob, cox3, cox2, cox1, nad3), (3rd codon positions of a8, nad2, nad6), and (3rd codon
positions of nad1, nad4L, nad4, nad5). The third codon positions are the most likely to reveal any
underlying evolutionary directions, as they are expected to be least subject to selective constraints.
Interestingly, the four protein coding genes that are encoded on the minority strand (nad1, nad4L,
nad4, nad5) were partitioned together, while those encoded on the majority strand were also
50
partitioned together (although they were further divided into two separate partitions). This suggests
that the underlying mutational model is different for the two strands of the mitochondrial genome, as
has been reported in mammals (Reyes et al., 1998). Finally, of the two partitions that contained 3rd
codon positions of genes encoded on the majority strand, a8, nad2 and nad6 were grouped together,
and have been reported to have the highest evolutionary rates in mammals and other insects (Li et al.,
2012; Saccone et al., 1999). Together, these observations indicate that PartitionFinder is able to
discover superior partitioning schemes for the phylogenetic analysis of mt genome data – they
produce more likely trees and may be more biologically realistic. This suggests that it may be useful
to reinvestigate previously analysed datasets, ones that were produced prior to the availability of
PartitionFinder.
51
CHAPTER 4:Coexistence of minicircular and a highly
rearranged mtDNA molecule suggests that
recombination shapes mitochondrial genome
organization
Mao M, Austin AD, Johnson NF, Dowton M (2014) Coexistence of minicircular and a highly
rearranged mtDNA molecule suggests that recombination shapes mitochondrial genome
organization. Mol Biol Evol. 31:636-644. doi:10.1093/molbev/mst255.
4.1 Introduction
The mitochondrial (mt) genome is one of the most highly used sources of molecular markers for
reconstructing phylogeny, both through alignment of orthologous sequences (Simon et al., 1994) and
through comparison of gene position (Boore et al., 1998). Analysis of aligned sequences indicates
that there are inherent biases in the number and types of nucleotide substitutions that occur, and that
incorporating this information into phylogenetic analysis improves the power of the analysis (Hillis
et al., 1994). To appreciate whether these patterns are general or specific to the taxon being studied,
it is also important to understand the mechanism of nucleotide substitution (Bakker et al., 2000). For
the same reasons, it is critical to understand the mechanism of gene rearrangement. But knowledge
on the mechanism and biases of gene rearrangement has remained elusive, because they occur much
more sporadically. Information on the mechanism and frequency of different types of rearrangement
can be best inferred by characterizing lineages with high rates of gene rearrangement (Dowton and
Austin, 1999).
52
There are three main models proposed to explain gene rearrangement: duplication/random loss
(Moritz et al., 1987; San Mauro et al., 2006), duplication/nonrandom loss (Lavrov et al., 2002) and
recombination (Dowton and Campbell, 2001). In the duplication/random loss model, slipped-strand
mispairing or inaccurate termination during replication can cause duplication of part of the mt
genome. The supernumerary gene copies are subsequently eliminated due to selection, which will
either restore or alter the original gene order. The duplication/random loss model is consistent with
the observed gene rearrangements in the vertebrates (San Mauro et al., 2006). However, it cannot
explain gene inversions and long-range translocations, which are common in invertebrate mt
genomes (Dowton et al., 2002). A defining feature of the duplication/nonrandom loss model is the
duplication of the entire mt genome, resulting in a dimeric molecule linked head to tail. Such a
dimeric molecule would contain two copies of transcriptional promoters. Mutation to one copy of
the promoters would result in the genes under the control of the disabled promoter becoming
pseudogenes; eventually these would be eliminated. After rearrangement, genes with identical
transcriptional direction will be clustered in the mt genome. However, the duplication/nonrandom
loss model also cannot explain gene inversions and translocations with different transcriptional
direction to genes close to the translocation point. Recombination was considered to be absent in
animal mtDNA based on early studies. For example, recombinant haplotypes of mtDNA were not
detected in somatic cell hybrids; sequestration of mtDNA molecules into clusters prevents physical
contact of unrelated molecules and there is also evidence that excision repair activity and crossover
products are absent in mammalian mitochondria (Clayton et al., 1974; Satoh and Kuroiwa, 1991;
Zuckerman et al., 1984). However, both direct and indirect evidence of animal mtDNA
recombination has been demonstrated more recently. In 1996, the end products of homologous
53
recombination were detected in human mitochondria (Thyagarajan et al., 1996). Lunt and Hyman
(1997) provided the most convincing evidence by characterizing the end products of recombination
in the nematode Meloidogyne javanica. A number of subsequent studies also provided evidence of
recombination in other animal species (Awadalla et al., 1999; D'Aurelio et al., 2004; Hoarau et al.,
2002; Kraytsberg et al., 2004; Ladoukakis and Zouros, 2001; Ujvari et al., 2007). However, it is not
yet widely accepted that mt recombination occurs (e.g., Jorde and Bamshad, 2000; Kivisild and
Villems, 2000; Kumar et al., 2000; Parsons and Irwin, 2000), while there is very little information on
how frequently it occurs.
The occurrence of recombination in animal mtDNA has substantial impact on evolutionary studies
that utilize mt genes. First, the traditional methods for phylogenetic analysis are based on the
assumption that animal mtDNA does not recombine; ignoring recombination might mislead
phylogenetic reconstruction (Rokas et al., 2003; Slate and Gemmell, 2004). Second, it offers an
intriguing understanding of how mt genes might rearrange. As the duplication/random loss model
cannot provide a full explanation of gene rearrangements across the Metazoa, recombination might
explain the gene inversions and long-range translocations that are often observed in invertebrate mt
genomes (Dowton and Campbell, 2001). The key component of this mechanism is the introduction
of double-strand breaks (DSBs) in the mt circle, with subsequent rejoining of the broken ends. Two
recent studies provided support for this mechanism: DSBs were demonstrated to play an important
role in the generation of mtDNA rearrangements in mice (Bacman et al., 2009). A more recent study
speculated that the presence of mtMUTS in the octocoral mt genome might promote recombination
which accounts for the gene inversions observed in that genome (Brockman and McFadden, 2012).
Here, we report the discovery of a minicircular mtDNA molecule in the megaspilid wasp
54
Conostigmus sp., similar to that reported by Lunt and Hyman (1997) in the nematode M. javanica.
We used a similar approach to investigate the presence of the end products of recombination: the
maxicircle and minicircle. We detected such end products, although we caution that polymerase
errors can produce a range of apparent recombination products. Furthermore, the possibility that the
minicircle comes from nuclear copies of mt sequences (numts; nuclear pseudogenes) is eliminated in
our study. We then investigate the structure of the minicircles produced and develop a model of
recombination that is consistent with the sequence data. We suggest that the presence of minicircles
is associated with the most highly rearranged invertebrate mt genomes (the Hymenoptera and
Phthiraptera) (Shao et al., 2009), lending credibility to the notion that recombination is an important
component of the mt genome rearrangement mechanism.
DNA extraction
Genomic DNA was extracted from 100% ethanol preserved specimens of Conostigmus sp. (collected
from Mount Barker, South Australia) using the “salting out” protocol (Aljanabi and Martinez 1997).
The DNA was resuspended in 100 μl of fresh TE solution (1 mM Tris–HCl, 0.1 mM EDTA [pH 8])
and stored at 4°C.
Amplification, sequencing, and annotation of the entire mt genome
Initially, small gene fragments were amplified and sequenced using either published universal
primers (Simon et al. 1994; Simon et al. 2006) or using primers designed from consensus
hymenopteran mt sequences. Using the sequence information obtained, specific primers were
designed for the amplification of the remaining regions. Short and long PCRs were performed using
55
BIOTAQ™ DNA Polymerase (Bioline, Australia) and Takara LA Taq (Takara Biomedical, Japan),
respectively, following the manufacturers’ instructions. For short PCRs, cycling condition consisted
of 94°C for 2 min, 35 cycles of 94°C for 30 sec, 45°C-65°C for 1 min, 72°C for 2 min, and a final
elongation for 10 min at 72°C. Long PCRs were performed with the following conditions: 94°C for
2 min, 30 cycles of 94°C for 1 min, 45°C-65°C for 30 sec, 68°C for 12 min, and a final elongation
for 10 min at 68°C. All PCR products were resolved by agarose gel electrophoresis and purified with
ExoSAP-IT® (GE Healthcare, Bucks, HP8 4SP, UK). The primer-walking method was used for
direct sequencing, using the ABIPRISM®BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied
Biosystems, Australia). Amplicons that were difficult to sequence directly were ligated into the
pGEM-T Easy Vector System (Promega, Madison, Wisconsin) and cloned into Escherichia coli
JM109 cells (Promega, Madison, Wisconsin). The sequences of both strands were determined for the
entire mt genome. Genes were identified using methods as previously described (Mao et al. 2012).
Amplification and sequencing of minicircles and the maxicircle
To detect the existence of minicircles, two specific, outward-facing primers (85-ATF and 85-ATR)
were designed, based on the A+T-rich region sequence, following the strategy of Lunt and Hyman
(Lunt and Hyman 1997). PCR amplification was carried out using Takara LA Taq. PCR products
were purified by agarose gel electrophoresis and cloned as described above. A total of 3-5 clones per
product were sequenced. The putative maxicircle was amplified using the primers 85-MR1 and
85-36R1 followed by a nested PCR with two internal primers 85-MR2 and 85-36R2. The PCR
product was purified and sequenced directly. The primers are indicated in Figure 4.1.
56
Figure 4.1 Organizations of the entire mt genome, maxicircle and minicircles that were initially characterized by
amplification. A. Organization of the entire mt A+T-rich region of Conostigmus sp. Different repeats are shown in
different colours. PCR primer sites and orientations are indicated by arrows. Breakpoint junctions are indicated by
black arrows. Regions with hatching indicate non-repeat regions. B. The real minicircles (L2) from different
individuals and the artifactual minicircles (L1, S1, and S2) generated by PCR errors. C. Organization of the
maxicircle A+T-rich region.
4.3 Results
The mt genome of Conostigmus sp.
The entire mt genome of Conostigmus sp. is 16,315 bp long. The overall A+T content is 82.9%. Like
other insect mt genomes, 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and the A+T-rich
region were identified. A series of gene rearrangements are evident relative to the putative ancestral
pancrustacean mt genome (Cook, 2005). Although tRNA genes are highly rearranged among wasps
(Dowton et al., 2009b), the protein-coding genes are highly conserved (but see Oliveira et al., 2008;
Xiao et al., 2011). In Conostigmus sp., 11 tRNA and 2 protein-coding genes are rearranged (Fig. 4.2).
Cox1 and nad2 are translocated into two junctions (Cox3-nad3 and rrnL-rrnS), respectively.
Interestingly, the gene orientations are almost identical to that of the ancestral organization, except a
57
single tRNA inversion of trnW.
Figure 4.2 Mitochondrial genome organization of Conostigmus sp., compared with the ancestral pancrustacean mt
genome organization (Cook 2005). tRNA genes are indicated by single-letter amino acid codes, L1, L2, S1, and S2
denote trnLCUN, trnLUUR, trnSAGN, and trnSUCN. Genes are transcribed from left to right except those indicated by
underlining. Gene movements, relative to the ancestral organization, are indicated with arrows.
The A+T-rich region is 1,447 bp long with an A+T content of 86.8%. It can be divided into three
subsections (Sec I, Sec II and Sec III) (Fig. 4.1A). Sec I contains seven copies of tandem repeat A
(the last one is a partial copy), nine copies of tandem repeat B, and one copy of non-tandem repeat C
and D. In the second subsection, several conserved elements (poly T stretch, [TA (A) ]n-like stretch,
TATA motif and stem-loop structure) can be identified, which are putatively involved in the initiation
of replication and transcription (Zhang and Hewitt, 1997). Sec III possesses four copies of tandem
repeat B and the second copy of non-tandem repeat C and D. D overlaps with trnG by 12 bp.
Initial characterization of the putative maxicircle and minicircles of Conostigmus sp.
The presence of distinct repeats within the A+T-rich region was reminiscent of those described by
Lunt and Hyman (Lunt and Hyman, 1997) in the nematode M. javanica, in which they characterized
both minicircular and maxicircular products of recombination. For this reason, we designed outward
facing amplification primers (85-ATF and 85-ATR, Fig. 4.1A) within the A+T-rich region in an
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attempt to amplify any minicircles that might be present in Conostigmus sp. A short extension time
should preclude the amplification of the entire mt genome, while any amplicons produced from the
entire mt genome would be easily distinguishable (based on their size of 16,100 bp) by agarose gel
electrophoresis. Thus, any amplicons smaller than 16,100 bp would be produced from minicircles. In
one reaction, four different sizes of minicircles (L1, L2, S1 and S2) were detected (Fig. 4.3A). To
test whether these putative minicircles could be amplified from a range of individuals, we amplified
them using the same primers in another two individuals caught at the same location (85B and 85C).
Four and three PCR products (L1 was lacking in 85C) were obtained respectively, with the sizes of
the various amplicons consistent with the minicircles from 85A (data for 85B-L1, 85B-L2 and
85C-L2, see supplementary Fig. 1A,
http://mbe.oxfordjournals.org/content/suppl/2013/12/11/mst255.DC1; data not shown for 85B-S1,
85B-S2, 85C-S1 and 85C-S2). We obtained sequence data from the most strongly amplified product
(L2) from each individual, the two shorter products (S1 and S2) from 85A, and the longest product
(L1) from 85B (Fig. 4.1B). However, we were not able to purify and sequence all products from
each individual, due to the low levels of some amplicons in some reactions. For example, we failed
to purify and sequence L1 from 85A.
After amplification of these putative minicircles, we attempted to amplify the maxicircle that would
be formed as the product of reciprocal recombination. If reciprocal recombination was responsible
for production of the minicircle, we should be able to detect a maxicircle; together, the minicircle
and maxicircle sequences should precisely match the sequence of the entire mt genome. To detect
the maxicircle, we designed primers that would amplify the A+T-rich region of the entire mt genome
(85-MR1 and 85-36R1, see Fig. 4.1A). Any maxicircles would be evident by the presence of shorter
59
than expected amplicons (as they would be missing the section encompassed by the minicircle). The
PCR amplification was performed and the only amplicon that was produced was one that represented
the A+T-rich region of the entire mt genome. To investigate whether there were low levels of
maxicircles present, we designed two internal primers (85-MR2 and 85-36R2) and performed nested
PCR. In this experiment, two PCR products, representing the A+T-rich region of the entire mt
genome and the maxicircle, were obtained. Sequence analysis of the maxicircle amplicon indicated
that it did correspond to the A+T-rich region of the entire mt genome (but lacking the minicircle
fragment), although two sites differed with the corresponding sequence of the A+T-rich region of the
entire mt genome (Fig. 4.1C). These sites were not close to the breakpoint junction. The inability to
amplify the maxicircle from genomic DNA, despite its smaller size compared with the entire mt
genome, might suggest that the subgenomic circles are not formed by reciprocal recombination. We
attempted to design a primer that straddles the putative breakpoint junction of the maxicircle in order
to perform reactions that would only amplify the maxicircle. However, this was unsuccessful due to
the lack of any sequence differences between the maxicircle breakpoint junctions. These junctions
occurred at two perfect (i.e., without any mismatches) microhomologies (21 bp) of non-tandem
repeat D, with one complete copy of repeat D kept in the maxicircle after formation. The breakpoint
junction primer would therefore anneal at three places: to the two copies of repeat D in the entire mt
genome, and the repeat D of the maxicircle (Supplementary Fig. 2,
http://mbe.oxfordjournals.org/content/suppl/2013/12/11/mst255.DC1).
We then carried out sequence analysis of the most strongly amplified PCR product L2 amplified
from 85A, representing the minicircle. The sequence of L2 corresponded to the fragment deleted
from the mt genome, based on the maxicircle sequence information. It comprised Sec II and part of
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Sec I and Sec III, although some variation existed when compared with the A+T-rich region of the
entire mt genome. In Sec II, the main difference was the length of the “TA” repeats. In Sec III, the
first copy of repeat B contained one site substitution while the last three copies were smaller; repeat
B is 10 bp long in the entire mt genome, but the last three copies are 9, 7, and 7 bp in the putative
minicircle. In addition, repeat D in Sec I was shorter, only containing the last 14 bp of that repeat
and with one site substitution. The breakpoint junctions occurred at two imperfect microhomologies
(14 bp) of non-tandem repeat D (Supplementary Fig. 2,
http://mbe.oxfordjournals.org/content/suppl/2013/12/11/mst255.DC1). This observation indicates
that the minicircle and maxicircle sequences do not precisely match the entire mt genome. This
suggests that the subgenomic circles are not formed by reciprocal recombination, unless the
recombination occurred some time ago, with the subgenomic circles accumulating mutations since
that time. Alternatively, the level of variation might be consistent with the notion that direct,
precisely matching repeat sequences are not necessary for the repair of DSBs (Bacman et al., 2009;
Mita et al., 1990; Samuels et al., 2004; Srivastava and Moraes, 2005).
We then compared the sequence of the fragments of L2 amplified from each individual. They were
mostly identical, with the main difference among them being the length of the TA repeats in Sec II.
The fragments S1 and S2 were 58% and 22% of the length of L2 in 85A. L1 was 50 bp longer than
L2 in 85B and most of the length difference was due to the number of TA repeats (21 repeats). All
the alignment data are shown in supplementary figure 1A and 1B
(http://mbe.oxfordjournals.org/content/suppl/2013/12/11/mst255.DC1).
61
Figure 4.3 Size separations of amplicons on 1% agarose gels. A. Putative minicircular amplicons obtained using
outward facing primers and genomic DNA (extracted from individual 85A). Primers were 85-ATF and 85-ATR. B.
minicircular PCR artifact obtained from plasmid DNA containing the A+T-rich region of the entire mt genome using
primers 85-ATF and 85-ATR. Although the size of the amplicon is similar to L2, sequencing indicated that the two
amplicons were distinct. C. minicircular amplicon obtained from genomic DNA extracted from individual 85A using
primers 85-ATF2 and 85-ATR. M, molecular weight marker (lambda DNA digested with EcoRI and HindIII).
Are the minicircles amplification artifacts?
As the A+T-rich region contained a complex series of repeats, the possibility remained that some of
these putative minicircles were artifacts of amplification. For example, PCR jumping, in which the
newly synthesized DNA strand jumps from one repeat to another, could produce small amplicons
that resembled those that would be produced by minicircles (or maxicircles). To investigate whether
the putative minicircles were artifacts caused by PCR jumping, we used plasmid DNA containing
cloned fragments of the A+T-rich region of the entire mt genome. In this way, we could examine
whether amplification of a template known not to contain minicircles could produce smaller
amplicons (i.e., artifactual minicircles), while sequencing would indicate whether they coincided
precisely with the putative minicircle amplicons characterized above (L1, L2, S1, and S2). In the
first set of experiments, we used a plasmid containing the A+T-rich region of the entire mt genome
as template, together with the same primers used to produce the minicircle amplicons (L1, L2, S1,
62
and S2). This reaction produced a single amplicon of size ~600 bp (Fig. 4.3B), but sequencing of this
fragment indicated that it was 639 bp and not similar to any of the minicircle amplicons. It contained
one copy of repeat C and D and nine copies of repeat B – this organization was not evident in either
the entire mt genome or in any of the minicircle amplicons. The amplification of a unique product
was surprising as we expected that molecules amplified from the cloned A+T-rich region should also
be amplified from genomic DNA: both templates contain the same sequence. We then investigated
whether the cloned A+T-rich region had rearranged during bacterial growth, but the sequence
precisely resembled the full-length A+T-rich region. This does not rule out the possibility that the
cloned DNA contained a small proportion of rearranged molecules, but we think it is more likely that
PCR jumping occurred during the amplification of the cloned A+T-rich region to a greater degree
compared with the genomic DNA amplification. This may be attributable to the much higher
concentration of target DNA. In the amplification utilizing the cloned A+T-rich region, the target is
not diluted by nuclear DNA, whereas in the genomic DNA amplification, the target (mt DNA)
represents only a fraction of the total DNA.
To investigate whether the presence of minicircles might also lead to PCR jumping, we used another
plasmid DNA containing the strongest amplified fragment 85A-L2. If PCR jumping did not occur,
we would expect to produce a single amplicon using the same PCR primers and conditions as used
in the original amplification. However, this amplification produced four bands, which corresponded
(in size) to each of the putative minicircles. Two of these amplicons were subcloned and sequenced
and found to be identical to the sequences of 85A-L2 and 85A-S1. We consider this strong evidence
that S1 is an artifact, produced by PCR template switching between the repeats present on the
minicircle. Although we could not purify and sequence the band corresponding to L1 and S2, the
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coincidence in size leads us to suspect that L1 and S2 are also artifacts of PCR jumping.
To further test whether the stongest amplicon (L2) was a PCR artifact, we performed two
experiments. First, we designed a primer (85-ATF2) that straddled the putative “breakpoint
junction” sequence and together with 85-ATR performed an amplification using genomic DNA (Fig.
4.1B). The breakpoint junction primer should only anneal to a minicircle, as this sequence is absent
in the entire mt genome. A single sharp band was obtained (Fig. 4.3C) and the sequence data showed
that it was identical to the corresponding part of L2. In the second experiment, we used the plasmid
containing the entire A+T-rich region as template, together with the same primer pair to perform
amplification. As expected, no PCR product was obtained, indicating that the breakpoint junction
primer does not misprime somewhere in the A+T-rich region. The two experiments showed that L2
is unlikely to be an artifact.
Elimination of the possibility of numts
numts commonly exist in metazoans (Bensasson et al., 2001). In our protocol, the amplification of
minicircles was carried out using genomic DNA. Therefore, the possibility that the minicircle came
from numts cannot be excluded. In order to investigate this possibility, we used a primer pair
(85-ATF2 and 85-MRF2) that would amplify the remainder of L2, if it was present on a minicircle.
These primers are inward facing on a minicircle but would be outward facing on a nuclear
chromosome. As expected, this reaction produced an amplicon with sequence identical to a section
of sec II of the A+T-rich region and, together with L2, conceptually comprises a circular molecule of
about 700 bp. The amplified fragment length varied slightly among different clones (see alignment
data in supplementary Fig. 1C,
64
http://mbe.oxfordjournals.org/content/suppl/2013/12/11/mst255.DC1). These differences might be
caused by polymerase error or represent minor variations present on different minicircles. The use of
outward-facing primers reduces the risk of amplifying numts, but does not completely exclude it. If a
numt is present as a tandem copy, outward facing primers would produce an amplicon. Indeed, if a
tandemly repeated numt existed which had a junction that precisely matched the breakpoints
identified in the putative minicircle L2, this template would produce amplicons with both of the pairs
of outward-facing primers described earlier (85-ATF and 85-ATR and 85-ATF2 and 85-MRF2).
However, a tandemly repeated numt would not produce the heterogeneity observed between clones
(all copies would be the same) nor the variation in repeat B and D that was observed in the
85-ATF/85-ATR amplification. Although the existence of a tandemly repeated numt cannot be
absolutely excluded, we consider its existence unlikely.
4.4 Discussion
Importance of control reactions
In this study, we report convincing evidence of the product of recombination (the minicircle) in the
mt genome of the parasitic wasp Conostigmus sp. However, we found that it was critical to establish
appropriate control reactions to identify and characterize artifacts produced by PCR jumping. We
suggest that this is especially important when repeats exist within the DNA template. Our
experiments indicated that it was necessary to perform control amplifications using both the isolated
(cloned) A+T-rich region and the putative minicircle in order to identify all non-biological products
of amplification. PCR artifacts were especially prevalent when using the cloned minicircle fragment,
suggesting that this sequence is more prone to PCR jumping. The main difference between these two
65
control sequences is the presence of the breakpoint junction sequence in the minicircle fragment.
Finally, our results suggest that it is critical to use breakpoint junction primers – primers that straddle
the putative breakpoint junction – in order to firmly establish the biological nature of putative
minicircles. Once the biological orgin of the minicircle was confirmed, it was also necessary to
demonstrate that the minicircle was derived from a circular (mt) molecule, as the existence of numts
might interfere with our identification of real recombination end products.
Possible explanations for low copy number of the maxicircle
The maxicircle detected in this study appeared to be present at very low copy number, as it could
only be detected by nested PCR. A low copy number of the maxicircle was also observed in human
tissues (Kajander et al., 2000). However, we interpret the existence of the maxicircle in our study
with some caution. As it could only be detected after nested PCR, we cannot exclude the possibility
that it was caused by PCR error. If, as we suspect, the minicircle contains the origin of replication
(discussed later), the absence of an origin of replication in the maxicircle would explain its presence
at very low copy number.
Comparison with minicircles from other Metazoa
The discovery of minicircles in a hymenopteran expands the number of taxonomic groups in which
minicircles have been characterized. Previously they have been characterized in humans (Holt et al.,
1988), the Phthiraptera (Cameron et al., 2011; Shao et al., 2009), and in nematodes (Armstrong et al.,
2000; Gibson et al., 2007; Lunt and Hyman, 1997), although the characteristics of the minicircles
vary between these groups. In humans, minicircles are generated by partial mt genome deletion, and
these minicircles coexist with the entire mt genome. In the Phthiraptera, three types of minicircles
66
have been identified: the first type is similar to the minicircles in humans (Cameron et al., 2011), the
second type has multiple genes and a short non-coding region (Cameron et al., 2011), and the third
type has one to three genes and a large non-coding region (Cameron et al., 2011; Shao et al., 2009).
In the nematodes, the minicircle of M. javanica is excised from the entire mt genome and coexsits
with both its reciprocal partner (maxicircle) and the entire mt genome (Lunt and Hyman, 1997);
minicircles found in the potato cyst nematode range from 6 to 9 kb (Armstrong et al., 2000; Gibson
et al., 2007). Some of the potato cyst nematode mincircles are mosaics, comprising multigenetic
fragments derived from other minicircles. The minicircle reported in the present study is most
similar to those reported in the nematode M. javanica, and the discovery of minicircles in four
taxonomically divergent groups indicates that they are likely to occur much more commonly than is
currently appreciated.
We found that there were both similarities and differences between the minicircle of Conostigmus sp.
and the minicircle reported by Lunt and Hyman (Lunt and Hyman, 1997) in the nematode M.
javanica. The minicircles were similar in that they were 1) both excised from the A+T-rich region of
the entire mt genome and 2) comprised of different repeats flanked by unique sequences. The
differences were mainly in two aspects. First, the breakpoints in the mt genome of Conostigmus sp.
occurred in two copies of a non-tandem repeat, and imperfect microhomologies (14 bp) could be
found around the breakpoints (supplementary Fig. 2,
http://mbe.oxfordjournals.org/content/suppl/2013/12/11/mst255.DC1). In contrast, only short (3 bp)
or no direct repeats could be found around the breakpoints in the entire mt genome of the nematode
M. javanica. Second, nucleotide deletion occurred upstream of the second breakpoint in the
minicircles of all individuals examined in this study, when compared with the entire mt genome. No
67
such deletions were evident in M. javanica. Two candidate explanations might be responsible for the
nucleotide variation within an individual. First, the minicircles were not recently formed but instead
were generated long ago and transmitted to subsequent generations. Another explanation for the
nucleotide variation is that mismatching might be tolerated during recombination as perfect
microhomologies are not necessary for the repair of DSBs. Both explanations might also be
responsible for the observation that the minicircles are highly similar in related individuals.
Unfortunately, it is very difficult to experimentally distinguish between these two scenarios.
We suspect that the minicircles contain origin of replication information, and that these smaller
molecules are likely to replicate faster than larger ones (Diaz et al., 2002). This could explain the
higher levels of minicircle compared with the maxicircle.
Possible mechanism yielding the minicircle and maxicircle
The mechanism that produces mtDNA minicircles is still unclear. The characterization of a suite of
minicircles, from evolutionarily divergent organisms, is perhaps the best approach for identifying the
key mechanistic components. Thus far, the production and repair of DSBs appears to be associated
with the existence of sublimons (ΔmtDNA) in humans (Krishnan et al., 2008). Recent research of
rodent mtDNA showed that DSBs promoted recombination, resulting in the formation of sublimons
(Bacman et al., 2009; Srivastava and Moraes, 2005).
Mitochondria have been shown to have an effective system of DSB repair in Drosophila (Morel et
al., 2008). Krishnan et al. (Krishnan et al., 2008) proposed that 3’→5’exonucleases could generate
single-stranded regions at DSBs, and these exposed single-strand regions could subsequently anneal
at regions containing microhomologous sequences, and that this would result in the formation of
68
sublimons. We speculate that the minicircle and maxicircle of Conostigmus sp. were probably
generated by a similar mechanism. However, in order to generate both a minicircle and a maxicircle,
we propose that two breakpoints must occur simultaneously near or within the direct repeats (14 bp)
(Fig. 4.4). After the minicircle sequence was excised, another pair of microhomologous sequences
(up to 21 bp, the sizes varies depending on the position of the breakpoints) could be detected around
thebreakpoints (supplementary Fig. 2,
http://mbe.oxfordjournals.org/content/suppl/2013/12/11/mst255.DC1), which probably facilitated the
formation of the maxicircle. Ligase III has been identified to play an important role in maintaining
mtDNA integrity (Gao et al., 2011); the ends of the linearized DNA around the breakpoints were
probably joined by ligase III.
Figure 4.4 Model for the generation of minicircle and maxicircle mtDNA by recombination during repair of DSBs.
Direct repeats which facilitate the formation of minicircle and maxicircle mtDNA are shown in red and purple regions,
respectively.
Direct link between intra-mitochondrial recombination and mt gene rearrangement
The Hymenoptera is now the second insect order after the Phthiraptera in which minicircles have
been detected (Shao et al., 2009). Coincidently, both groups possess the most rearranged mt genomes
among insects. We suggest that there might be a direct link between these two phenomena. Although
69
intra-mitochondrial recombination has been considered as a possible mechanism for gene
rearrangement in animal mt genomes (Dowton and Campbell, 2001), there has been very little direct
evidence to support this. One reason for this is that the evidence, in the form of excised partial mt
genomes, direct repeats, or inverted repeats, may be erased soon after (in evolutionary terms) the
recombination event (Brockman and McFadden, 2012). The presence of minicircles, combined with
the highly rearranged mt genome reported in this study, supports the notion that recombination is an
important component of the mt gene rearrangement mechanism. Our working hypothesis is that the
generation of minicircles is a crucial first step in the rearrangement mechanism, but that these
minicircles may have to be long-lived (i.e. contain an origin of replication). The integration of these
minicircles back into the full-length mt genome, via homologous recombination, then generates gene
rearrangements. This hypothesis not only gives a good explanation to the existence of minicircles
but also accommodates the existence of the non-tandem repeat fragments that are found in some taxa,
such as those whose mt genome contains two or three copies of the control regions (Kumazawa et al.,
1996; Kurabayashi et al., 2008; Mueller and Boore, 2005). These duplicated control regions have
been postulated to be generated and maintained by recombination (Kurabayashi et al., 2008; Mueller
and Boore, 2005).
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CHAPTER 5: Evolutionary dynamics of the
mitochondrial genome in the Evaniomorpha
(Hymenoptera) – a group with an intermediate rate of
gene rearrangement
Mao M, Gibson T, Dowton M (2014) Evolutionary dynamics of the mitochondrial genome in the
Evaniomorpha (Hymenoptera) - a group with an intermediate rate of gene rearrangement. Genome
Biol Evol. 6, 1862-1874. doi:10.1093/gbe/evu145.
5.1 Introduction
Knowledge of the forces that shape the organization of the animal mitochondrial (mt) genome has
suffered from a lack of suitable model systems. This is because the rate of gene rearrangement in
most animal mt genomes is generally extremely slow; for example most vertebrates have identically
arranged mt genomes (Boore, 1999). The ideal model system would provide multiple gene
rearrangements for comparison (in order to identify common themes), but in a lineage that is not
rearranging so frequently that hidden and convergent rearrangements obscure the interpretation of
evolutionary events. In the insects, a number of higher level lineages have been identified with
accelerated rates of mt gene rearrangement – the Hymenoptera (Dowton and Austin, 1999) and the
hemipteroid orders (Shao et al., 2001) are two examples. Within these rapidly rearranging lineages,
taxonomic sampling at lower levels may be all that is required to identify good model groups; that is,
ones in which the evolutionary trajectory of genome reorganization is straightforward to interpret. In
the present study, we investigated whether one group of the Hymenoptera (the Evaniomorpha) might
71
provide such a model system.
The Hymenoptera (sawflies, wasps, ants and bees) is one of the most important components of insect
diversity. As the third largest insect order, it is estimated to contain more than 140,000 extant species,
placed into two traditional suborders (Symphyta and Apocrita) (Huber, 2009). The Apocrita has long
been considered as a natural group and comprises more than 90% of the Hymenoptera (Huber, 2009;
Sharkey, 2007). Many apocritan species play valuable roles in biological control, ecosystem and
production of commercial products (LaSalle and Gauld, 1993).
Traditionally, the Apocrita is subdivided into two groups, the Aculeata (stinging wasps) and the
Parasitica (parasitoid wasps), with the Aculeata now widely accepted as being derived from within
the Parasitica (Sharkey, 2007). The reconstruction of a robust phylogeny for the Apocrita has long
been of great interest to hymenopteran systematists (reviewed by Sharkey, 2007). In 1988, Rasnitsyn
proposed a fully resolved phylogenetic hypothesis of higher level hymenopteran relationships based
on morphological and fossil evidence (Rasnitsyn, 1988). Despite the use of non-cladistic
methodology, Rasnitsyn’s research remains influential and sets a stage for current research of
hymenopteran phylogeny. In his hypothesis, the Apocrita was divided into four lineages, the
Ichneumonomorpha, the Vespomorpha (Aculeata), the Proctotrupomorpha and the Evaniomorpha
(Fig. 5.1). The Ichneumonomorpha and Aculeata have long been recovered as natural groups (Castro
and Dowton, 2006; Dowton and Austin, 2001; Dowton et al., 1997; Heraty et al., 2011; Klopfstein et
al., 2013; Rasnitsyn and Zhang, 2010; Sharkey et al., 2011; Vilhelmsen et al., 2010) and the
monophyly of the Proctotrupomorpha has been supported with several comprehensive studies
(Castro and Dowton, 2006; Heraty et al., 2011; Klopfstein et al., 2013; Sharkey et al., 2011).
However, the monophyly of the Evaniomorpha (including the Ceraphronoidea, Evanioidea,

72
Megalyroidea, Stephanoidea and Trigonalyoidea) remains unclear from both morphological and
molecular analyses. In morphological analyses, a subsequent numerical cladistic analysis of
Rasnitsyn’s (1988) data failed to retrieve the Evaniomorpha (Ronquist et al. 1999). Gibson (1999)
suspected that the mesocoxal articulatory structure, which supported the monophyly of the
Evaniomorpha according to Rasnitsyn (1988), was a retained symplesiomorphy rather than a
synapomorphy (Gibson 1999). Moreover, Rasnitsyn himself proposed that the Evaniomorpha was
not monophyletic and divided it into three lineages (Rasnitsyn and Zhang, 2010). Most molecular
analyses recovered the Evaniomorpha as paraphyletic, with the Aculeata nested within the
Evaniomorpha. Further, the sister group of the Aculeata varied among different analyses (Castro and
Dowton, 2006; Heraty et al., 2011; Klopfstein et al., 2013; Sharkey et al., 2011). Within the
Evaniomorpha, there is little consensus on the phylogenetic relationships among the superfamilies
due to a lack of reliable morphological characters and the limited number of molecular markers.
Figure 5.1 Simplified interpretation of the phylogenetic relationships among the major lineages within the Apocrita
from Rasnitsyn (1998).
Prior to this study, there were only four evaniomorph mt genomes available in GenBank,
representing three superfamilies [Evania appendigaster (Evanioidea: Evaniidae), Pristaulacus
compressus (Evanioidea: Aulacidae), Schlettererius cinctipes (Stephanoidea: Stephanidae) and
Conostigmus sp. (Ceraphronoidea: Megaspilidae)] (Dowton et al., 2009a; Mao et al., 2014a; Wei et
al., 2010b; Wei et al., 2013). Each of these mt genomes displayed a relatively small number of
73
changes to their mt genome organization, when compared with the ancestral
pancrustacean/hymenopteran organization, and these rearrangements involved both tRNA genes and
(to a lesser extent) protein-coding genes. Schlettererius had evidence of two tRNA gene
rearrangements (Dowton et al., 2009b), Pristaulacus had evidence of one protein-coding gene (nad1)
rearrangement and two tRNA gene rearrangements (Wei et al., 2013), Evania had evidence of four
tRNA gene rearrangements (Wei et al., 2010b), whereas Conostigmus had 2 protein-coding genes
rearranged (cox1, nad2), and 10 tRNA genes rearranged (Mao et al., 2014a). Thus this group
represents a good candidate model system in which to investigate the dynamics of mt genome
rearrangement, as individual rearrangements might be inferred through denser taxonomic sampling.
For this reason, in this study we present four new mt genomes for representatives of four
evaniomorph superfamilies, Megalyra sp. (Megalyroidea: Megalyridae), Orthogonalys pulchella
(Trigonalyoidea: Trigonalyidae), Ceraphron sp. (Ceraphronoidea: Ceraphronidae) and Gasteruption
sp. (Evanioidea: Gasteruptiidae). We also use these entire mt genomes to further investigate
relationships among the Evaniomorpha and Aculeata. Because of the likely paraphyletic nature of
the Evaniomorpha, we include the Aculeata in order to examine the phylogeny within a natural
group.
5.2 Materials and Methods
DNA extraction
The collection details for each study species are listed in Table 5.1. Genomic DNA was extracted
from 100% ethanol preserved specimens using the ‘salting out’ protocol (Aljanabi and Martinez,
1997). The DNA was resuspended in 100 μl of fresh TE solution (1 mM Tris–HCl, 0.1 mM EDTA
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[pH 8]) and stored at 4°C.
Table 5.1 Species sequenced in this study, collection data and GeneBank accession numbers.
Species Library reference Family Superfamily Collection locality Accession number

Ceraphron sp. M247 Ceraphronidae Ceraphronoidea California, USA KJ570858
Gasteruption sp. M19 Gasteruptionidae Evanioidea Auburn, South Australia KJ619460
Megalyra sp. M272 Megalyridae Megalyroidea Wistow, South Australia KJ577600
Orthogonalys pulchella M16 Trigonalyidae Trigonalyoidea Clarke County, Washington, USA KJ619461
Mt genome amplification, sequencing, and annotation
Short gene fragments were amplified and sequenced using a range of universal insect mt primers
(Simon et al., 1994; Simon et al., 2006) and primers that had been previously designed from
consensus hymenopteran mt sequences. Using the sequence information obtained, taxon-specific
primers were designed for each sample to amplify the remaining regions. PCR and sequencing
reactions were conducted as previously described (Mao et al., 2014a). The sequences of both strands
were determined for the entire mt genomes of Ceraphron sp., Gasteruption sp., and O. pulchella, In
addition, we sequenced both strands for all coding regions and most of the A+T-rich region of
Megalyra sp., but failed to get the full double stranded sequence for the entire A+T-rich region due
to an array of 50 bp tandem repeats. This array contained 20 repeats, and as a result, no unique
internal sequencing primers could be designed.
Raw sequences were assembled into contigs in ChromasPro Ver 1.33 (Technelysium Ltd., Tewantin,
Australia). tRNA genes were identified using tRNA-scan SE 1.21 (lowelab.ucsc.edu/tRNAscan-SE/)
(Lowe and Eddy, 1997) and ARWEN 1.2 (http://130.235.46.10/ARWEN/) (Laslett and Canbäck,
2008). ORFinder (www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to identify protein-coding genes,
specifying the invertebrate mt genetic code. The start and stop codons of some genes were corrected
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according to the boundaries of tRNA genes and through alignment with other hymenopteran mt
sequences. rRNA genes were identified using BlastN and the ends of genes were assumed to extend
to the boundaries of the neighboring tRNA or protein-coding genes. However, the boundary between
rrnL and rrnS was difficult to define in Megalyra sp., as there is no gene between them. In this case,
we determined this boundary through sequence comparison with published hymenopteran mt rRNA
sequences.
Nucleotide composition, gene rearrangement, and repeat analyses
The A+T content was determined for the major strand of each of the four mt genomes by MEGA5
(Tamura et al., 2011). In order to investigate whether gene inversions influenced the nucleotide
compositional biases, we also measured the AT and GC skews for some protein-coding genes (nad2,
cox1, nad6, and cob) and two rRNA genes of 8 evaniomorph taxa and 12 aculeate taxa. These genes
were inverted in at least one of the four new mt genomes (see below). The formulae used were
AT-skew = (A - T) / (A + T) and GC-skew = (G - C) / (G + C) (Perna and Kocher, 1995).
Gene rearrangement analyses were conducted with CREx via the freely available CREx web server
(http://pacosy.informatik.uni-leipzig.de/crex) (Bernt et al. 2007). We compared gene orders using a
dissimilarity measurement – number of breakpoints and visually inspected the output diagram to
identify shared, derived gene rearrangements.
Direct and inverted repeats longer than 12 bp were identified for the A+T-rich region and the entire
mt genome of each evaniomorph taxa using UGENE v.1.13.1 (Okonechnikov et al., 2012). We
calculated the number of different size repeats around the breakpoints and in the A+T-rich region to
investigate whether any particular size of repeat facilitate gene rearrangement.
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Sequence alignment and phylogenetic analysis
A total of 24 taxa were analyzed in this study, including 8 evaniomorph taxa, 12 aculeate taxa, and 4
symphytan taxa. Nucleotide sequences for each of the 13 protein-coding genes and the 2 rRNA
genes were imported into separate files using MEGA5 and aligned using Muscle (Edgar, 2004) or
MAFFT (Katoh et al., 2005). For the protein-coding genes (excluding the stop codons), an amino
acid alignment was generated first for each gene in Muscle as implemented within MEGA5, or
MAFFT at the freely available TranslatorX server (http://translatorx.co.uk/) (Abascal et al., 2010). A
nucleotide alignment was then inferred from the amino acid alignment. The alignment parameters
for all genes in Muscle were the default settings, which have been specified in a previous study (Mao
et al., 2012). For the MAFFT alignment of the rRNA genes, we used the G-INS-i algorithm as
implemented in the MAFFT web server (http://mafft.cbrc.jp/alignment/server/) (Katoh et al., 2005).
MAFFT (G-INS-i) has been shown to be more accurate than other programs (Golubchik et al., 2007).
No regions were excluded from the rRNA alignments. Individual gene alignments were
concatenated prior to phylogenetic analysis.
The best partitioning schemes and corresponding nucleotide substitution models were determined
with PartitionFinder version 1.0.1 (Lanfear et al., 2012) using the Bayesian Information Criterion
(BIC) and a heuristic search algorithm. A total of 41 data blocks were predefined (3 codon positions
of 13 protein-coding genes + 2 rRNA genes). Maximum Likelihood and Bayesian approaches were
employed to infer phylogenetic trees. Maximum Likelihood analyses were conducted with RAxML
via the available RAxML BlackBox server (http://embnet.vital-it.ch/raxml-bb/) (Stamatakis et al.,
2008). The GAMMA model of rate heterogeneity was employed for all partitions. The “Maximum
likelihood search” and “Estimate proportion of invariable sites” boxes were selected, with a total of
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100 bootstrap replicates performed. Bayesian analyses were conducted with MrBayes v. 3.2.2
(Ronquist et al., 2012a) via the online CIPRES Science gateway portal (Miller et al., 2010). The
Markov chain Monte Carlo (MCMC) process was set so that four chains (three heated and one cold)
ran simultaneously. Four separate runs using unlinked partitions (unlink statefreq = all; unlink
revmat = all; unlink shape = all; unlink pinvar = all; prset applyto = all; ratepr = variable) for a total
of 1,000,000 generations were performed, with sampling every 100 generations. Stationarity for each
run was assessed by importing the parameter files into Tracer v. 1.5 (Rambaut and Drummond,
2009).
Rogue taxon identification
‘Rogue’ taxa, defined as those that have an unstable position in topological trees (Wilkinson, 1996),
can obscure relationships that are consistently recovered during bootstrap and bayesian analyses
(Aberer et al., 2013). In order to reveal those relationships that were consistently recovered by our
analyses, we conducted rogue taxon filtering using the approach implemented in RogueNaRok
(http://rnr.h-its.org/rnr) (Aberer et al., 2013). The RAxML bootstrap tree sets and Bayesian tree sets
(excluding burn-in) were employed to perform these analyses.
5.3 Results and Discussion
General features of mt genomes
Three complete and one nearly complete mt genomes were sequenced for this study: Ceraphron sp.
(14,947 bp), Gasteruption sp. (17,884 bp), O. pulchella (17,277 bp) and Megalyra sp. (18,996 bp).
For Megalyra sp., we were able to estimate the size of the genome with a reasonable degree of
precision, because imperfect copies exist in the array of 50 bp tandem repeats, for which we failed to
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get the full double stranded sequence. Each genome contains all of the 37 genes commonly found in
animal mt genomes (Boore, 1999). In addition, a second copy of trnE was identified in the O.
pulchella mt genome. As the shortest hymenopteran complete mt genome to date, the Ceraphron sp.
mt genome is extremely compact with a total of only 105 bp of short non-coding regions (excluding
the A+T-rich region). The short non-coding regions in Megalyra sp., Gasteruption sp. and O.
pulchella are 345 bp, 909 bp and 919 bp long, respectively. The four mt genomes have a similar A+T
content, ranging from 80% (Ceraphron sp.) to 83.8% (O. pulchella). The AT and GC skew analysis
showed that gene inversions had no influence on nucleotide compositional biases (data not shown).
Three of the conventional start codons (ATA, ATG or ATT) could be assigned to all of the
protein-coding genes. Most tRNA genes have a typical cloverleaf structure. The exceptions are trnS1
in all four species, trnS2 in Ceraphron sp. and trnR in Ceraphron sp. and Gasteruption sp. All of
these tRNA genes lack the D-stem pairings in the dihydrouridine (DHU)-arm. The missing D-stem
has been commonly noted for the trnS1 gene in insects (Mao et al., 2012; Sheffield et al., 2008; Yang
et al., 2013), while to our knowledge, it has not been previously reported in trnS2 in insect mt
genomes. The missing D-stem in trnR is likely a common feature of hymenopteran mt genomes. In
addition to the two mt genomes reported here, it was also found in another two evaniomorph taxa
(Conostigmus sp. and S. cinctipes) (Dowton et al., 2009a; Mao et al., 2014a) and in two sceliond mt
genomes (Ceratobaeus sp. and Idris sp.) sequenced in our laboratory (Mao and Dowton, 2014).
Furthermore, the 3’ end of the aminoacyl acceptor stem of trnR overlaps with the downstream gene
in Ceraphron sp., Megalyra sp. and O. pulchella. The truncated aminoacyl acceptor stem is probably
restored to conventional structure by extensive tRNA editing (Dowton and Austin, 1999; Lavrov et
al., 2000; Masta and Boore, 2004; Segovia et al., 2011). The considerable difference in genome size
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among the four evaniomorph taxa is mainly due to variation in the size of the A+T-rich region,
which ranges from 692 bp (Ceraphron sp.) to 3,871 bp (Megalyra sp.). One or more series of tandem
repeats could be found in the Megalyra sp., O. pulchella, and Gasteruption sp. mt genomes, whereas
only three short non-tandem repeats are present in the Ceraphron sp. mt genome (Fig. 5.2 and
Supplementary Fig. 1A, http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evu145/-/DC1).
Most notably, two copies of the A+T-rich region exist in the Gasteruption sp. mt genome, which are
separated by trnL2. The second copy is 77.7% of the length of the first copy and the length difference
is mainly due to the variation in repeat number of an 11 bp microsatellite sequence (Supplementary
Fig. 1B, http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evu145/-/DC1). Similarly, the
major portion of the A+T-rich region in the O. pulchella mt genome is also duplicated
(Supplementary Fig. 1C, http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evu145/-/DC1).
The presence of duplicated A+T-rich regions (control regions) has been reported in a diverse range
of taxa, such as mantellid frogs (Kurabayashi et al., 2008), Amazona parrots (Eberhard et al., 2001)
and Australasian Ixodes ticks (Shao et al., 2005). It is proposed that the presence of two control
regions may be advantageous and be maintained either through stabilizing selection or through gene
conversion (Eberhard et al., 2001).
The four mt genome organizations are shown in Figures 5.3-5.5. All of them possess dramatic gene
rearrangements when compared with the ancestral pancrustacean mt genome organization (Cook,
2005), which is also thought to represent the ancestral organization of the hymenopteran mt genome
(Mao et al., 2012). Remarkably, each of the newly sequenced mt genomes has protein-coding or
rRNA gene rearrangement(s), a feature which has not been commonly reported in the Hymenoptera
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[only 8 of the 38 available mt genomes (this number excludes congenerics) have protein-coding or
rRNA gene rearrangements] (Castro et al., 2006; Dowton et al., 2009b; Mao et al., 2014a; Oliveira et
al., 2008; Wei et al., 2010a; Wei et al., 2013; Xiao et al., 2011).
Figure 5.2 Structure of the A+T-rich region in the Ceraphron sp., Gasteruption sp., Megalyra sp. and Orthogonalys
pulchella mt genomes. TR1-N and NTR1-N indicate the distinct tandem repeats and non-tandem repeats in each
genome, respectively. The 80 bp repeat in the O. pulchella mt genome, which was difficult to classify as either
tandem or non-tandem, is labeled as R1. The length and copy number of each repeat are indicated above each repeat.
The small repeats that nest within the large repeats are not shown. The width of different boxes is not proportional but
indicative of the size of a repeat within a particular genome. Different repeats in each mt genome are indicated with
different colors. Boxes with asterisks underneath represent a partial copy of a repeat.
The mt genome of Ceraphron sp.
In Ceraphron sp., two protein coding genes (nad2 and nad6) and 10 tRNA genes have rearranged
relative to their ancestral positions. The tRNA gene rearrangements mainly occur at the nad3-nad5
junction and around the A+T-rich region. Four tRNA genes (trnA, trnR, trnN, and trnS1) have moved
from the nad3-nad5 junction to the tRNA gene cluster downstream of the A+T-rich region. trnE has
also moved out of the nad3-nad5 junction, and we propose that it might be involved in two gene
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rearrangement events. First, it moved to the tRNA gene cluster downstream of the A+T-rich region
together with the other four tRNA genes, then it inverted and moved to the nad1-trnL1 junction
together with nad2. This is a more parsimonious scenario than independent movement with
inversion of both genes (trnE and nad2) to the same gene junction. We also compared the mt
genome organization of Ceraphron sp. with another ceraphronoid taxon, Conostigmus sp. (Fig. 5.3),
the sequence of which was reported in a previous study (Mao et al., 2014a). In both ceraphronoids,
the trnQ, trnN, trnS1, and trnV genes have moved to the tRNA gene cluster upstream of the cox1
gene, whereas the trnW gene has inverted. The nad2 gene has rearranged in both taxa but into
different positions. The rearrangement of nad2 has also been reported in two chalcidoid taxa,
Nasonia and Philotrypesis, in which nad2 has also moved into different positions (Oliveira et al.,
2008; Xiao et al., 2011). The most striking gene rearrangement in both ceraphronoids is that the two
rRNA genes are separated by protein-coding genes (nad6 in Ceraphron sp. and nad2 in Conostigmus
sp.) instead of trnV lying between them, which has not been observed in other Hymenoptera. We
consider that the existence of different protein-coding genes between the two rRNA genes might be
useful markers to resolve the internal relationships of the Ceraphronoidea.
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Figure 5.3 Mitochondrial genome organization of two taxa from the Ceraphronoidea, Ceraphron sp. (present study)
and Conostigmus sp. (Mao et al. 2014), compared with the ancestral pancrustacean/hymenopteran mt genome
organization. tRNA genes are indicated by single-letter amino acid codes, L1, L2, S1 and S2 denote trnLCUN, trnLUUR,
trnSAGN and trnSUCN, respectively. Genes are transcribed from left to right except those indicated by underlining.
Different types of gene movements, relative to the ancestral organization, are indicated with different colors: blue
represents long-distance translocation; red represents long-distance translocation and inversion; purple represents
local translocation; green represents local inversion.
The mt genome of Gasteruption sp.
The Gasteruption sp. mt genome possesses 11 gene rearrangements (two protein-coding genes and
nine tRNA genes) and a duplication of the A+T-rich region (Fig. 5.4) relative to the ancestral
hymenopteran. The cox1 gene has inverted and moved into a position upstream of the A+T-rich
region. The nad2 gene has also inverted, but remains in its ancestral location. Among the rearranged
tRNA genes, most of them have inverted and moved to positions remote from their ancestral
locations, although trnY has inverted, but remains in its ancestral location (downstream of nad2). In
addition, trnQ has moved to a position downstream of nad2. The existence of duplicate A+T-rich
regions separated by trnL2 is consistent with the duplication/random loss model (Moritz et al., 1987).
A parsimonious explanation is the cox1 and trnL2 genes inverted and moved to the position upstream
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of the A+T-rich region simultaneously (cox1-trnL2→trnL2-cox1), then a duplication of cox1, trnL2
and the A+T-rich region followed by random deletions of the redundant copy of the cox1 and trnL2
genes occurred. An important evidence for this explanation is that two copies of a 10 bp
complementary repeat exist at the beginning of rrnS and at the end of trnL2 (AAAAGTATTT in rrnS
and TTTTCATAAA in trnL2). We consider this short repeat may promote the cox1 and trnL2
inversion and long-range movement into the region between rrnS and A+T-rich region via
recombination (Dowton and Campbell, 2001; Mao et al., 2014a). We then compared the mt genome
organization of Gasteruption sp. with another two evanioid taxa, E. appendigaster and P.
compressus (Fig. 5.4). The gene positions are relatively conserved in E. appendigaster and P.
compressus with only four and three gene rearrangements, respectively, when compared with the
ancestral pancrustacean/hymenopteran. Although two tRNA genes (trnS2 and trnW) have inverted in
both Gasteruption sp. and Evania appendigaster, no mt gene rearrangements are shared among the
three taxa. Similarly, trnS2 is no longer adjacent to cob in all three taxa, but it has different, derived
positions. Thus, among the three sequenced evanioid mt genomes, we identified 18 independent
gene rearrangements, indicating that these could be extremely useful characters for assessing
phylogeny in this superfamily.
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Figure 5.4 Mitochondrial genome organization of three taxa from the Evanioidea: Gasteruption sp. (present study),
Evania appendigaster (Wei et al., 2010b) and Pristaulacus compressus (Wei et al., 2010b). Genes are transcribed
from left to right except those indicated by underlining. Different types of gene movements, relative to the ancestral
organization, are indicated with different colors: blue represents long-distance translocation; red represents
long-distance translocation and inversion; purple represents local translocation; green represents local inversion.
The mt genomes of Megalyra sp. and O. pulchella
Figure 5.5 shows the mt genomes of Megalyra sp. and O. pulchella sequenced in this study.
Megalyra sp. is from the superfamily Megalyroidea, whereas O. pulchella is from the superfamily
Trigonalyoidea. The sister relationship of these two superfamilies has been recovered by some
molecular analyses (including this study, see below) (Castro and Dowton, 2006; Dowton and Austin,
2001; Dowton et al., 1997; Heraty et al., 2011; Klopfstein et al., 2013), so we illustrate them in the
one figure to facilitate comparisons. Both of them possess dramatic gene rearrangements when
compared with the ancestral pancrustacean/hymenopteran. In Megalyra sp., there are eight gene
rearrangements. The most striking rearrangement is the one in which the two rRNA genes have
inverted and moved into the nad2-cox1 junction. Inverted rRNA genes have been reported in other
insect orders, the Phthiraptera and Thysanoptera (Cameron et al., 2007a; Cameron et al., 2011;
Covacin et al., 2006; Shao and Barker, 2003). Interestingly, these four studies reported that both
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rRNA genes were inverted, similar to our observations in the Megalyra sp. mt genome. We suspect
that there might be an advantage to having the two rRNA genes encoded on the same strand in
insects. One candidate mechanism responsible for this phenomenon is the transcriptional feedback
system regulating mtDNA replication. In this mechanism, the initiation of mtDNA replication is
hypothesized to be positively correlated with the transcription rate, with the latter negatively
regulated by the accumulation of gene products. Therefore, mtDNA replication is indirectly
regulated by the amount of synthesized products (Axel and Thomas, 2014). The rate of rRNA
synthesis was found to be up to 60-fold higher than mRNA synthesis (i.e. of protein-coding genes) in
humans due to the transcription termination factor binding mtDNA immediately downstream of the
two rRNA genes (Martin et al., 2005). A significant difference between the transcription levels of
rRNA and mRNA was also found in Drosophila (Torres et al., 2009). We suspect that the
hymenopteran mt genome might also have a higher rate of rRNA synthesis. Therefore, the
mechanism of transcriptional feedback system regulating mtDNA replication would confer an
advantage to maintaining both rRNA genes on the same strand, as regulation of the synthesis of both
rRNA genes would occur concomitantly. The retention of the rRNA genes on the minor strand in
other available evaniomoph mt genomes indicates that the inversion occurred after the divergence of
the Megalyroidea. In O. pulchella, 4 protein coding genes (nad2, cox1, nad6, and cob) and 10 tRNA
genes have rearranged relative to the ancestral positions. nad2 and cox1 have moved into the
position upstream of the A+T-rich region and the nad3-nad5 junction, respectively. nad6 and cob
have inverted and swapped positions (nad6-cob→cob-nad6). The gene boundary cob-nad6 was also
characterized in Venturia canescens (Ichneumonoidea) and Cotesia vestalis (Ichneumonoidea)
(Dowton et al., 2009b; Wei et al., 2010a), similar to our observations in O. pulchella. However,
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these two superfamilies (Trigonalyoidea and Ichneumonoidea) are not closely related, indicating that
this arrangement has likely evolved on two or three occasions independently. Therefore, it is
probably one of the few examples of convergent evolution of gene order involving protein-coding
gene rearrangements. Two tRNA genes (trnQ and trnY) have inverted in both Megalyra sp. and O.
pulchella, but they are in different, derived positions. In Megalyra sp., both trnQ and trnY remain in
their ancestral locations, whereas in O. pulchella, they have moved to the position downstream of
rrnS. Furthermore, trnH is no longer between nad5 and nad4, but the derived positions are different
in the two taxa.
Figure 5.5 Mitochondrial genome organization of Megalyra sp. (Megalyroidea) (present study) and Orthogonalys
pulchella (Trigonalyoidea) (present study). The sister relationship of these two superfamilies is supported by present
study and some other molecular studies. Genes are transcribed from left to right except those indicated by underlining.
Different types of gene movements, relative to the ancestral organization, are indicated with different colors: blue
represents long-distance translocation; red represents long-distance translocation and inversion; purple represents
local translocation; green represents local inversion.
Evolutionary dynamics of evaniomorph mt genomes
A total of 64 gene rearrangements were identified in the seven evaniomorph mt genomes, including
4 local translocations (within a tRNA gene cluster and without inversion), 32 gene inversions and 49
long-range movements (some genes were involved in two rearrangement events). The local
translocations are readily explained by the duplication/random loss model (Moritz et al., 1987),
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whereas the dominant rearrangement events (inversion and long-range movement) are more
consistent with the intramolecular recombination mechanism (Dowton and Campbell, 2001; Mao et
al., 2014a). In this mechanism, the repeat fragments located around the breakpoints may play an
important role in facilitating the recombination (Mao et al., 2014a). The A+T-rich region (control
region) has been suggested as a “hot spot” of recombination (Kurabayashi et al., 2008). Among the
64 gene rearrangements, there are 43 rearrangements that occurred close to the A+T-rich region. A
series of repeat sequences detected in each evaniomorph mt genome A+T-rich region may have
played a role in the gene rearrangements, via recombination (Table 5.2).
Table 5.2 The number of repeats (including direct and inverted repeats) within the A+T-rich region and gene
rearrangements identified for seven evaniomorph taxa.
Species 12-20 bp 21-50 bp 51-100 bp >100 bp Close* Not close* Inversion Long range Local
Ceraphron sp. 10 8 0 0 11 1 5 10 0
Conostigmus sp. 23 0 9 2 5 7 1 11 1
Evania appendigaster 35 31 0 0 4 0 3 3 1
Gasteruption sp. 50 0 0 2 7 4 10 7 0
Megalyra sp. 84 31 0 4 5 3 6 6 0
Pristaulacus 37 0 0 0 3 0 0 2 1
Orthogonalys pulchella 21
compressus 2 5 2 8 6 7 10 1
Note: *close/not close refers to whether the gene rearrangements occurred close to or far from the A+T-rich region.
The definition of ‘close to the A+T-rich region’ is: (1) when a tRNA gene moves to or moves out from a position
between the rrnS-nad2 gene junction, or (2) when a tRNA moves together with rrnS or nad2.
There have been few studies on the association of repeats with rearrangements in the mt genome. In
a recent study on the plastid genome (Weng et al., 2014), repeats were strongly associated with the
breakpoints in the rearranged genomes. In particular, large repeats (>20 bp and >60 bp) were
significantly correlated with the degree of genome rearrangement. In order to better understand the
relationship between repeats and gene rearrangements, we conducted a similar analysis for the
A+T-rich region and the entire mt genome among the seven evaniomorph taxa. However, no
significant correlations between the size of repeats and gene rearrangements could be detected (the
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number of different size of repeats in the A+T-rich region is listed in table 5.2). We suspect that this
might be due to the high degree of divergence between the taxa studied here, with the number and
type of repeats that are present at the time of rearrangement being obscured by subsequent evolution.
The utility of gene rearrangements for assessing phylogeny in the Evaniomorpha
Gene rearrangement characters have been suggested as reliable markers for deducing phylogenetic
relationships (Boore and Brown, 1998; Dowton et al., 2002). To explore the potential phylogenetic
signals in the evaniomorph mt gene orders, such as shared, derived gene rearrangements, we visually
inspected each pairwise comparison of gene orders in the output diagrams from CREx. A large
number of individual gene rearrangements were detected between the evaniomorph mt genomes.
However, no shared, derived gene rearrangements could be identified. This result indicates that the
frequency of gene rearrangements might still be too high to be useful for assessing higher-level
relationships. We investigate this point further in the following section.
Table 5.3 Breakpoint numbers of the evaniomorph, hemipteroid and nematoceran mt genomes relative to the ancestral
mt genome organization.
Species Number of breakpoints Accession number Reference

Hemipteroids
Triatoma 0 NC_002609 Dotson and Beard 2001
Heterodoxus 35 NC_002651 Shao et al. 2001
Thrips 30 AF335993 Shao and Barker 2003
Lepidoscopid 15 AF335994 Shao et al. 2003
Diptera: Nematocera
Anopheles 5 NC_000875 Mitchell et al. 1993
Culicoides 0 NC_009809 Matsumoto et al, unpublished
Cramptonomyia 0 JN861747 Beckenbach 2012
Tipula 0 JN861743 Beckenbach 2012
Sylvicola 0 JN861752 Beckenbach 2012
Paracladura 21 JN861751 Beckenbach 2012
Arachnocampa 2 JN861748 Beckenbach 2012
Mayetiola 12 GQ387648 Beckenbach and Joy 2009
Hymenoptera: Evaniomorpha
Megalyra 16 KJ577600 Present study
Gasteruption 14 KJ619460 Present study
Evania 10 FJ593187 Wei et al. 2010b
Pristaulacus 5 KF500406 Wei et al. 2013
Conostigmus 16 KF015227 Mao et al. 2014
Ceraphron 18 KJ570858 Present study
Orthogonalys 21 KJ619461 Present study
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Rates of gene rearrangement in the Evaniomorpha
Do the Evaniomorpha have an intermediate rate of gene rearrangement? To answer this question, we
calculated the breakpoint numbers of the evaniomorph, hemipteroid (hemipteran orders; Hemiptera,
Phthiraptera, Psocodea, Thysanoptera) and nematoceran (Diptera: Nematocera; crane flies, gnats,
and mosquitoes) mt genomes relative to the ancestral organization of insects. The hemipteroids
(excluding the Hemiptera) have been identified as a group with a high rate of mt gene rearrangement
(e.g. Shao et al., 2001, 2003), whereas most Nematocera have a low rate of mt gene rearrangement
(e.g., Beckenbach, 2012). As presented in Table 5.3, the breakpoint numbers of the seven
evaniomorph taxa range from 5 to 21, whereas the numbers of the three hemipteroid taxa (i.e.,
excluding Triatoma from the Hemiptera) are high (15-35), with most nematocerans low (0-5),
although Paracladura is an obvious exception. These numbers confirm the intermediate rate of gene
rearrangement of the Evaniomorpha. However, the inability to identify shared, derived gene
rearrangements indicates that the rate of the gene rearrangement is still too high to be reliably used
for reconstructing phylogenetic relationships. Therefore, increasing taxonomic sampling, particularly
in those evaniomorph groups with a relatively lower rate of gene rearrangement might be more
useful to establish phylogenetic relationships. Within the Evaniomorpha, the three evanioid taxa
yield the lowest breakpoint numbers, which suggests the Evanioidea is the most promising candidate
for further analyses. Although the monophyly of the Evanioidea was not recovered by some previous
research (Castro and Dowton, 2006; Dowton and Austin, 2001), two most recently comprehensive
studies robustly supported its monophyly (Heraty et al., 2011; Klopfstein et al., 2013).
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Previous phylogenetic analyses of hymenopteran mt genome data by our group has established that
uncontroversial relationships are only recovered with partitioned, model-based (Bayesian) analyses
of nucleotide data (Dowton et al., 2009a). Analysis of amino acid sequences using either maximum
parsimony or Bayesian approaches failed to recover these uncontroversial relationships, as did
analysis of nucleotide sequence data using the maximum parsimony criterion. Although our earlier
studies also found that the exclusion of third codon positions markedly improved the recovery of
these relationships, more recently we have found that the use of PartitionFinder (Lanfear et al., 2012)
now facilitates the inclusion of third codon positions (Mao and Dowton, 2014). PartitionFinder tends
to group proteins into the strand on which they are encoded, an approach that we did not use in our
earlier studies. For this reason, we analyzed the present dataset using model-based analyses of
nucleotide data which was partitioned by PartitionFinder. Nevertheless, in order to assess how
sensitive our phylogeny was to the analytical approach, we analyzed our dataset using eight
analytical approaches, which are outlined below.
We analyzed two taxon datasets (with and without rogue taxon deletion) using two alignment
approaches (Muscle and MAFFT) and two phylogenetic approaches (Maximum likelihood and
Bayesian inference). Our initial dataset contained all of the available evaniomorph and aculeate
species. The trees yielded a number of expected relationships with high support (posterior
probabilities 0.98-1.00 in Bayesian analyses and bootstrap proportions 57-100 in maximum
likelihood analyses). For example, the Ceraphronoidea, Apoidea, Formicidae and Vespidae were
consistently recovered as monophyletic (Fig. 5.6 and Supplementary Fig. 2,
http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evu145/-/DC1). However, some
traditionally monophyletic groups failed to be recovered in all of the analyses. For example, the two
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chrysidoid genera (Cephalonomia and Primeuchroeus) failed to group together; Evania was placed
within the Aculeata and grouped as a sister of Radoszkowskius. This latter relationship was also
reported in a recent mt phylogeny of the Hymenoptera (Kaltenpoth et al., 2012). We then
investigated whether some taxa were being placed in variable positions during the bootstrap and
Bayesian analyses, and as a result were obscuring other, more stable relationships (Wilkinson, 1996).
We prefer this objective approach to taxon pruning, rather than basing taxon exclusion upon, for
example, inconsistency with previous phylogenetic hypotheses. Therefore, we conducted rogue
taxon identification on all of the tree sets using RogueNaRok. For the RAxML bootstrap tree sets,
Evania appendigaster and Primeuchroeus spp. were consistently identified as rogue taxa, whereas
only Primeuchroeus spp. was identified in Bayesian tree sets. For consistency between analyses, we
removed the concatenated sequences corresponding to the two rogue taxa (Evania and
Primeuchroeus) and repeated the sequence alignment (as removal of rogue taxa might impact on the
alignment) and phylogenetic analyses. The phylogenetic resolution was improved compared with the
initial analyses. For example, a monophyletic Aculeata was consistently recovered in all analyses.
Therefore, our discussion below focuses on the trees generated by the dataset with these two taxa
removed.
The general topologies were broadly congruent across the four analyses with similar levels of
support for major clades (Fig. 5.7 and Supplementary Fig. 3,
http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evu145/-/DC1). The two alignment
approaches had no effect on topology and limited effect on nodal support (Fig. 5.7 and
Supplementary Fig. 3A, http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evu145/-/DC1).
When the two phylogenetic inference approaches are compared, there is only one topological
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difference, which was caused by the variable positions of Radoszkowskius (Vespoidea) and
Cephalonomia (Chrysidoidea). In Bayesian analyses, the two taxa grouped together and this clade
was sister to Apoidea + the remaining Vespoidea (Fig. 5.7 and Supplementary Fig. 3A,
http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evu145/-/DC1). However, in maximum
likelihood analyses, Radoszkowskius was recovered as the sister group to the remaining Aculeata and
Cephalonomia formed a clade with three formicid taxa (Camponotus, Pristomyrmex and Solenopsis)
(Supplementary Fig. 3B and C,
http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evu145/-/DC1). Nodal support was
generally stronger for the Bayesian than the maximum likelihood analyses as has been commonly
noted (Cameron et al., 2012; Nelson et al., 2012). The data matrix and the two Bayesian trees in Fig.
5.6 and 5.7 have been deposited in TreeBASE (Accession number S16039, http://treebase.org/).
Figure 5.6 Bayesian analysis of the initial taxon dataset (without rogue taxon deletion) based on mitogenomic
sequences including 13 protein-coding genes, and two rRNA genes. All the Symphyta, Evaniomorpha and Aculeata
were recovered as paraphyletic grades. Posterior probabilities are shown at each node.
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Figure 5.7 Bayesian analysis of the reduced taxon dataset (with rogue taxon deletion) based on mitogenomic
sequences including 13 protein-coding genes, and two rRNA genes. Both the Symphyta and Evaniomorpha were
recovered as paraphyletic grades. Posterior probabilities are shown at each node.
In the pruned dataset, there are 22 hymenopteran taxa, with four taxa coming from the Symphyta.
Our analyses strongly supported the Cephoidea (Cephus) as sister to Orussoidea + Apocrita, which is
congruent with previously morphological studies (Vilhelmsen, 2001; Vilhelmsen et al., 2010). The
Orussoidea (Orussus) was recovered as sister to the Apocrita with high nodal support.
Within the Apocrita, the Aculeata was consistently recovered as monophyletic. Ignoring the unstable
positions of Radoszkowskius and Cephalonomia, the internal relationships are highly congruent with
our previous study (Mao et al., 2012). Compared with the previous study, there are two new
representatives: Camponotus from the Vespoidea and Philanthus from the Apoidea. As expected,
Camponotus formed a clade with another two vespoid taxa: Pristomyrmex and Solenopsis. All these
three taxa are from the same family Formicidae. Philanthus was recovered at the base of the
Apoidea.
The Evaniomorpha was recovered as paraphyletic, with the Aculeata nested within them. Of the
representatives from the Evaniomorpha, the Stephanoidea was placed as the sister group to the
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remaining Evaniomorpha+Aculeata. The sister relationship of the Ceraphronoidea and Evanioidea
was also highly supported in each analysis. The Trigonalyoidea was recovered as sister to the
Megalyroidea, and this clade was sister to the Aculeata. A paraphyletic Evaniomorpha with respect
to the Aculeata has commonly been recovered in previous molecular studies (Castro and Dowton,
2006; Dowton and Austin, 2001; Heraty et al., 2011; Klopfstein et al., 2013; Sharkey et al., 2011).
However, the likely sister group to the Aculeata remains controversial. In our previous studies, the
Aculeata were generally recovered among a clade with the Stephanoidea, Trigonalyoidea and
Megalyroidea, but with weak support (Castro and Dowton, 2006; Dowton and Austin, 2001). A
recent analysis of combined morphological and molecular data supported a sister group of Aculeata
+ Evanioidea, but this clade was not supported by the morphology-only tree or individual gene trees
(Sharkey et al., 2011). Additionally, some recent molecular analyses recovered the same sister group
to the Aculeata as in this study, but the results were sensitive to the alignment approach (Heraty et al.,
2011; Klopfstein et al., 2013). In contrast, in this study the Aculeata + (Trigonalyoidea +
Megalyroidea) clade was insensitive to variations in alignment approach and phylogenetic inference
approach. We consider this clade to be the best supported hypothesis, based on current evidence. The
phylogenetic relationships among evaniomorph superfamilies are also far from settled. Since
Rasnitsyn (1988) placed the Stephanoidea within the Evaniomorpha, there has been no robust
morphological or molecular evidence to support this affiliation (Rasnitsyn, 1988). Instead, most
subsequent analyses placed the Stephanoidea as the most basal apocritan lineage (Dowton and
Austin, 1994; Heraty et al., 2011; Huber, 2009; Peters et al., 2011; Sharkey et al., 2011; Vilhelmsen
et al., 2010; Whitfield, 1992). Our results supported the basal position of the Stephanoidea, but failed
to give a clear indication to the affiliation of this superfamily. The sister relationship between the
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Trigonalyoidea and Megalyroidea has been consistently proposed in molecular analyses (Castro and
Dowton, 2006; Dowton and Austin, 2001; Dowton et al., 1997; Heraty et al., 2011; Klopfstein et al.,
2013). However, recent morphological analyses favor the Ceraphronoidea as the sister to the
Megalyroidea based on mesosomal characters (Vilhelmsen et al., 2010). Our results confirmed the
Trigonalyoidea + Megalyroidea clade with robust molecular support and yielded a novel sister
relationship of the Ceraphronoidea and Evanioidea. The inclusion of other taxa is required to further
test this novel relationship.
To date, most molecular phylogenies on the internal relationships of the Apocrita have relied on
relatively short mt and nuclear gene fragments. The few studies that have used the much larger mt
genome to assess this group have done so with relatively few taxa – usually one representative from
each superfamily. Our study is the first attempt to use the entire mt genome, and a denser taxonomic
sampling, to reconstruct phylogeny. Nevertheless, we do so for just two of the four apocritan groups,
the Evaniomorpha and Aculeata. The molecular phylogenetic hypothesis presented here provides a
reliable basis for future analyses.
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CHAPTER 6: Higher-level phylogeny of the
Hymenoptera inferred from mitochondrial genomes
This chapter is slightly modified from a paper that was submitted for publication in Molecular
Phylogenetics and Evolution and is currently in review:
Mao M, Gibson T, Dowton M. Higher-level phylogeny of the Hymenoptera inferred from
mitochondrial genomes.
6.1 Introduction
The Hymenoptera, comprising of sawflies, wasps, bees and ants, is one of the most speciose insect
orders. They live in all terrestrial and some aquatic habitats (Huber, 2009). The Hymenoptera
conventionally comprises two suborders, the Symphyta and the Apocrita, with 27 superfamilies (9
superfamilies in the Symphyta and 18 in the Apocrita) (Aguiar et al., 2013). Members of the
Symphyta are more primitive, with phytophagous lifestyle. The Orussidae is the only parasitoid
symphytan family (Sharkey, 2007). More than 90% of the described hymenopteran species belong to
the Apocrita (Huber, 2009). As one of the most biologically diverse suborder of insects, the Apocrita
exhibits a rich spectrum of lifestyles including parasitoidism, predation, omnivory, mycophagy and
secondary reversals to phytophagy (Huber, 2009; New, 2012).
Robust phylogenetic relationships could provide a framework to understand the evolution of the
various biologies exhibited by the Apocrita (Dowton and Austin, 2001). However, the higher-level
relationships within the Apocrita remain contentious (Sharkey, 2007). Based on morphological and
fossil evidence, Rasnitsyn (1988) proposed a new infraorder system, which formed a framework for
subsequent research of hymenopteran phylogeny. In this system, the Apocrita was divided into four
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infraorders, the Ichneumonomorpha, the Vespomorpha (Aculeata), the Proctotrupomorpha and the
Evaniomorpha, with the Ichneumonomorpha sister to the Aculeata and the Proctotrupomorpha sister
to the Evaniomorpha. The monophyly of the Ichneumonomorpha and Aculeata was consistently
supported by subsequent morphological and molecular analyses (Castro and Dowton, 2006; Dowton
and Austin, 1994, 2001; Dowton et al., 1997; Heraty et al., 2011; Klopfstein et al., 2013; Ronquist et
al., 1999; Sharkey et al., 2011; Vilhelmsen et al., 2010). However, the sister relationship between the
two infraorders was only recovered by some morphological and early molecular analyses (Dowton
and Austin, 1994; Dowton et al., 1997; Rasnitsyn and Zhang, 2010; Vilhelmsen et al., 2010). Instead,
most of the later molecular analyses supported the Aculeata nested within the paraphyletic
Evaniomorpha and a sister relationship between the Ichneumonomorpha and the Proctotrupomorpha
(Dowton and Austin, 2001; Heraty et al., 2011; Klopfstein et al., 2013; Sharkey et al., 2011).
The Proctotrupomorpha (including the Proctotrupoidea, Cynipoidea, Diaprioidea,
Mymarommatoidea, Platygastroidea and Chalcidoidea) and Evaniomorpha (including the
Ceraphronoidea, Evanioidea, Megalyroidea, Stephanoidea and Trigonalyoidea) were two novel
concepts in Rasnitsyn’s infraorder system. The clade Evaniomorpha has not been consistently
recovered in both morphological and molecular analyses (Castro and Dowton, 2006; Gibson, 1999;
Heraty et al., 2011; Klopfstein et al., 2013; Rasnitsyn and Zhang, 2010; Ronquist et al., 1999;
Sharkey et al., 2011). For example, most recent analyses supported the Stephanoidea as the most
basal apocritan lineage (Heraty et al., 2011; Peters et al., 2011; Sharkey et al., 2011; Vilhelmsen et al.,
2010). Furthermore, recent molecular analyses recovered the Evaniomorpha as paraphyletic relative
to the Aculeata (Castro and Dowton, 2006; Heraty et al., 2011; Klopfstein et al., 2013; Sharkey et al.,
2011). Similarly, the monophyly of the Proctotrupomorpha was not supported by early
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morphological or molecular analyses. In morphological analyses, Ronquist et al. (1999) reanalyzed
Rasnitsyn’s (1988) data using numerical cladistic method and the trees showed that the
Ceraphronoidea fell among the Proctotrupomorpha, close to the Chalcidoidea and Platygastroidea
(Ronquist et al., 1999). An early molecular analysis conducted by Dowton et al. (1997) using 37 taxa
based on 16S rRNA gene also failed to retrieve a monophyletic Proctotrupomorpha, as the
Cynipoidea was placed as a relatively basal apocritan lineage. But the reason for this might be the
limited taxon samplings (only two cynipoid representatives) (Dowton et al., 1997). Later, Dowton
and Austin (2001) performed multiple analyses with a dramatically increased taxon dataset and more
molecular markers (16S rRNA, COI and 28S rRNA). However, the Heloridae (Proctotrupoidea) was
recovered outside the Proctotrupomorpha in most of these analyses. Interestingly, when the
morphological characters were included, the Ceraphronoidea was placed within the
Proctotrupomorpha (Dowton and Austin, 2001). Although later and more comprehensive molecular
analyses consistently recovered the Proctotrupomorpha as monophyletic, the internal relationships
remain elusive (Castro and Dowton, 2006; Heraty et al., 2011; Klopfstein et al., 2013; Sharkey et al.,
2011). For example, Sharkey (2007) separated the Diaprioidea from the Proctotrupoidea as a new
superfamily based on previous molecular evidence (Castro and Dowton, 2006; Dowton and Austin,
2001), but the monophyly of the Diaprioidea was not well supported by later molecular analyses
(Heraty et al., 2011; Klopfstein et al., 2013).
The entire mitochondrial (mt) genome is a popular molecular marker used in phylogenetic studies
mainly because of its maternal inheritance and higher rate of nucleotide substitution compared with
the nuclear DNA (Moritz et al., 1987). In addition, some other mt genome characters, such as gene
rearrangements, also provide useful phylogenetic information (Boore and Brown, 1998). However,
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poor representation of a broad range of lineages restricts the evolutionary utility of the mt genome.
Among the insect orders, hymenopteran mt genomes are not well represented (Cameron, 2014).
Therefore, the main purpose of the present study is to increase our understanding of the phylogeny
of the Hymenoptera by extending the taxonomic range of available mt genome sequences. Here, we
present three new mt genomes for representatives of three hymenopteran superfamilies: Ibalia
leucospoides (Cynipoidea), Monomachis antipodalis (Diaprioidea) and Pelecinus polyturator
(Proctotrupoidea). The mt genomes from the superfamilies Cynipoidea and Diaprioidea are
sequenced for the first time. We conducted phylogenetic analyses using these new mt genomes
together with the previously published mt genomes of the Hymenoptera. For the first time, we
present analyses of mt genome data from representatives of every extant apocritan superfamily, with
the exception of Mymarommatoidea. A range of analytical approaches were employed. In addition,
we compared the organization of the newly sequenced mt genomes with other Proctotrupomorpha
taxa and with the putative ancestral organization for the Hymenoptera, in order to identify any
shared, derived gene rearrangements.
6.2 Materials and Methods
DNA extraction
The collection details for each species sequenced for this study are listed in Table 6.1. All specimens
were stored at 4°C in the UOW wasp alcohol library before extraction. Genomic DNA was extracted
from 100% ethanol preserved specimens using the ‘salting out’ protocol (Aljanabi and Martinez,
1997). The DNA was resuspended in 100 μl of fresh TE solution (1 mM Tris–HCl, 0.1 mM EDTA
[pH 8]) and stored at 4°C.
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Table 6.1 Species sequenced in this study, collection data, and GeneBank accession numbers.
Species Library reference Family Superfamily Collection locality Accession number

Ibalia leucospoides M4 Ibaliidae Cynipoidea Mt Gambier, South Australia KJ814197
Monomachis antipodalis M241 Monomachidae Diaprioidea ACT, Australia KM104168
Pelecinus polyturator M22 Pelecinidae Proctotrupoidea Ottawa, Canada KM104167
Mt genome amplification, sequencing, annotation, and bioinformatic analysis
PCR amplifications and sequencing reactions were conducted as previously described (Mao et al.,
2014a). Briefly, short fragments were amplified and sequenced using a range of universal insect
mitochondrial primers (Simon et al., 1994; Simon et al., 2006) and primers that had been previously
designed from consensus hymenopteran mt sequences. Using the sequence information obtained,
taxon-specific primers were designed for each sample to amplify the remaining regions by long PCR.
The primer walking method was employed to determine the complete, double stranded sequence for
each long PCR product.
Raw sequences were assembled into contigs in ChromasPro Ver 1.33 (Technelysium Ltd., Tewantin,
Australia). tRNA genes were identified using tRNA-scan SE 1.21 with a cove cutoff score of 5
(lowelab.ucsc.edu/tRNAscan-SE/) (Lowe and Eddy, 1997) and ARWEN 1.2
(http://130.235.46.10/ARWEN/) (Laslett and Canbäck, 2008). ORFinder
(www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to identify protein-coding genes, specifying the
invertebrate mt genetic code. The start and stop codons of some genes were corrected according to
the boundaries of tRNA genes and through alignment with other hymenopteran mt sequences.
Ribosomal RNA genes were identified by sequence comparison with published hymenopteran mt
ribosomal RNA sequences.
Nucleotide compositions were determined using MEGA5 (Tamura et al., 2011). The AT and GC
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skews were measured for the major (J) strand of each genome. The formulae used were AT-skew =
(A - T) / (A + T) and GC-skew = (G - C) / (G + C) (Perna and Kocher, 1995).
Sequence alignment and phylogenetic analysis
Nucleotide sequences for each of the 13 protein-coding genes and the 2 rRNA genes were imported
into separate files using MEGA5 (Tamura et al., 2011) and aligned using Muscle (Edgar, 2004) or
MAFFT (Katoh et al., 2005). For the protein-coding genes (excluding the stop codons), an amino
acid alignment was generated first for each gene in Muscle as implemented within MEGA5, or
MAFFT at the freely available TranslatorX server (http://translatorx.co.uk/) (Abascal et al., 2010). A
nucleotide alignment was then inferred from the amino acid alignment. The alignment parameters
for all genes in Muscle were the default settings, which have been specified in a previous study (Mao
et al., 2012). For the MAFFT alignment of the rRNA genes, we used the G-INS-i algorithm as
implemented in the MAFFT web server (http://mafft.cbrc.jp/alignment/server/) (Katoh et al., 2005).
Individual gene alignments were concatenated prior to phylogenetic analysis.
The best partitioning schemes and corresponding nucleotide substitution models were determined
with PartitionFinder version 1.0.1 (Lanfear et al., 2012) using the Bayesian Information Criterion
(BIC) and a heuristic search algorithm with branch lengths linked and unlinked, respectively. Two
data blocks were predetermined for analyses: 41 data blocks [2 rRNA genes + 3 codon positions of
13 protein-coding genes (rrnS + rrnL + PCG123)] and 28 data blocks [2 rRNA genes + 1st and 2nd
codon positions of 13 protein-coding genes (rrnS + rrnL + PCG12)]. Maximum Likelihood and
Bayesian approaches were employed to infer phylogenetic trees. Analyses were performed using
MrBayes v. 3.2.2 (Ronquist et al., 2012a) and RAxML v. 8.0.9 (Stamatakis, 2014) via the online
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CIPRES Science gateway portal (Miller et al., 2010). For Maximum likelihood analyses, a total of
1000 bootstrap replicates were performed with the GTRGAMMA substitution model applied to all
partitions. For Bayesian analyses, the Markov chain Monte Carlo (MCMC) process was set so that
four chains (three heated and one cold) ran simultaneously. Four separate runs using unlinked
partitions for a total of 1,000,000 generations were performed, with sampling every 100 generations.
Stationarity for each run was assessed by importing the parameter files into Tracer v. 1.5 (Rambaut
and Drummond, 2009).
6.3 Results and Discussion
General features of mt genomes
One complete mt genome of I. leucospoides (17,212 bp) and two nearly complete mt genomes of P.
polyturator (14,896 bp) and M. antipodalis (14,066 bp) were sequenced and have been deposited in
GenBank (Table 6.1). We were unable to amplify a portion of the A+T-rich region for P. polyturator
and the entire A+T-rich region, three tRNA genes (trnI, trnM, and trnQ) and part of nad2 and rrnS
for M. antipodalis. All 37 genes typical of animal mt genomes were identified in both I. leucospoides
and P. polyturator mt genomes (Boore, 1999). However, unlike most animal mt genomes, the I.
leucospoides mt genome contains two extra trnM genes and inverted, duplicated A+T-rich regions.
The three trnM genes are highly similar, with 92% – 97% sequence identities. They are separated by
35 bp and 41 bp short non-coding regions. The two inverted copies of the A+T-rich region are 457
bp and 848 bp long, respectively. The first copy is a perfect inverted repeat of the major portion of
the second copy. The presence of duplicated A+T-rich regions (control regions) has been reported in
a diverse range of lineages, such as mantellid frogs (Kurabayashi et al., 2008), Amazona parrots
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(Eberhard et al., 2001), plague thrips (Shao and Barker, 2003) and Australasian Ixodes ticks (Shao et
al., 2005). However, to our knowledge, the inverted, duplicated A+T-rich regions have not been
previously reported in other animals. The 100% sequence identity indicates that (1) the inverted
duplication occurred very recently; or (2) the two inverted A+T-rich regions might evolve in concert
as has been reported for several duplicated control regions (Eberhard et al., 2001; Kurabayashi et al.,
2008).
Table 6.2 Characteristics of the three mitochondrial genomes sequenced in this study.
Species Length (bp) A+T content (%) AT-skew GC-skew

Ibalia leucospoides 17217 86.3 -0.032 -0.012
Monomachis antipodalis 14066 83.8 -0.045 0.138
Pelecinus polyturator 14896 81.2 -0.031 -0.341
Some general characteristics of the three genomes are given in Table 6.2. As widely reported in other
hymenopteran mt genome, the nucleotide base composition is similar for the three mt genomes with
a strong AT bias (81.2% – 86.3%). Each of the three mt genomes exhibits negative AT-skews for the
J strand, ranging from -0.045 to -0.031. The GC-skews of the J strand in the I. leucospoides and P.
polyturator mt genomes are negative (-0.012 and -0.341, respectively) while the M. antipodalis mt
genome shows a positive GC-skew (0.138). It has been proposed that the reversal of strand
asymmetry might be caused by inversion of the replication origin (Hassanin et al., 2005). All of the
protein-coding genes start with the typical ATA, ATG or ATT codon and stop with the completed
(TAA or TAG) or truncated (TA or T) termination codons. Several short non-coding regions were
identified in each mt genome. The P. polyturator mt genome contains a total of 219 bp of short
non-coding regions with the largest one of 59 bp between nad4L and trnT genes. The sizes of the
short non-coding regions within the M. antipodalis mt genome are highly variable, ranging from 1
bp to 298 bp. A total of 982 bp of short non-coding regions was identified in the I. leucospoides mt
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genome; the largest one (256 bp) contains an array of 9 bp tandem repeats (TTATTAATC or
TTATTAATT) present in 29 copies (the last one is a partial copy).
Figure 6.1 Mitochondrial genome organization of Ibalia leucospoides, Pelecinus polyturator, and Monomachis
antipodalis, compared with the ancestral pancrustacean/hymenopteran mt genome organization. tRNA genes are
indicated by single-letter amino acid codes, L1, L2, S1 and S2 denote trnLCUN, trnLUUR, trnSAGN and trnSUCN,
respectively. Genes are transcribed from left to right except those indicated by underlining. Gene movements, relative
to the ancestral organization, are indicated with arrows.
The three mt genome organizations are shown in Figure 6.1. Several gene rearrangements were
revealed in each mt genome when compared with the organization of the ancestral
pancrustacean/hymenopteran (Cook, 2005; Mao et al., 2012). In M. antipodalis, three tRNA genes
(trnS2, trnY, and trnV) have rearranged relative to their ancestral positions. All of them have inverted
and moved to the cox1 – cox2 gene junction. The derived gene rearrangements were not found in
other proctotrupomorph taxa, indicating they occurred during the divergence of Monomachis. There
are four tRNA gene rearrangements in the P. polyturator mt genome relative to the ancestral
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pancrustacean/hymenopteran: trnQ has swapped position with trnI; trnM has moved to the cob –
nad1 gene junction; trnE and trnF have inverted and swapped positions. We then compared the
organization of P. polyturator with another species of the Proctotrupoidea, Vanhornia eucnemidarum
(Castro et al., 2006), to investigate whether any of the tRNA gene rearrangements were shared
within this superfamily. However, no shared gene rearrangements could be identified in the two taxa.
Although trnM is no longer in the tRNA gene cluster upstream of nad2 in both taxa, it has different,
derived positions. In V. eucnemidarum, it has inverted and moved to the nad1 – rrnL gene junction.
We could not compare the position of trnI and trnQ as they failed to be amplified in the V.
eucnemidarum mt genome. The I. leucospoides mt genome has extensive rearrangements involving
15 tRNA genes as well as 7 protein-coding genes. The most striking one is a large inversion of a
group of genes from trnE to trnS2, genes from this group were found in three separate, derived
positions. trnE and trnF remain in their ancestral positions; genes from nad5 to cob have moved to a
position downstream of rrnS; trnS2 has moved to a position downstream of the A+T-rich region. In
addition, the inverted copy of the A+T-rich region lies next to trnE. We propose that the A+T-rich
region might have first duplicated and moved to the position downstream of trnS2 via a
duplication/random loss model (Moritz et al., 1987), then become inverted with the trnE – trnS2 gene
block simultaneously via recombination (Dowton and Campbell, 2001; Mao et al., 2014a). The
breakpoints occurred in the nad3-nad5 and cob-nad1 gene junctions. This explanation is not only a
more parsimonious scenario than several independent inversion events, but also consistent with the
hypothesis that the A+T-rich region is important for the formation of long-lived minicircles during
recombination (Mao et al., 2014a). The inverted trnE – trnS2 gene block is unique when compared
with other proctotrupomorph mt genomes. However, this is not the first report of large gene block
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inversion in the Proctotrupomorpha. In previous studies, large inversions of cox1-cox3 or cox1-nad3
gene blocks have been reported in three chalcidoid wasps (Oliveira et al., 2008; Xiao et al., 2011).
Interestingly, the breakpoints in these mt genomes also occurred in the nad3-nad5 gene junction. We
suspect that there might be some structural characteristics in this region that facilitate the occurrence
of double strand breaks, and that repair of these breaks might lead to gene rearrangement (Mao et al.,
2014a).
Table 6.3 Summary of the major clades recovered by different dataset and analytical approach. Support values are
shown for each recovered relationship (Posterior probability for Bayesian analyses and bootstrap proportion for
maximum likelihood analyses). Abbreviations: -, not recovered; PCG123, 1st, 2nd and 3rd codon positions of 13
protein-coding genes; PCG12, 1st and 2nd codon positions of 13 protein-coding genes; BI, Bayesian analysis; ML,
maximum likelihood analysis; Link, branch lengths = linked in PartitionFinder; Unlink, branch lengths = unlinked in
PartitionFinder; rEV, the remaining Evaniomorpha; PR, Proctotrupomorpha; IC, Ichneumonomorpha; Cy, Cynipoidea;
Di, Diaprioidea; Ch, Chalcidoidea.
rrnS+rrnL+PCG123 rrnS+rrnL+PCG12
BI_Muscle BI_MAFFT ML_Muscle ML-MAFFT BI_Muscle BI_MAFFT ML_Muscle ML-MAFFT
Clade
Link Unlink Link Unlink Link Unlink Link Unlink Link Unlink Link Unlink Link Unlink Link Unlink
Orussoidea + Apocrita 0.91 0.74 1 1 - - 78 86 1 1 1 1 61 64 86 90
Stephanoidea + the remaining Apocrita 1 1 0.96 0.93 86 89 77 79 1 1 0.99 0.98 76 75 70 76
(Evaniidae + Aculeata) + (rEV+PR+IC) 0.92 1 0.98 - - - - - 0.72 0.8 - 0.82 34 34 - -
Aculeata - 0.48 - - 51 47 - - 0.7 - - - - - - -
Evanioidea + Aculeata - - - - - - - - - - 0.88 - - - 17 -
Apoidea 1 1 1 1 100 99 100 99 1 1 1 1 100 100 99 100
Chrysidoidea 0.34 - 0.63 0.42 - - - - - - - - - - - -
Trigonalyoidea + Megalyroidea 1 1 1 1 100 100 100 100 1 1 1 1 100 100 100 100
Ichneumonomorpha 1 1 1 1 100 100 100 100 1 1 1 1 100 100 100 100
(Trigonalyoidea + Megalyroidea) + IC 1 0.98 1 1 85 84 85 81 1 1 1 1 60 57 78 76
Ceraphronoidea + Evanioidea 0.62 0.79 0.53 - - - - - 0.89 0.64 - 0.63 - 22 - -
Ceraphronoidea + Platygastroidea - - - 0.63 48 46 53 56 - - - - 50 - - 47
Proctotrupomorpha - - - - - - - - 0.9 0.63 0.69 0.65 - 33 43 -
Ceraphronoidea + Proctotrupomorpha - - - - - - - - - - 0.88 - - - 32 -
(Ceraphronoidea + Evanioidea) + PR - - - - - - - - 0.78 0.58 - - - 17 - -
Cynipoidea + Proctotrupoidea - - - - 17 20 37 36 - - - - - - - 26
Diaprioidea + Chalcidoidea 0.99 0.85 1 0.98 74 70 92 87 0.9 0.69 0.98 0.74 66 57 78 72
Cynipoidea + (Diaprioidea + Chalcidoidea) 1 1 1 1 - - - - 0.99 1 1 1 50 50 42 -
Proctotrupoidea + (Cy + (Di + Ch)) - - - - - - - - 1 1 1 0.98 68 67 63 -
Proctotrupoidea + Platygastroidea 0.66 0.94 0.66 - - - - - - - - - - - - -
107
Phylogenetic analyses were performed using 48 complete or partial mt genomes, representing 14
apocritan and 3 symphytan superfamilies. We analyzed two datasets (rrnS + rrnL + PCG123 and rrnS
+ rrnL + PCG12) using two alignment approaches (Muscle and MAFFT), two partition schemes
(branch lengths = linked and unlinked) and two phylogenetic approaches (Maximum likelihood and
Bayesian inference). Previous phylogenetic studies of mt genome data have indicated that Bayesian
analyses were superior to parsimony analyses (Castro and Dowton, 2007; Dowton et al., 2009a). For
this reason, we do not conduct parsimony analyses in the present study. A total of 11 topologies of
the relationships among the apocritan superfamilies were produced by the 16 separate analyses. As
shown in Table 6.3, some clades were consistently recovered by all analyses. However, the
relationships between the four infraorders and within the Proctotrupomorpha and Evaniomorpha
vary widely between datasets and across analytical approaches. Our recent study found that the use
of PartitionFinder facilitated the inclusion of 3rd codon positions when investigating the phylogeny
of more closely related taxa (Mao and Dowton, 2014). However, the taxa included here are more
remotely related, and excluding 3rd codon positions produced topologies more consistent with other
analyses of higher-level relationships. For example, a monophyletic Proctotrupomorpha was only
recovered by the rrnS + rrnL + PCG12 dataset (Fig. 6.2). The analytical approaches also influenced
the topology and the level of nodal support. We discuss this in more detail below.
Outgroup and basal relationships of the Apocrita
Our analyses generally support the Orussoidea as the sister group to the Apocrita, which is consistent
with previous morphological and molecular analyses (Klopfstein et al., 2013; Rasnitsyn, 1988;
Vilhelmsen, 2001; Vilhelmsen et al., 2010). Two maximum likelihood analyses of the rrnS + rrnL +
PCG123 dataset aligned by Muscle recovered the clade (Orussoidea + Cephoidea) + Apocrita, but
108
the nodal supports for the sister group of Orussoidea + Cephoidea were very low (BP = 32 / 42).
Within the Apocrita, the Stephanoidea was consistently placed as the most basal apocritan lineage
with high nodal support, a placement that conflicts with Rasnitsyn’s infraorder system (1988), but
consistent with a number of subsequent studies (Heraty et al., 2011; Peters et al., 2011; Sharkey et al.,
2011; Vilhelmsen et al., 2010).
Figure 6.2 Bayesian analysis of the Muscle alignment dataset (branch lengths = linked in PartitionFinder) based on
mitogenomic sequences including first and second codon positions from 13 protein-coding genes, and the two rRNA
genes. Posterior probabilities are shown at each node.
Aculeata and Ichneumonomorpha
The Aculeata was recovered as monophyletic in four Muscle-aligned analyses. It failed to be
109
retrieved in other analyses due to the placement of the non-aculeate genus Evania, which grouped as
a sister of Radoszkowskius (Aculeata: Mutillidae). This relationship has been recovered in some
analyses in our previous study and a recent mitochondrial phylogeny of the Hymenoptera
(Kaltenpoth et al., 2012; Mao et al., 2012). In our previous study, a monophyletic Aculeata was
recovered when excluding 3rd codon positions. By contrast, excluding 3rd codon positions did not
improve the resolution of a monophyletic Aculeata here. Within the Aculeata, the Apoidea was
strongly supported as monophyletic and placed within a paraphyletic Vespoidea, which is in
agreement with previous morphological and molecular analyses (Debevec et al., 2012; Heraty et al.,
2011; Pilgrim et al., 2008; Sharkey et al., 2011; Vilhelmsen et al., 2010). The two chrysidoid taxa
(Cephalonomia and Primeuchroeus) were also nested within the Vespoidea, but they failed to group
together in most of the analyses. Apparently, a better taxon sampling is needed to provide more
stable resolution. Two vespoid families, Vespidae and Formicidae, were monophyletic across all
analyses.
The Ichneumonomorpha was consistently and strongly supported in our analyses. The internal
relationships were robust to the variation of datasets and analytical approaches and highly congruent
with our mt genome previous study (Mao et al., 2012).
Rasnitsyn (1988) proposed a sister relationship between the Aculeata and the Ichneumonomorpha
based on the presence of ovipositor valvilli and distinct articulation involving a pair of propodeal
condyles. However, this sister relationship was not supported by a subsequent cladistic analysis of
his data (Ronquist et al., 1999). Although early molecular studies using 16S rRNA data retrieved the
clade Aculeata + Ichneumonomorpha (Dowton and Austin, 1994; Dowton et al., 1997), most of the
later molecular analyses recovered the Aculeata nested within the Evaniomorpha and grouped the
110
Ichneumonomorpha and the Proctotrupomorpha together (Dowton and Austin, 2001; Heraty et al.,
2011; Klopfstein et al., 2013; Sharkey et al., 2011). Our analyses consistently recovered a novel
clade with high nodal support – (Trigonalyoidea + Megalyroidea) + Ichneumonomorpha, while the
position of the Aculeata within the Apocrita remains variable. Most of the Bayesian analyses (except
rrnS + rrnL + PCG123_MAFFT_Unlink and rrnS + rrnL + PCG12_MAFFT_Link analyses)
supported the Evaniidae + Aculeata as sister to the remaining Evaniomorpha + Ichneumonomorpha
+ Proctotrupomorpha. Two analyses of the rrnS + rrnL + PCG12 dataset aligned by MAFFT
recovered a close relationship between the Evanioidea and the Aculeata, but the nodal support was
weak (PP = 0.88 and BP = 17).
Evaniomorpha
The Evaniomorpha is the most controversial concept in Rasnitsyn’s infraorder system (1988). The
monophyly and internal relationships remain problematic in both morphological and molecular
analyses (summarized in Mao et al., 2014b). Our recent mitochondrial phylogeny based on 8
evaniomorph and 12 aculeate taxa robustly recovered the Trigonalyoidea + Megalyroidea as the
sister group to the Aculeata and a sister relationship between the Ceraphronoidea and the Evanioidea
(Mao et al., 2014b). The sister group relationship of Trigonalyoidea + Megalyroidea was strongly
confirmed in our present analyses, but they were recovered as the sister group to the
Ichneumonomorpha rather than the Aculeata. The clade Ceraphronoidea + Evanioidea was generally
supported by Bayesian analyses, indicating this relationship is sensitive to the phylogenetic inference
approach.
Proctotrupomorpha
111
The Proctotrupomorpha was generally recovered as monophyletic by the rrnS + rrnL + PCG12
dataset (except BI_Muscle_Unlink and ML_MAFFT_Unlink analyses). The Ceraphronoidea +
Evanioidea clade or the Ceraphronoidea was placed as the sister group to the Proctotrupomorpha.
These two sister relationships are at odds with most previous molecular studies that found a sister
relationship between the Ichneumonomorpha and the Proctotrupomorpha (Dowton and Austin, 2001;
Heraty et al., 2011; Klopfstein et al., 2013; Sharkey et al., 2011). In the remaining analyses, the
Platygastroidea fell outside the Proctotrupomorpha and grouped with the Ceraphronoidea in six
maximum likelihood and one Bayesian analyses. This is not unprecedented, as the sister group of
Platygastroidea + Ceraphronoidea has been proposed by Ronquist et al. (1999) based on
morphological characters and supported by some analyses based on molecular and morphological
data in Dowton and Austin (2001). But the difference is that the Ceraphronoidea fell within the
Proctotrupomorpha in these two studies (Dowton and Austin, 2001; Ronquist et al., 1999). Within
the Proctotrupomorpha, the Diaprioidea was consistently recovered as the sister group to the
Chalcidoidea. The Diaprioidea is a relative new superfamily separated from the Proctotrupoidea by
Sharkey (2007) based on molecular evidence (Castro and Dowton, 2006; Dowton and Austin, 2001).
The placement of this superfamily was unstable in early molecular analyses (Dowton and Austin,
2001). However, later analyses supported a sister relationship with the Chalcidoidea (Castro and
Dowton, 2006; Heraty et al., 2011). Our results supported this sister relationship with moderate to
high nodal support. The support values were higher in the analyses using the rrnS + rrnL + PCG123
dataset, indicating the 3rd codon positions contributed phylogenetic signal to support this relationship.
There is little consensus concerning the placement of the Cynipoidea in previous analyses. Rasnitsyn
(1988) placed the Cynipoidea as a sister group to the Diapriidae + Monomachidae + Austroniidae
112
clade (Rasnitsyn, 1988). Early molecular analyses favored the Cynipoidea as a relatively basal
apocritan lineage (Dowton et al., 1997). A later molecular study based on expanded taxon sampling
and molecular data recovered the Cynipoidea as a part of the Proctotrupomorpha, but its placement
was highly variable (Dowton and Austin, 2001). Although recent molecular analyses generally
recovered a sister relationship of Cynipoidea + Platygastroidea (Castro and Dowton, 2006; Dowton
and Austin, 2001; Heraty et al., 2011; Klopfstein et al., 2013), a recent morphological study
supported a close relationship between the Cynipoidea and the Diapriidae (Vilhelmsen et al., 2010).
Contrary to previous studies, our results recovered the Cynipoidea as a sister group to the
Proctotrupoidea (5 maximum likelihood analyses) or to the clade Diaprioidea + Chalcidoidea (all
Bayesian analyses + 3 maximum likelihood analyses). The support values for the clade Cynipoidea +
(Diaprioidea + Chalcidoidea) were much higher than the clade Cynipoidea + Proctotrupoidea. The
placement of the Proctotrupoidea was also unstable in our analyses. Except for the clade Cynipoidea
+ Proctotrupoidea, the remaining analyses recovered it as a sister group to the Cynipoidea +
(Diaprioidea + Chalcidoidea) (7 analyses based on the rrnS+rrnL+ PCG12 dataset) or
Platygastroidea (3 Bayesian analyses based on the rrnS+rrnL+ PCG123 dataset).
6.4 Conclusions
Although our study provides the most comprehensive mitochondrial phylogeny of the higher-level
relationships within the Hymenoptera, many relationships are far from resolved, especially within
the Proctotrupomorpha and the Evaniomorpha. Considering the limited representatives of mt
genomes in each hymenopteran superfamily, a denser taxon sampling is needed for future studies.
For example, only one cynipoid taxon–I. leucospoides from a small family Ibaliidae–was present
in our analyses. Further taxon sampling from other cynipoid families, such as the two largest
113
families Figitidae and Cynipidae, should be included to clarify the placement of the Cynipoidea
within the Proctotrupomorpha. Further taxon sampling might also prove useful to provide
phylogenetic signal in regions of the topology where internodes are relatively short. As shown in Fig.
6.2, the internode of the clade Proctotrupoidea + (Cynipoidea + (Diaprioidea + Chalcidoidea))
recovered in our analyses was very short at the base when compared with external branches. In
recent comprehensive studies, a close relationship among the Proctotrupoidea, Chalcidoidea,
Diaprioidea and Mymarommatoidea was strongly recovered (Heraty et al., 2011; Klopfstein et al.,
2013; Sharkey et al., 2011). Therefore, inclusion of taxa from the Mymarommatoidea might be a
good choice to provide signal in these problematic areas.
114
CHAPTER 7: General Conclusions
7.1 Main outcomes
This study provides fresh insight into the mt genomes of the Hymenoptera and their utility for the
recovery of higher-level phylogeny. Eleven complete or nearly complete mt genomes representing
eight apocritan superfamilies were sequenced, which dramatically expanded the cladistic diversity of
the mt genome dataset of the Hymenoptera. With these new mt genomes, a more accurate estimate of
the degree of gene rearrangements was obtained for two major apocritan lineages: the Evaniomorpha
and Proctotrupomorpha. Work was also undertaken to investigate the possible mechanisms for gene
rearrangement. The most important finding was the coexistence of minicircular and a highly
rearranged mtDNA in the megaspilid wasp Conostigmus sp., which provided evidence to support the
notion that recombination is an important aspect of the gene rearrangement mechanism. The first mt
genome phylogeny of the Apocrita with a complete representation of superfamilies (excluding
Mymmaromatoidea) was conducted with a range of phylogenetic analytical approaches. Some robust
and highly supported relationships were recovered, indicating that the mt genome is a useful marker
to resolve higher-level relationships within the Hymenoptera.
7.2 Recommendations and future research
7.2.1 Increasing taxon sampling of the Apocrita
The expanded taxon sampling facilitated the investigation of mt gene rearrangements and their
utility to resolve phylogenetic relationships within the Hymenoptera. However, no shared, derived
gene rearrangements could be identified between and within the superfamilies of the Evaniomorpha
and Proctotrupomorpha, indicating the frequency of gene rearrangements of these groups is too high
115
to be useful for assessing relationships between families. By contrast, comparison of mt genome
organizations within the subfamily Scelioninae suggested that gene rearrangements have much
potential as phylogenetic markers at the subfamily level. Therefore, additional work should be
focused on obtaining denser taxonomic sampling at the subfamily level for the investigation of gene
rearrangements. The groups with a relatively lower rate of gene rearrangement would be a good
starting point, such as the evanioid families in the Evaniomorpha and the diaprioid families in the
Proctotrupomorpha.
This study provided the first comprehensive mitochondrial phylogeny of the higher-level
relationships within the Apocrita. However, the taxon sampling used in this study was still limited,
with no representatives from the Mymmaromatoidea and no more than three representatives from
each of the evaniomorph superfamilies and the remaining proctotrupomorph superfamilies. The
limited taxon sampling resulted in the position and monophyly of some superfamilies remaining
unresolved, such as the Evanioidea, Cynipoidea and Platygastroidea. Future taxonomic sampling
would need to be included in a more comprehensive study. Representative taxa from the
Mymmaromatoidea would be the first option. Additionally, increasing taxon sampling in those
superfamilies, whose position was variable across analytical approaches, is also essential.
The long PCR amplification of complete mt genomes is the most widely used tool in insects. In this
study, taxon-specific primers were designed for each sample to improve the efficiency of the large
fragment amplification. However, large gene (protein-coding or rRNA genes) rearrangements in
some mt genomes brought some difficulties to the amplification. Thus, different primer
combinations were tested, which was a time-consuming process. Furthermore, the area surrounding
the A+T-rich region has proven difficult to amplify in the Hymenoptera (Castro and Dowton, 2006;
116
Dowton et al., 2009a). Although most of the mt genomes in this study were amplified entirely
through optimizing PCR conditions, designing more specific primers for the hymenopteran mt
genomes or using nested PCR, the entire mt genome sequence for Monomachis antipodalis
(Diaprioidea) and Pelecinus polyturator (Proctotrupoidea) failed to be amplified. The missing data
in some mt genomes may not have a significant impact on phylogenetic analysis (Dowton et al.,
2009a), but it has been shown to hinder the identification of shared, derived gene rearrangements in
some groups in this study, such as the Chalcidoidea (Chapter 2) and Proctotrupoidea (Chapter 6). So
it is also important to obtain the entire mt genome (at least the complete coding region) for further
taxon sampling. The recently developed "Next-Generation Sequencing" (NGS) technologies, such as
Roche and Illumina, provide a robust high-throughput, low-cost platform for de novo sequencing of
mt genomes, either from specific amplicons or directly from total genomic DNA. These technologies
can rapidly obtain large amounts of mt genomes from multiple individuals in one reaction and
substantially improve coverage and sequencing depth. Furthermore, a mapping assembly approach,
MITObim, for reconstructing mt genomes directly from raw NGS data has also been developed
recently. This approach is applicable for specific assembly of mitochondrial genomes from mixed
samples containing different species (Hahn et al., 2013). NGS technologies have already been used
for generating densely sampled trees from whole mt genomes in two large insect orders, the
Coleoptera and Lepidoptera (Gillett et al., 2014; Timmermans et al., 2014). Future studies could
therefore consider using NGS technologies to construct large dataset of hymenopteran mt genomes.
7.2.2 Incorporating genome-based collection of markers
In Chapter 5, topologies reconstructed with taxa from the Evaniomorpha and Aculeata robustly
supported the Trigonalyoidea + Megalyroidea as the sister group to the Aculeata. However, this
117
sister relationship collapsed when the Proctotrupomorpha and Ichneumonomorpha were included to
reconstruct the apocritan phylogeny in Chapter 6. The conflicting results demonstrate the limits of
the present dataset to resolve higher-level relationships of the larger and more diverse Hymenoptera
(or Apocrita). In addition to a broader taxon sampling mentioned above, incorporation of other
phylogenetic markers would also be beneficial to improve the resolution in further studies. A recent
phylogenetic analysis of relationships among families and higher groupings of the Diptera provides a
good example. This study reconstructed the evolution of a major branch of the fly tree of life based
on 14 nuclear loci, complete mt genomes and 371 morphological characters(Wiegmann et al., 2011).
So far, a very limited number of nuclear markers, such as 18S, 28S and EF-1α, have been used for
the reconstruction of hymenopteran phylogeny (Castro and Dowton, 2006; Heraty et al., 2011).
Sharanowski et al.’s (2010) research based on EST data was a good attempt to explore a large
number of molecular loci (24 ESTs) to resolve relationships among hymenopteran superfamilies
(Sharanowski et al., 2010). However, the taxon sampling was limited in their analyses and the
topologies were sensitive to analytical methods. Given the advantage of NGS technologies, further
studies could consider incorporating genome-based collection of markers from large taxon sampling.
In particular, nuclear protein-coding genes would be promising candidates that could extend our
knowledge of higher-level relationships of the Hymenoptera.
7.3 Conclusions
This PhD research has made a major progress in understanding the evolution of hymenopteran mt
genomes and their utility as phylogenetic markers. The large number of gene rearrangements
detected here has facilitated the investigation of the mechanisms of gene rearrangement, which will
further promote more realistic analyses of gene rearrangement characters in the Hymenoptera and
118
other invertebrate groups. This work has also provided a reliable basis for future phylogenetic
studies on the Hymenoptera, which will rely on increased taxon sampling and genomic sampling.
119
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University of Wollongong

Mitochondrial genomes and their utility for the

Research Online is the open access institutional repository for the

School of Biological Sciences

Doctor of Philosophy, in the School of Biological Sciences, University of Wollongong, is wholly my

qualifications at any other academic institution.

18th August 2014

Thesis Certification ............................................................................................................................ ii

List of Tables .................................................................................................................................... viii

List of Figures .................................................................................................................................... ix

List of Abbreviations ......................................................................................................................... xi

Abstract ............................................................................................................................................ xiv

List of publications ......................................................................................................................... xvii

Statement of Contribution ............................................................................................................ xviii

CHAPTER 1: General Introduction................................................................................................. 1

1.1 Preamble ............................................................................................................................. 1

1.3.1 Mechanisms of gene rearrangement .................................................................................... 5

1.3.2 Gene rearrangements as phylogenetic markers ................................................................... 8

1.4 Higher-level phylogenetic relationships within the Hymenoptera ................................. 9

1.4.1 Suborder Symphyta ............................................................................................................. 9

1.4.2 Suborder Apocrita ............................................................................................................. 10

1.5 Conclusions ....................................................................................................................... 13

2.1 Introduction ...................................................................................................................... 17

DNA extraction, PCR amplification, and sequencing ................................................................ 19

Sequence analyses ...................................................................................................................... 20

Sequence alignment ................................................................................................................... 21

Phylogenetic analysis ................................................................................................................. 22

2.3 Results and discussion...................................................................................................... 23

Genome size and gene content ................................................................................................... 23

Codon usage and amino acid composition ................................................................................. 25

Translation initiation and termination codons ............................................................................ 26

Transfer RNA genes ................................................................................................................... 27

Ribosomal RNA genes ............................................................................................................... 27

Non-coding regions .................................................................................................................... 28

Genome organization ................................................................................................................. 29

Phylogenetic analysis and considerations .................................................................................. 31

2.4 Conclusions ....................................................................................................................... 34

3.1 Introduction ...................................................................................................................... 35

DNA extraction .......................................................................................................................... 36

Amplification, sequencing, and annotation of entire mt genomes ............................................. 37

Sequence alignment ................................................................................................................... 38

Genetic divergence and phylogenetic analysis ........................................................................... 39

3.3 Results and discussion...................................................................................................... 41

Genome size and gene content ................................................................................................... 41

Protein-coding genes .................................................................................................................. 42

Transfer RNA and Ribosomal RNA ........................................................................................... 43

Noncoding regions ..................................................................................................................... 44

Genome organizations ................................................................................................................ 46

Ka/Ks ratios among the three Scelionidae taxa .......................................................................... 48

CHAPTER 4: Coexistence of minicircular and a highly rearranged mtDNA molecule suggests

4.1 Introduction ...................................................................................................................... 52

DNA extraction .......................................................................................................................... 55

Amplification, sequencing, and annotation of the entire mt genome ......................................... 55

Amplification and sequencing of minicircles and the maxicircle .............................................. 56

4.3 Results ............................................................................................................................... 57

The mt genome of Conostigmus sp. ........................................................................................... 57

Are the minicircles amplification artifacts? ............................................................................... 62

Elimination of the possibility of numts ...................................................................................... 64

4.4 Discussion .......................................................................................................................... 65

Importance of control reactions.................................................................................................. 65

Possible explanations for low copy number of the maxicircle ................................................... 66

Comparison with minicircles from other Metazoa ..................................................................... 66

Possible mechanism yielding the minicircle and maxicircle ...................................................... 68