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The characTerizaTion of larval homology: TranscripTomic insighTs
inTo The origin and evoluTion of animal
life cycles
William ludden haTlebergb.a. (honors)
A thesis submitted for the degree of Doctor of Philosophy atThe
University of Queensland in 2017
School of Biological Sciences
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Abstract
The majority of animals have a complex biphasic life cycle
characterized by distinctive larval and adult
body plans. Since the 1800s, the origins of marine larvae have
puzzled embryologists, resulting in a
long and convoluted history of theoretical literature, which
largely focus on two central questions: (1)
are extant larvae or adults more representative of the ancestral
animal body plan, and (2) how many
times did marine larvae evolve across metazoan phyla. Despite
the ecological and developmental
importance of this developmental mode, there is still no
concrete evidence for when or how the biphasic
life cycle evolved. However, recent studies predict that biphasy
is an ancient synapomorphic trait of
all metazoans, suggesting that ontogenies divided into distinct
larval and adult phases represent the
ancestral animal state. Therefore larval body plans are likely
to be homologous in most animal phyla.
In the present age of genomic data, these speculative theories
have yet to be proven or refuted by
empirical evidence. Therefore, in the present study, I employ
novel computational methodology to
re-examine established conceptual frameworks for life cycle
evolution to create a unified model for
animal body plan evolution.
Here, I use the basal marine sponge, Amphimedon queenslandica,
as a foundational case study for
life cycle evolution. Because the majority of bioinformatic
tools were developed in and optimized for
classic vertebrate model systems, I first evaluate the validity
of computational methods for functional
gene characterization in an early branching non-model system. I
find that functional characterization
methods such as gene ontology (GO) are overly-specific and
largely variable between annotation
methods, and propose a new pipeline to effectively extract
biological meaning from a non-model
ontogeny. Using these tools, I characterize the pelagobenthic
transition from free-swimming larvae to
sessile adults in A. queenslandica. I find that larval and adult
transcriptomes largely employ a shared
transcriptional toolkit that is primarily composed of ancient
pre-metazoan genes. However, I also find
evidence for phase-specific regulatory modules characterized by
the unequal distribution of gene age.
Specifically, I show that the larval transcriptome is enriched
in older, pre-metazoan genes, while the
adult transcriptome is largely composed of novel, lineage
specific innovations.
To place these findings from A. queenslandica in an evolutionary
context, I acquired comparable
datasets from five other biphasic metazoan lineages across the
animal tree including: the coral, Acropora
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digitifera, the mollusc, Haliotis asinina, the hemichordate,
Balanoglossus misakiensis, the sea urchin,
Strongylocentrotus purpuratus, and the ascidian, Herdmania
momus. Through this comparative approach,
I find that the majority of the genes that are significantly
differentially expressed during metamorphosis
are lineage-specific innovations. However, many of these
co-expressed, taxonomically restricted genes
appear to be regulated by conserved transcription factors in
multiple species. Taken together, these
findings provide the first genomically informed framework for
the origins of animal biphasy. Here
I discuss the implications of these results in light of
historical hypotheses for larval evolution, and
propose a novel conceptual framework for animal life cycle
evolution.
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Declaration by author
This thesis is composed of my original work, and contains no
material previously published or written
by another person except where due reference has been made in
the text. I have clearly stated the
contribution by others to jointly-authored works that I have
included in my thesis.
I have clearly stated the contribution of others to my thesis as
a whole, including statistical assistance,
survey design, data analysis, significant technical procedures,
professional editorial advice, and any
other original research work used or reported in my thesis. The
content of my thesis is the result of
work I have carried out since the commencement of my research
higher degree candidature and does
not include a substantial part of work that has been submitted
to qualify for the award of any other
degree or diploma in any university or other tertiary
institution. I have clearly stated which parts of
my thesis, if any, have been submitted to qualify for another
award.
I acknowledge that an electronic copy of my thesis must be
lodged with the University Library and,
subject to the policy and procedures of The University of
Queensland, the thesis be made available for
research and study in accordance with the Copyright Act 1968
unless a period of embargo has been
approved by the Dean of the Graduate School.
I acknowledge that copyright of all material contained in my
thesis resides with the copyright holder(s)
of that material. Where appropriate I have obtained copyright
permission from the copyright holder
to reproduce material in this thesis.
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Publications during candidature
Conference Abstracts
Hatleberg, W. L., B. M. Degnan, S. M. Degnan. (2016) Genomic
insight into the evolution of lar-val body plans. Society for
Molecular Biology and Evolution (SMBE), Gold Coast, QLD,
Australia
de Mendoza, A., W. L. Hatleberg, U. Technau, K. Pang, B. M.
Degnan, R. Lister. (2016) Early evolution and dynamics of DNA
methylation in animals. Society for Molecular Biology and
Evolu-tion (SMBE), Gold Coast, QLD, Australia
Hatleberg W. L., S. M. Degnan, B. M. Degnan. (2016) One genome,
two body plans: how do lar-val and adult gene expression profiles
differ in the sponge Amphimedon queenslandica? Society for
Comparative and Integrative Biology (SICB), Portland, OR, USA
Papers
Hall, M. R., K. M. Kocot, K. W. Baughman, S. L.
Fernandez-Valverde, M. E. A. Gauthier, W. L. Hatleberg, A.
Krishnan, C. McDougall, C. A. Motti, E. Shoguchi, T. Wang, X.
Xiang, M. Zhao, U. Bose, C. Shinzato, K. Hisata, M. Fujie, M.
Kanda, S. F. Cummins, N. Satoh, S. M. Degnan, and B. M. Degnan.
(2017) The crown-of-thorns starfish genome as a tool for biocontrol
of a coral reef pest. Nature. 544: 231-234.
Publications included in this thesis
None.
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Contributions by others to the thesis
Bernard M. Degnan and Sandie M. Degnan contributed to the
conception and design of this research,
advised on the analysis and interpretation of data, and provided
critical feedback on the drafting of
the thesis.
Selene L. Fernandez-Valverde contributed to the data analysis of
Chapter 2, performing the BLIND
clustering analyses, producing the lists of differentially
expressed genes, and creating the Blast2GO/
InterPro annotation (around 10% of the total data analysis).
Andrew Baird of James Cook University and Sabrina Kaul-Strehlow
from the University of Vienna
performed the necessary fieldwork to acquire the Acropora sp.
and Balanoglossus samples used in
Chapter 4. Carmel McDougall assisted with the fieldwork and
collection of Haliotis asinina.
Library preparation and transcriptome sequencing in Chapter 4
were conducted by Macrogen Inc.,
South Korea.
Statement of parts of the thesis submitted to qualify for the
award of another degree
None.
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Acknowledgements
I originally arrived in Australia thinking that I would only be
here for a year and a half to complete a
Masters degree. Now, looking back on the last three and a half
years of my life, I realize how many
people I need to thank for making my stay in this remarkable
country so extraordinary.
First and foremost, I would like to thank Bernie and Sandie
Degnan for giving me the opportunity to
work in this lab and encouraging me to switch from a Masters to
a PhD. I am very grateful for both
the independence you’ve granted me as well as the much-needed
guidance to keep me focused and
on track. Together you have taught me to become a more thorough
scientist and rigorous thinker.
Your continuing encouragement and support of my interest in
historical and philosophical biology
has allowed me to pursue the PhD project I had been dreaming of
ever since I first learned about the
bizarre and beautiful world of larval evolution.
This project would also not have been possible without the
support of the American Australian
Association, which provided me with the funding for my first
year in Australia, as well as the grants
awarded to Bernie Degnan and Sandie Degnan by the Australian
Research Council, which allowed me
to continue my research for the remainder of my PhD. I would
also like to acknowledge the University
of Queensland for providing me with a UQ International
Scholarship, as well as the School of Biological
Science for the opportunity to travel to the Society of
Comparative and Integrative Biology (SICB)
meeting in January 2016.
To complete this thesis, I have had to stand on the shoulders of
some true giants. I could not have
completed this project without the foundation and framework that
was provided to me by so many
people. I came to Australia hoping to learn bioinformatics, and
I could never have taken on this
computational project without the continuing help of countless
people. I am particularly indebted to
Selene Fernandez-Valverde, as well as my fellow PhD students,
particularly Andrew Calcino, Federico
Gaiti, and Laura Grice. I am also grateful to Felipe Aguilera
for his help with Phylostratigraphy, Kevin
Kocot for his help with OrthoMCL, and Maely Gauthier for her
constant willingness to help with
software installations (and for being so patient with me when I
couldn’t parse files!).
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Although my stint in the wet lab was brief, I am grateful to
everyone who helped me with my bench
work, especially Federico for all of his guidance with ChIP-Seq,
Carmel McDougall for her exhaustive
knowledge about every protocol, and Kerry Roper for keeping the
lab running like a well-oiled machine.
Additionally, I would like to thank Gemma Richards, and Federico
for providing me with comments
on my drafts, and a special thanks to Laura for being my
aesthetics consultant, proof-reader, and all
around support team for my PhD.
My two trips to Heron Island for fieldwork are among my fondest
memories in Australia. Therefore, I
would like to thank the Heron Island staff, particularly Liz and
Dani for all of their help, and Maureen
for reassuring my mom far away in the US that I was safe during
the cyclone. Special thanks to Carmel
for making my first trip collecting and spawning abalone and
Herdmania go smoothly and enjoyably,
as well as my field work partner for the second trip, Tahsha,
for all of her help in the lab, being my
snorkel buddy, and always partaking in midnight chocolate breaks
(if only we’d found those Steggles!).
I am also indebted to Sabrina Kaul-Strehlow from the University
of Vienna for sharing Balanoglossus
with me, and Andrew Baird of James Cook University for providing
me with coral samples. Without
these samples, Chapter 4 wouldn’t have been the same.
More than anything, PhDs are a test of perseverance, and I could
not have done it without the never-
ending support of my family and friends. Above all else, I need
to thank my family back home for
whole-heartedly supporting me in my move across the world – I
love you all the way to Australia and
back. To my “Australian family”, Roger, Jenny, Jess, Ben, and
Mia: I cannot thank you enough for
adopting me into your amazing family and including me in your
lives – you’ve shown me so much
kindness; words cannot express how grateful I am. To my friends
back home, particularly Lauren
“Boots” Xenakis and Sara Powers: thank you for your
unconditional reassurance that I am doing the
right thing.
To all of my friends in Australia: for the last 3.5 years, the
Degnan office was largely my whole world.
Coming alone to a country where I didn’t know a soul, I was
lucky enough to learn that I didn’t need
to look much farther than the desks around me. While labs are
often transitory places, each member,
both past and present, has helped make Brisbane an enjoyable
place to spend the past few years of my
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life: Ben, Fede, Katia, Eunice, Jabin, Shun, Andrew, Romy, Lisa,
Aude, Xueyan, Gemma, Markus,
Kerry, Maely, Kevin, Arun, Laura, Tahsha, Simone, and Bec. I
will never forget all of the good times
we had at Friday drinks, spontaneous coffees, and pizza parties
in the park. Also, to all of my non-lab
friends: Cait, Aowen, Steve, Eddie, Andrew, and Caitlin – thank
you!
I would also like to give a shout out to the people at the UQ
swimming pool, in particular Jae and
Sarah for encouraging me to join the swim squad and being a ray
of sunshine at 5:00am. You’ve kept
me balanced and taught me that I don’t need to be ‘sedentary by
choice’.
Finally, I would like to give special thanks to Laura, my fellow
co-founder of ‘Milkshake Fridays’,
for all of our culinary adventures, teaching me the joys of Kate
Bush, and always being there when I
needed you (Uni-Halo forever); Simone, for your continual
encouragement, friendship, and reminders
to eat my vegetables; Tahsha, for our amazing weekends at
Caloundra and your unwavering support
and positivity; and Bec, for teaching me to become a morning
person and get to the pool on time,
Wednesday movie club, and enabling my coffee addiction. The
lists go on and on - I will truly miss
you all!
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Keywords
Evolution, development, larvae, biphasic, life cycle,
transcriptome, genome, body plan
Australian and New Zealand Standard Research Classifications
(ANZSRC)
060305, Evolution of Developmental Systems, 60%060102,
Bioinformatics, 20%060408, Genomics, 20%
Fields of Research (FoR) Classification
0603, Evolutionary Biology, 60%0601, Biochemistry and Cell
Biology, 20%0604, Genetics, 20%
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Table of conTenTschapTer 1 - inTroducTion: philosophical and
hisTorical paradigms for life cycle evoluTion 23
1.1 Diversity of animal life cycles 23
1.1.1 ‘Primary’ ciliated larval types 23
1.1.2 Similarities between primary larvae 25
1.1.3 Planktotrophy vs. lecithotrophy, the loss of feeding
larvae, and the evolution of direct
development 27
1.1.4 Reacquisition of indirect development and the evolution of
‘secondary’ larvae 29
1.1.5 How did larvae evolve? 30
1.2 Life cycle evolution: a historical dilemma 31
1.2.1 Early theories: from parallelism to recapitulation 31
1.2.2 Balfour and Garstang 32
1.2.3 Recapitulation and the ‘Terminal Addition Hypothesis’
33
1.2.4 Intercalation hypothesis 34
1.2.5 Ancient synapomorphy and the adaptive decoupling
hypothesis 36
1.3 Aims of this study 37
1.3.1 The Amphimedon queenslandica model system: a foundational
case study 38
1.3.2 Aim 1: Gleaning biological meaning from transcriptomes of
non-model organisms 39
1.3.3 Aim 2: Analysis of the genomic orchestration of biphasy in
A. queenslandica 40
1.3.4 Aim 3: Is metamorphosis conserved across the metazoan
phyla? 40
chapTer 2 - gleaning biological meaning from TranscripTomes of
non-model organisms:
assessmenT of cel-seq developmenTal expression profiles in The
sponge Amphimedon queenslAndicA
41
2.1 Abstract 41
2.2 Introduction 42
2.3 Results 43
2.3.1 BLIND clustering largely places transcriptomes in expected
temporal order based on
morphology 44
2.3.2 BLIND clustering reveals distinct ‘transcriptional’ blocks
45
2.3.3 Different gene ontology (GO) annotation methods provide
drastically different GO
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annotations for the A. queenslandica genome 48
2.3.4 Gene ontology (GO) enrichment analyses of DEG lists
accentuate the methodological bias
between annotations 49
2.3.5 Protein domain-based (Pfam and InterPro) gene ontology
(GO) annotations are more
effective at recovering known G-protein coupled receptors
(GPCRs) than BLAST-based annotations
51
2.3.6 BLAST-based gene ontology (GO) annotations are more
effective at recovering putative
transcription factors (TFs) than protein domain-based
annotations 52
2.3.7 Gene ontology (GO) enrichment recovers the temporal
expression of known differentially
expressed genes (DEGs) 54
2.3.8 KEGG and GOSlim methodologies are in accordance with one
another for key
developmental pathways 56
2.4 Discussion 59
2.4.1 Certain drastic life cycle transitions, such as the
emergence from maternal brood chamber,
are not marked by large-scale transcriptional changes 59
2.4.2 GOSlim provides a conservative method to computationally
characterize non-model life
cycles 61
2.4.3 The efficiency of each GO annotation depends on the
biology of the candidate gene 63
2.4.4 Gene ontology (GO) enrichments largely corroborate the
temporal expression patterns of
candidate genes 65
2.4.5 GOSlim enrichments and KEGG are often in accordance with
observed biological function
67
2.4.6 Computational recommendations: To avoid methodical bias,
multiple annotations must be
used to infer gene function in silico in a non-model organism
68
2.5 Conclusions 69
2.6 Methods 70
2.6.1 Identification of transcriptional blocks and differential
expression analyses 70
2.6.2 Genome annotation using gene ontology (GO), orthology, and
GO enrichment 70
2.6.3 KEGG annotation and pathway reconstruction 71
chapTer 3 - The orchesTraTion of biphasy in The sponge,
Amphimedon queenslAndicA, and The
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evoluTion of complex life cycles 73
3.1 Abstract 73
3.2 Introduction 73
3.3 Results 75
3.3.1 Pelagic and benthic transcriptomes are transcriptionally
distinct 75
3.3.2 Amphimedon queenslandica larvae and adults are primarily
using the same genes at similar
expression levels, therefore, the majority of larval genes are
not turned ‘OFF’ in adulthood 76
3.3.3 Differentially expressed genes (DEGs) remaining highly
expressed throughout the life cycle
are enriched in pre-metazoan traits, suggesting that both body
plans rely on core set of ancient genes
77
3.3.4 Distinct phase- and stage-specific coexpression clusters
reinforce the transcriptional
dissimilarity between pelagic and benthic phases 80
3.3.5 Larval and juvenile transcriptomes are more similar in
gene age than the reproductive adult
81
3.4 Discussion 83
3.4.1 Pelagic and benthic transcriptomes share a core set of
highly expressed genes of ancient
origin, suggesting that biphasy is a synapomorphic trait of the
Metazoa 83
3.4.2 Despite this core set of genes, similarity in overall
transcriptional profile correlate with body
plan and ecology rather than with organismal size or complexity,
supporting the adaptive decoupling
hypothesis 85
3.4.3 Gene age correlates with organismal size and complexity
instead of ecological phase,
consistent with theories for developmental constraint and the
evolution of gene regulatory networks 86
3.5 Conclusions 87
3.6 Methods 88
3.6.1 Differential Expression Analysis 88
3.6.2 Quartile analysis 88
3.6.3 Gene Annotation and Analysis 89
3.6.4 Gene Age Analyses 90
chapTer 4 - comparaTive insighT inTo The evoluTion of animal
life cycles 91
4.1 Abstract 91
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4.2 Introduction 92
4.3 Results 94
4.3.1 The majority of genes differentially expressed across
metamorphosis do not have orthologs
in the other sampled species 95
4.3.2 Despite the lack of orthology between differentially
expressed genes (DEGs) in each species,
conserved transcription factors (TFs) are involved in
metamorphosis across phyla 98
4.3.3 Novelty is widespread throughout the metamorphic temporal
coexpression network (TCN)
in sponges and sea urchins 100
4.3.4 Comparison of sponge and sea urchin temporal coexpression
networks (TCNs) suggest that
conserved transcription factors regulate phylum-specific
batteries 103
4.4 Discussion 105
4.4.1 Metamorphosis is characterized by phylum-specific genes:
evidence for convergence? 106
4.4.2 Conserved transcription factors (TFs) are differentially
expressed across metamorphosis in
all six sampled taxa 109
4.4.3 Temporal coexpression networks (TCNs) and the ‘mode’ of
evolutionary change 110
4.5 Conclusions 112
4.6 Methods 113
4.6.1 Sample collection 113
4.6.2 Library preparation and sequencing 114
4.6.3 Transcriptome assembly and gene prediction 114
4.6.4 Differential expression analysis 115
4.6.5 Orthology and gene age analyses 115
4.6.6 Protein domain analyses 116
4.6.7 Network analysis 116
chapTer 5 - discussion: shifTing paradigms in The evoluTion of
animal life cycles – The
incorporaTion of genomic daTa seTs inTo hisTorical concepTual
frameworks 119
5.1 Overview of findings: How does biphasy operate on a genomic
level? 120
5.2 Theoretical and evolutionary implications: When and how did
biphasy evolve? 122
5.2.1 Periodization and adaptive decoupling 122
5.2.2 Homology vs. convergence: When did biphasy evolve? 125
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5.2.3 Terminal addition vs. intercalation: How did biphasy
evolve? 128
5.3 Synthesis of findings: A modified hypothesis for the
evolution of biphasy 129
5.4 Conclusions and looking forward 132
references 133
appendices 159
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lisT of figuresFigure 1.1 Diversity and phylogeny of metazoan
life cycles 24
Figure 1.2 Schematic flowchart illustrating the progression of
historical theoretical hypotheses for the origin
and evolution of animal life cycles 32
Figure 1.3 Nielsen’s theory of terminal addition 34
Figure 1.4 Implications of the intercalation hypothesis 35
Figure 1.5 Possible scenarios for the evolution of biphasic life
cycles 37
Figure 1.6 Life cycle of the demosponge, Amphimedon
queenslandica 39
Figure 2.1 The A. queenslandica life cycle can be divided into
discrete transcriptional ‘blocks,’ which allow
for the characterization of ontogenetic changes in lieu of
traditional morphological stages 46
Figure 2.2 Gene ontology (GO) annotations of the A.
queenslandica genome and lists of differentially
expressed genes (DEGs) are variable between three methods 48
Figure 2.3 Enrichment of GOSlim terms varies between annotation
methods 50
Figure 2.4 Certain gene ontology (GO) annotations are more
successful at identifying G-protein coupled
receptors than others in the A. queenslandica genome 51
Figure 2.5 Gene Ontology (GO) does a poor job recovering
transcription factors (TFs) identified by
orthology to known human TFs in the A. queenslandica genome
53
Figure 2.6 GO enrichments still support key changes in
expression patterns for critical genes of interest 55
Figure 2.7 GO enrichments for signal transduction are supported
by KEGG analyses 57
Figure 2.8 Comparison of GO enrichment and KEGG analyses for the
apoptosis / cell death pathway 58
Figure 2.9 Proposed workflows to bioinformatically characterize
gene function across the ontogeny of a non-
model system 60
Figure 3.1 The life cycle of A. queenslandica 76
Figure 3.2 Expression quartile analysis of differentially
expressed genes (DEGs) 77
Figure 3.3 Phylostratigraphy (PS) enrichment analysis for each
quartile expression profile 78
Figure 3.4 Evolutionary conservation of quartile expression
profiles 79
Figure 3.5 Differentially expressed genes (DEGs) divided into
eight co-expressed ‘gene suites’ 81
Figure 3.6 Phylostratigraphy (PS) analysis of the A.
queenslandica transcriptome and individual gene suites
82
Figure 4.1 Gain/loss tree of HomologGroups (HGs) used in the
inference of gene age 96
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Figure 4.2 Characterization of differentially expressed genes
(DEGs) during the metamorphosis of six animal
species 97
Figure 4.3 Temporal coexpression network (TCN) for Amphimedon
queenslandica metamorphosis (late
embryogenesis through feeding juvenile stages), highlighting
gene age distribution 101
Figure 4.4 Temporal coexpression network (TCN) for
Strongylocentrotus purpuratus metamorphosis
(precompetent larva through young juvenile stages), highlighting
gene age distribution 102
Figure 4.5 Comparison of sponge (Amphimedon queenslandica) and
sea urchin (Strongylocentrotus
purpuratus) metamorphic temporal coexpression network (TCN)
based on shared differentially expressed
(DE) transcription factors (TFs) 105
Figure 5.1 Walter Garstang’s theory of parallel ontogenies
(1922): “The real phylogeny of Metazoa has never
been a direct succession of adult forms, but a succession of
ontogenies or life-cycles” 124
Figure 5.2 Evolutionary hypothesis for the evolution of extant
animal life cycles 130
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lisT of TablesTable 4.1 Shared TFs that are differentially
expressed across metamorphosis in multiple taxa 99
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lisT of appendicesAppendix 2.1 BLIND ordered samples indicating
morphological stage and transcriptional block 159
Appendix 2.2 Identification of differentially expressed gene
lists 159
Appendix 2.3 Differentially expressed lncRNAs 160
Appendix 2.4 Differentially expressed genes across the A.
queenslandica life cycle 160
Appendix 2.5 List of enriched biological process (BP) GO terms
for each annotation method (Blast2GO/
InterPro, Trinotate-BLAST, and Trinotate-pfam), including
subsequent overlap analyses 161
Appendix 2.6 Total GO Enrichments for biological process (BP)
are more variable between annotation
methods than GOSlim, with little overlap between all three
methodologies 161
Appendix 2.7 List of enriched GOSlim terms for each annotation
method (Blast2GO/InterPro, Trinotate-
BLAST, and Trinotate-pfam), including subsequent overlap
analyses 162
Appendix 3.1 Additional quartile overlap 164
Appendix 3.2. Gene ontology enrichment of each differentially
expressed gene suite 164
Appendix 3.3 Functional characterization of each gene suite
165
Appendix 3.4 Additional phylostratigraphy 166
Appendix 4.1 RNA-Seq Statistics for Acropora ‘dig-gem’, Haliotis
asinina, Balanoglossus misakiensis, and
Herdmania momus 167
Appendix 4.2 Transcriptome assembly (Trinity), gene annotation
(TransDecoder), and differential expression
analysis (RSEM/edgeR) statistics 167
Appendix 4.3 Fixed phylogenetic tree used in the parsimony
analyses of gene age for all species 168
Appendix 4.4 Dollo Parsimony (gain/loss) of HomologGroups
168
Appendix 4.5 Pfam domain expansion for differentially expressed
genes of six metazoan taxa 168
Appendix 4.6 Species-specific, bilaterian and deuterostome Pfam
domain enrichments 168
Appendix 4.7 Species-specific, bilaterian and deuterostome Pfam
domain enrichment examples 169
Appendix 4.8 All HomologyGroups (HGs) containing transcription
factor orthologs 169
Appendix 4.9 HomologyGroups (HGs) containing differentially
expressed transcription factors 169
Appendix 4.10 HomologyGroups (HGs) containing differentially
expressed transcription factor orthologs
shared between the sea urchin and sponge (extended version)
169
Appendix 4.11 FastOrtho configuration settings 170
Appendix 4.12 Accession information for temporal coexpression
networks 170
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Abbreviation DefinitionAd Acropora digitifera (gem-type)Dig-gem
Acropora digitifera (gem-type)Aq Amphimedon queenslandicaAqu2.1
Amphimedon queenslandica gene model version 2.1ARC Australian
Research CouncilBLAST Basic Local Alignment Search Toolblastp
Protein BLASTBLIND Basic linear index determination of
transcriptomesBm Balanoglossus misakiensisBP Biological processbp
Base pairCC Cellular componentCel-seq Cell Expression by Linear
amplification and SequencingChIP-Seq Chromatin Immunoprecipitation
Sequencingdb DatabaseDEG Differentially expressed geneDNase
DeoxyribonucleaseDOWN Significantly downregulateddpf Days post
fertilizationdps Days post settlementE-value Expect valueevo-devo
Evolutionary developmental biologyFDR False discovery rateGEO Gene
expression omnibusGO Gene ontologyGO-ID Gene ontology
identifierGPCR G-protein coupled receptorGRN Gene regulatory
networkgtf Gene transfer formatHa Haliotis asininaHG Homology
groupHGT Horizontal gene transferHm Herdmania momusHMMER
Biosequence analysis using profile hidden Markov modelshpf Hours
post fertilizationhpi Hours post induction
lisT of abbreviaTions
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KEGG Kyoto Encyclopedia of Genes and GenomesLCA Last common
ancestorlncRNA Long non-coding RNAMAPK Mitogen-activated protein
kinaseMF Molecular functionmRNA Messenger RNANCBI National Center
for Biotechnology InformationNO Nitric OxideOG Ortholog groupP-adj
Adjusted P-valueP-value Calculated probabilityPCA Principle
component analysisPolyA PolyadenylationPS Phylostratigraphy or
phylostrataQ QuartileRNA Ribonucleic acidRNA-seq RNA sequencingSp
Strongylocentrotus purpuratusSRA Sequence read archiveTCN Temporal
coexpression networkTF Transcription factorTGFβ transforming growth
factor-β TRG Taxonomically-restricted geneUP Significantly
upregulatedUQ University of Queenslandvsd variance stabilizing
distributionWnt WinglessWSP Wnt Signaling pathway
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chapTer 1 - inTroducTion: philosophical and hisTorical paradigms
for life cycle evoluTion
1.1 diversiTy of animal life cyclesAnimals display a wide
variety of life cycles, typified by two predominant strategies:
direct and indirect
development (Figure 1.1A). Direct developing species, including
most of the classic genetic and
developmental biology systems (mouse, zebrafish, C. elegans),
only have one body plan throughout
their life cycle. In contrast, indirect developing species
progress through two unique body plans – a
non-reproductive larva and a reproductive adult.
1.1.1 ‘Primary’ ciliated larval types
Among indirect developers, the majority of species have a
ciliated larval body plan, including poriferans
(sponges), cnidarians, protostomes (bryozoans, molluscs,
annelids), and deuterostomes (echinoderms,
and hemichordates). Sponges are widely considered to be one of
the earliest branching animal lineages
(Edgecombe et al. 2011) due to their relatively simple
morphology and lack of neuronal and muscular
cell types or a centralized gut. Despite this seemingly simple
adult body plan, the phylum Porifera
includes both indirect and direct developmental modes. This
includes multiple larval types, such as
amphiblastula and calciblastula in Calcarean sponges,
trichimella in Hexaxtinellida sponges, and
parenchymella, disphaerula, and coeloblastula in Demosponge taxa
(reviewed in Ereskovsky 2010,
Wörheide et al. 2012, Degnan et al. 2015). Based on recent
poriferan phylogeny, the parenchymella
larva is thought to be the ancestral larval type among sponges
(Wörheide et al. 2012).
Unlike the diversity of larval forms in poriferans, the majority
of cnidarian taxa in all classes (Anthozoa,
Schyphozoa, Cubozoa and Hydrozoa) have a similar bilayered and
monociliated planula larva,
characterized by an outer epidermis (ectoderm), an internal
gastrodermis (endoderm), and aboral
sensory cilia (Ruppert et al. 2004). This planula larva is
considered to be ancestral to all cnidarian
classes (Collins 2002). In contrast to sponges and cnidarians,
which display phylum-specific larval types,
protostomia-lophotrochozoa lineages (including Annelid, Mollusc,
Nemertean, and Platyhelminthe
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24
phyla) all share a common trochophore larva. This trochophore
larva is characterized by a ciliary band
called a prototroch, which is used for locomotion and feeding
(Damen and Dictus 1994). Likewise,
non-chordate deuterostomes (including hemichordates and
echinoderms) frequently have a dipleurula-
type larvae, including the tornaria larvae of hemichordates and
the dipleurula of echinoderms, which
are believed to share common origins (Nezlin 2000).
Porifera
Ctenophora
Cnidaria
Acoelomorpha
Mollusca
Echinodermata
Annelida
Crustacea
Arthropoda
Hemichordata
Urochodata
Chordata
Cephalochordata
Protostomes
Deuterostom
es
Indirect developing
Direct developingPrimary larva
Feeding Non-feeding Secondary larva
Feeding Non-feeding
= Indirect developing
= Direct developing
= Secondary larva
= Primary larva
= Loss of larval stage
A B
Direct developing
Figure 1.1 Diversity and phylogeny of metazoan life cycles. (A)
Phylogenetic distribution of primary developmental modes of the
major metazoan lineages. Indirect/pel-agobenthic/biphasic life
cycles (blue) are widespread throughout the animal kingdom.
However, some phyla have are direct developing (green). Gold lines
illustrate lineages with a ‘primary’ ciliated larva and red lines
indicate secondarily-derived larvae. (B) Dichotomous key of extant
larval types illustrating a proposed hypothesis for life cycle
evolution, where indirect development is believed to be ancestral.
Due to this inferred ancestral state, indirect developers are also
known as ‘primary’ larva (indicated in gold), which can be further
subdivided into feeding (planktotrophic) and non-feeding
(lecithotrophic) larval types. Alternatively, it is believed that
direct development evolved from an indirect developing ancestor
through the loss of the larval stage (indicated by the gray dashed
line). In some lineages, these direct developers independently
evolved another biphasic life cycle. These new larval types are
called ‘secondary’ (indicated in red) in contrast to the inferred
ancestral state of ciliated larvae. Like primary larvae, secondary
larvae can be further subdivided into feeding and non-feeding
larval types.
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Ch a p t e r 1: In t r o d u C t I o n
Many studies hypothesize that these four larval types (sponge
larva, cnidarian planula, trochophore
larvae, and dipleurula-like larvae) represent four independent
evolutionary origins of larval body plans
(Nielsen 1998, Hadfield 2000). Furthermore, it is hypothesized
that the biphasic life cycle, consisting of
a pelagic ciliated larva and a benthic adult is the ancestral
developmental mode in all phyla (Jägersten
1972). Hence, ciliated larvae are often called ‘primary’ larvae,
in reference to the supposed ancestry of
these larval types (Jägersten 1972; Figure 1.1B; also see
section 1.2). However, there are also multiple
similarities between these four larval types, suggesting that
primary larvae may also be homologous
(Nielsen and Nørrevang 1985, Degnan and Degnan 2006, Nielsen
2013).
1.1.2 Similarities between primary larvae
Perhaps the earliest hypotheses for larval homology was among
protostome species (Hatschek 1878,
Roule 1891), suggesting that the ubiquity of the trochophore
larval body across lophotrochozoans is
indicative of common ancestry among spiralians (more recently
this has been supported by Nielsen
and Nørrevang 1985). Additional studies have compared protostome
and deuterostome lineages in
search of evidence of common bilaterian origins for marine
larvae (Nielsen 1994, 1998). However,
these studies largely support the independent evolution of
trochophore larvae in protostomes (consistent
with Nielsen and Nørrevang 1985, Rouse 1999, 2000) and
dipleurula-like larvae in deuterostomes
(consistent with Nezlin 2000).
More recently, molecular techniques have been used to
investigate whether there may be more deeply
conserved processes patterning disparate larval types (e.g.
Jackson et al. 2005, Dunn et al. 2007, Rentzsch
et al. 2008, Mazza et al. 2010, Santagata et al. 2012, Marlow et
al. 2014). Specifically, studies have
examined the similarities of bilaterian larval gut development,
illustrating that expression patterns,
such as of Brachyury, otx, and goosecoid, are conserved between
protostome and deuterostome larvae
(Arendt et al. 2001). Unlike morphological studies, this
suggests that protostome and deuterostome
larvae share deeply conserved developmental mechanisms for
larval patterning, suggesting that larval
homology predates the bilaterian last common ancestor.
In this context, the apical organ is an ideal candidate to
investigate larval homology between phyla,
because it is found across all bilaterian taxa and is only
present in the larvae of indirect developing
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species (Nielsen 2005, Dunn et al. 2007, Lacalli 2008). In
ciliated protostome and deuterostome larvae,
the apical organ can be defined by a thickened epithelium at the
animal pole of the larvae, with a
characteristic cluster of elongated cilia (apical tuft), and
neuronal ganglion (Nielsen 2004, Byrne et al.
2007, Dunn et al. 2007). There is clear evidence that the larval
apical organ is responsible for sensing
surroundings and appears to be especially tied to larval
settlement in species of most indirect developers,
including cnidarian larvae (e.g. Rentzsch et al. 2008,
Conzelmann et al. 2013). However, it is unclear if
similarities of the apical neurons can be deemed homologous in
basal clades, such as the planula larvae
of cnidarians. Despite having a primitive nerve net as adults,
cnidarian larvae have very similar apical
morphology to trochophores, including an apical tuft (Chia and
Koss 1979, Sinigaglia et al. 2015).
Even without neurons, the anterior region of the sponge is
capable of distinguishing changes in light
intensity (Leys and Degnan 2001), and is suggested to be
important to larval settlement (Degnan and
Degnan 2010, Degnan et al. 2015, Nakanishi et al. 2015, Ueda et
al. 2016). Specifically, flask cells in
the anterior region of the A. queenslandica larvae have been
shown to respond to environmental cues
via calcium mediated signaling pathways (Nakanishi et al. 2015),
which in turn interact with Nitric
Oxide signaling pathways to initiate metamorphosis (Ueda et al.
2016). This suggests that the apical
region (posterior region in sponges) in ciliated primary larva
across the Metazoa might be involved
in environmental sensing. However, this may also be the result
of functional convergence (Dunn et
al. 2007).
There have also been a handful of molecular studies
investigating genes associated with the formation
of the larval apical region (most recently, Marlow et al. 2014).
These studies comparing expression
patterns between representatives of different phyletic lineages
indicate that some genes involved in
apical organ formation and patterning predate the eumetazoan
split (FGF - Rentzsch et al. 2008; COE
- Jackson et al. 2005; Six3/6 - Santagata et al. 2012, Marlow et
al. 2014), and some are specific to
bilaterians (Homeobrain, Rax, Orthopedia - Mazza et al. 2010).
However, based on gene regulatory
network inferences, Dunn et al. (2007) conclude that there is no
evidence for homology in apical organ
patterning between protostomes and deuterostomes, suggesting
that trochophore and dipleurula larvae
result from independent evolutionary events (Dunn et al. 2007).
Given the findings presented in this
section, it remains uncertain how many times ciliated larval
body plans arose (reviewed in Hadfield
2000), and under what mechanisms complex life cycles have
evolved (see section 1.2).
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Ch a p t e r 1: In t r o d u C t I o n
1.1.3 Planktotrophy vs. lecithotrophy, the loss of feeding
larvae, and the evolution of direct
development
Primary, ciliated larval types can be further subdivided into
feeding (planktotrophic) and non-feeding
(lecithotrophic) larval types (Figure 1.1B). Planktotrophic
larvae (which can be found in animal phyla
such as, Mollusca, Annelida, Echinodermata, and Hemichordata)
are capable of feeding via a larval
gut (Thorson 1950). Alternatively, lecithotrophic larvae, such
as all poriferan larva, most cnidarians,
some bryozoans, and some echinoderms, cannot feed until after
metamorphosis, when the adult feeding
structures have been established (Thorson 1950, Vance 1973).
Non-feeding larvae generally have a
shorter larval phase than planktotrophic species, the length of
which is dictated by finite energetic
resources from the maternally derived yolk (‘energetic burnout’:
Hadfield et al. 2001; Vance 1973).
Thus, planktotrophy versus lecithotrophy is often seen as an
adaptive trade-off (Marshall and Keough
2003): planktotrophic larvae are energetically cheap to produce,
allowing for a greater number of
offspring, yet, feeding larvae also experience a higher rate of
mortality prior to settlement (Thorson
1950).
This trade-off has multiple implications for the various
ecological roles marine larvae can play in an
animal life cycle (reviewed in Strathmann 1978b). For instance,
as many marine invertebrates have
sessile adult body plans, it is intuitively believed that
increasing time in the water column provides a
mechanism for gene flow and dispersal between populations
(Hedgecock 1986); however, this hypothesis
has recently come into question (Levin 2006, Sanford and Kelly
2011), as some studies suggest that
larvae may not be dispersing as far as originally thought
(Swearer et al. 2002, Palumbi 2004, Morgan
et al. 2009). Thus, as planktotrophic larvae are capable of
feeding, they are able to remain in the water
column longer than lecithotrophic species, potentially resulting
in greater dispersal and gene flow. In
contrast, lecithotrophic species, which are unable to travel as
far, have a higher rate of metamorphic
success. In addition to a role in dispersal, larvae play a
crucial role in site selection (Raimondi and
Keough 1990). If the larva does not choose an appropriate
location to settle, the organism cannot reach
sexual maturity (Pechenik 2006). Therefore many larvae have
evolved a tightly regulated mechanism
of site selection based on a myriad of environmental cues both
within and between species (reviewed in
Steinberg and Nys 2002, Hadfield 2011). As with dispersal, there
are noticeable differences in settlement
strategies between planktotrophic and lecithotrophic larvae, as
the former are capable of feeding until
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an appropriate site is found (Hadfield et al. 2001, Pechenik
2006). Alternatively, lecithotrophic larvae
are limited by the amount of yolk, and thus in the absence of an
appropriate settlement cue, must either
spontaneously settle or die (Hadfield et al. 2001).
While life history strategies were originally thought to be
dichotomous (Thorson 1950), some larva
display ‘facultative planktotrophy’ (e.g. Kempf and Hadfield
1985, Emlet 1986), and are capable of
taking advantage of both developmental modes (reviewed in Allen
and Pernet 2007). However, there
is increasing uncertainty whether these ‘non-traditional’
developmental modes represent evolutionary
intermediates between planktotrophy and lecithotrophy or have
been selected for due to some adaptive
advantage (Collin 2012). Regardless, the prevalence of
intermediate life history modes sheds light on
the potential evolutionary theories for the diversity of extant
life cycles. In the literature, it is assumed
that planktotrophy is ancient among bilaterians and thus the
loss of feeding larvae is a derived trait
(Strathmann 1978a, 1978b, Collin 2004, Collin et al. 2007).
Likewise, as the presence of a ciliated larva
is considered to be ancestral (Jägersten 1972), it is believed
that direct development is also a derived
trait that evolved from an indirect developing species
(Jägersten 1972, Strathmann 1978; reviewed
in Reitzel et al. 2006). In some phyla, loss of a feeding larval
phase is closely intertwined, and often
synonymous, with the shift from indirect to direct development
(e.g. Collin et al. 2007).
Many phyla contain both direct and indirect developing taxa,
including sponges, cnidarians, annelids,
arthropods, echinoderms, and chordates. Based on echinoderm
phylogeny, it is apparent that the ancestral
echinoderm was indirect developing, therefore direct development
has evolved multiple times in the
phyla through the loss of a planktotrophic larval stage
(Strathmann 1978b, Wray 1996). Additionally,
among the cnidarians, extant taxa display a highly diverse range
of developmental modes—each with
varying degrees of biphasy (Martin and Koss 2002). However the
majority of the four classes (Anthozoa,
Schyphozoa, Cubozoa and Hydrozoa) produce similar non-feeding,
ciliated planula larvae, suggesting
that the ancestral cnidarian had a pelagobenthic life cycle
(Collins 2002). Given that, metamorphosis
from a planula larva to a primary polyp in the anthozoan,
Nematostella vectensis, is a gradual process
with little tissue reorganization (Rentzsch et al., 2008),
indicating that the dramatic metamorphosis
seen in other cnidarian taxa was partially lost in this species
(Reitzel et al. 2006).
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Ch a p t e r 1: In t r o d u C t I o n
1.1.4 Reacquisition of indirect development and the evolution of
‘secondary’ larvae
In the past, it was thought that once the complex morphological
and genetic machinery needed to
create a planktotrophic larva were lost, they were unlikely to
be regained (Strathmann 1978a, 1978b).
However, reacquisition occurs in both brachiopods, which retain
larval morphology in juveniles, and
some gastropods, which retain planktotrophic feeding structures
in lecithotrophic larvae (Strathmann
1978b). Recent studies in gastropods indicate that the
likelihood of feeding reacquisition is related to
the amount of time that has lapsed since its loss, as the
feeding morphology in recently lecithotrophic
species is more likely to be retained (Collin 2004).
Furthermore, as both feeding and swimming
structures remain intact in these lecithotrophic larva, it has
been hypothesized that the reacquisition
of planktotrophy in these species may result from heterochronic
shifts in development pathways
(Collin et al. 2007). Other explanations for the reacquisition
of complex, developmental traits, such as
planktotrophy and/or indirect development, include the genetic
pleiotropy and the inherent modularity
of developmental pathways involved in morphogenesis (reviewed in
Collin and Miglietta 2008).
As the majority of developmental genes are used in multiple
morphogenetic pathways, Collin and
Miglietta (2008) argue that structures for larval feeding can be
readily reacquired over relatively short
evolutionary time because the genetic machinery required for
their establishment remain intact in other
genetic modules.
In addition to the reacquisition of a planktotrophic larva from
a lecithotrophic species, in some extreme
cases, it is believed that certain larval types (such as the
tadpole larva in ascidians and the cyprid larva
in crustaceans) evolved independently from a completely direct
developing ancestor (Jägersten 1972,
Hadfield 2000). Therefore, these larvae (deemed ‘secondary’ in
comparison to ‘primary’ ciliated larvae;
Figure 1.1B) were convergently reacquired from a species that
had previously (and permanently) lost its
ancestral ciliated larva. Hence, these ‘secondary’ larvae bear
little morphological resemblance to ciliated
larvae and are often characterized by distinct genetic
mechanisms underlying metamorphic transitions
(Hadfield 2000, Heyland and Moroz 2006). For instance, unlike
primary pelagobenthic life cycles,
where metamorphosis often occurs rapidly in response to external
settlement cues, secondarily derived
metamorphic events (such as in crustaceans,
caterpillar/butterfly and tadpole/frog) are characterized
by slow, hormonally-regulated transitions (Hadfield 2000,
Heyland and Moroz 2006).
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Ascidian tadpole larvae represent another example of a
secondarily derived larval body plan. Unlike
ciliated larvae, ascidian tadpoles are characterized by a
muscular tail and notochord that is reabsorbed
during metamorphosis into the benthic filter-feeding body plan
(Katz 1983). Due to the presence of
the notochord, and the basal position of cephalochordates among
chordates, ascidians are believed
to have evolved from a direct developing cephalochordate-like
ancestor (Satoh 2009). Thus, biphasy
and the adult filter-feeding adult body plan in ascidians are
likely to be derived. Interestingly, despite
being classified as ‘secondary’ larvae, ascidian larvae display
a significant number of similarities with
primary ciliated larvae (Hadfield et al. 2000). Like many
ciliated larvae, ascidian tadpoles display a
distinct period of larval competence (Degnan et al. 1997)
followed by a rapid metamorphic event
in response to exogenous settlement cues (Green et al. 2002).
This is consistent with the hypothesis
presented in Hadfield et al. (2001), which argues that
competence is a convergent trait that evolved to
minimize vulnerability during settlement/metamorphosis (Hadfield
2000). Additionally, certain signaling
pathways, such as Nitric Oxide appear to be employed in both
primary and secondary metamorphic
events, reinforcing convergence (Bishop and Brandhorst 2003,
2007, Comes et al. 2007, Bishop et al.
2008, Ueda and Degnan 2013, 2014, Ueda et al. 2016).
Alternatively, as hypothesized in the reacquisition
of planktotrophic larvae (Collin and Miglietta 2008), these
similarities may have also been preserved
from ancient (and previously lost) life cycles through intact
pleiotropic developmental pathways.
1.1.5 How did larvae evolve?
Classifications of marine larvae are inherently tied to
hypotheses for life cycle evolution. Therefore,
it is impossible to classify larvae without touching upon the
theoretical origins of each larval type.
However, the evolutionary history of animal life cycles remains
largely unclear. Given the incredible
diversity of marine larvae, studies have speculated that the
biphasic life cycle, including those with
primary or secondary larval types, is the result of multiple
episodes of convergent evolution (Nielsen
1998, Rouse 2000, Hadfield 2000, Dunn et al. 2007). However, the
prevalence of biphasy among extant
animal taxa suggests common ancestry of this complex
developmental trait (Degnan and Degnan 2006,
Mikhailov et al. 2009, Nielsen 2013). This dilemma lies at the
very heart of animal evolution, drawing
upon hundreds of years of theoretical embryology. Hence, the
driving force behind this thesis is to
expand upon these foundational speculative hypotheses, many of
which were conceived before the
dawn of modern genetics, using novel genomic and transcriptomic
approaches. In the following section,
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Ch a p t e r 1: In t r o d u C t I o n
I examine these hypotheses through the lens of historical
embryology in order to highlight how the
shifting paradigms for life cycle evolution have influenced the
current framework for larval evolution.
1.2 life cycle evoluTion: a hisTorical dilemma1.2.1 Early
theories: from parallelism to recapitulation
There is a long and complex history of speculative literature
surrounding the origins and evolution
of animal life cycles, beginning with Karl Ernst von Baer
(Figure 1.2). von Baer’s laws are a set of
observations, which arguably founded embryological theory and
modern evolutionary developmental
(‘evo-devo’) biology (reviewed in Abzhanov 2013). Simply stated,
these principles claim that all
animals develop from a simple or ‘general’ form to a more
complex one, and these general characters
are only found during early development. This conceptual model
is often described as a developmental
‘funnel’, where morphological and genetic complexity increases
in a linear fashion from the zygote
to the adult body plan. From an early stage of embryological
work, researchers noticed a similarity
between the increasing complexity during embryogenesis and the
increasing complexity in the
fossil record, establishing an early link between evolution and
development, deemed ‘parallelism’
(reviewed in Abzhanov 2013), which would become the original
framework for subsequent comparative
embryological and evolutionary theories.
Perhaps the most well known of these is Haeckel’s biogenetic
laws, which famously claim that ‘ontogeny
recapitulates phylogeny’ (Haeckel 1868, Abzhanov 2013). Haeckel
believed that each new phase is
built upon ancestral forms. Therefore, the embryo provides a
window into an animal’s ancestry, and
development depicts the evolutionary progression to the adult
body plan. Haeckel saw the similarity
between developing metazoans at the gastrula stage, and asserted
that the ancestor of all animals must
have resembled this form at some point in evolution, a
hypothetical ancestor he called the “gastraea”
(Love et al. 2008). This drastic embellishment on early theories
of parallelism perpetuated von Baer’s
developmental funnel, and ultimately became deeply engrained in
later concepts of evolutionary
developmental thinking (see section 1.2.3).
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1.2.2 Balfour and Garstang
While less well known than Haeckel (and significantly less
cited), 19th century embryologists, Francis
Balfour and Walter Garstang, made important advances in the
understanding of embryology (reviewed
by Brian Hall, 2000). Balfour’s treatises on embryology mark
important differences from Haeckel
(Balfour 1880, 1881). The first is that early development is not
‘immutable’ as Haeckel and von Baer
thought, therefore “not all embryonic features reveal ancestral
patterns” (Hall, 2000). In addition to
phenotypic variation in the adult form, Balfour noticed that
early development is often marked by
significant embryonic variations between phyla. This idea
highlights an important departure from the
developmental ‘funnel’ proposed by von Baer and Haeckel
(Abzhanov 2013), becoming a prototype
for the ‘hourglass model’ of evolution, which was later made
famous by the phylotypic stage (Duboule
1994, Raff 1996). The primary implication of the hourglass model
is that that morphological and
genetic divergence could occur in all stages of an ontogeny
except the phylotypic stage (Raff 1992).
von Baer
Haeckel
Balfour
Garstang Jägersten &Nielsen
Degnan &Degnan
Sly et al.
NielsenPhylotypicStage
Mikhailovet al
ontogney recapitulates phylogeny
Parallelism
Terminal addition
biphasy is synapomorphic
Intercalation
early embryogenesisis divergent
Developmental stagesevolve independently
In accordanceRejects
Inspired
Adaptivedecoupling
1.2.1
1.2.2
1.2.3
1.2.41.2.5
Developental ‘funnel’ model
Developental ‘hourglass’ model
Figure 1.2 Schematic flowchart illustrating the progression of
historical theoretical hypoth-eses for the origin and evolution of
animal life cycles.
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33
Ch a p t e r 1: In t r o d u C t I o n
In addition to Balfour, Walter Garstang continued to reform
Haeckel’s theories, even boldly claiming
that the “basis of this law is demonstrably unsound” (Garstang
1922). In particular, Garstang argued that,
“the real Phylogeny of Metazoa has never been the direct
succession of adult forms, but a succession
of ontogenies or life-cycles...so that every phase of the
life-cycle is modified in some way or other”,
thereby stressing that ontogeny does not recapitulate phylogeny,
but “creates” it (Garstang, 1922).
The implication of this statement is that while larvae and
adults evolve separately, it is the ontogeny
as a whole that is passed from generation to generation
(Garstang 1928). Like Balfour, this concept
paved the way for other alternative hypotheses for life cycle
evolution that were unconstrained by the
Haeckelian paradigm, including Nancy Moran’s Adaptive decoupling
hypothesis (Moran 1994; see
section 1.2.5).
1.2.3 Recapitulation and the ‘Terminal Addition Hypothesis’
Despite Garstang’s harsh criticism of recapitulation hypotheses,
in the latter part of the previous century,
there was a resurgence of Haeckel’s biogenetic laws, pioneered
by Jägersten and Nielsen (Jägersten
1972, Nielsen and Nørrevang 1985, Nielsen 2005, 2008, 2009,
2013). These theories proposed that
the similarities between larval forms are too great to ascribe
solely to convergent evolution. Therefore
proponents of this “terminal addition” theory hypothesized that
the last common ancestor was larva-
like, and the adult phase evolved later.
Early theories of terminal addition (the “Trochaea theory”; such
as Nielsen and Nørrevang 1985)
greatly resembled the original theories for the homology of
trochophore larvae (Hatschek 1878, Roule
1891), and therefore only extend to a protostome-lophotrochozoan
last common ancestor. However,
Nielsen’s most recent hypothesis (Figure 1.3) continues to
develop upon Haeckel’s initial theory of
recapitulation, expanding the scope to include deeper
relationships in metazoan evolution (Nielsen
2008, 2009, 2013). Nielsen proposes the last common ancestor
(LCA) of living metazoans was a
pelagic colonial choanoflagellate, the so-called
“choanoblastaea”, capable of feeding using peripheral
choanocytes. He hypothesizes that the origins of early
pelagobenthic life cycles involved the polarization
of the choanoblastaea, which settled on the pole lacking
choanocytes, leading to the internalization
and subsequent loss of locomotory function (Nielsen 2008).
Following the establishment of a biphasic
life cycle in early metazoans, Nielsen speculates that the adult
“sponge-like” phase was lost through
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34
neoteny, establishing a holopelagic ciliary particle feeder: the
gastraea. From the gastraea, an organized
nervous system evolved, resulting in a neurogstraea—a eumetazoan
ancestor which Nielsen suspects
to be similar to modern anthozoan larvae. Based on this
hypothesis, all larval types are homologous.
However, adult body plans (and therefore the biphasic life
cycle) evolved independently in cnidarian,
protostome, and deuterostome lineages, yet share the same
genetic toolkit that was present in a last
common pelagobenthic metazoan ancestor.
1.2.4 Intercalation hypothesis
In response to the increasingly accepted Gastraea hypothesis, an
opposing “Intercalation” theory has
also gained momentum, in which the last common ancestor (LCA) of
metazoans was a direct developing
benthic organism (Sly et al. 2003). In this scenario, larval
forms evolved through the intercalation
of new genes to the previously existing “adult-like” ontogeny.
In 2003, Sly et al. discussed how the
hypothetical adult-first hypothesis could have occurred (Figure
1.4A). By illustrating gene ontogenies
as colored arrows, Sly illustrates how a novel gene pathway
could eventually pattern a new larval
Gastraea
Sponges
Deuterostromes
Neurogastraea
Protostomes
Trochaea
Neoteny
Cnidarians
METAZOAEUMETAZOA
BILATERIA
LCA
Figure 1.3 Nielsen’s theory of terminal addition. The most
recent iteration of Haeckel’s recapitulation theory, where a
holobenthic choanoblastaea-like ancestor developed a biphasic life
cycle through the polarization and settlement of a choanoblastaea.
Once biphasy is established, the adult phase of the last common
ancestor was lost through the retention and subsequent sexual
maturation of the larval stage. The resultant species (dubbed,
‘gastraea’) is the last common ancestor of eumetazoans. The
gastraea precedes the ‘neurogastraea’ following the evolution of a
nervous system. Upon the evolution of the neurogastraea, cnidarians
and bilaterians split, and the trochaea is derived in the
proto-stome lineage. This theory postulates at least 4 evolutionary
events that lead to biphasic life cycles, implying that adult forms
are derived, while larval forms are homologous among all metazoan
lineages. Synthesized and adapted from Nielsen 2008, 2009.
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Ch a p t e r 1: In t r o d u C t I o n
body plan (thereby creating a new ontogeny), involving the
addition of a facultative developmental
sub-pathway. Through time, the new pathway gains constituency,
and a rudimentary “larval” form
is established. Eventually, the new facultative pathway is more
advantageous to the original gene
ontogeny, and a true larval form arises.
Sly et al. (2003) claim that intercalation theory is more
parsimonious than early terminal addition
theories, as the adult-first hypothesis can explain gene
homology between both adults and larval forms
with fewer incidence of convergent evolution (Figure 1.4B).
However, Sly et al.’s analysis of larval
evolution only explains the emergence of biphasy at the last
common ancestor of bilaterians. More
recently, Degnan and Degnan (2006) hypothesized the importance
of meiosis in the evolution of larval
forms (Degnan and Degnan 2006). There, they argue that once
gametes are released from the adult,
they effectively become a new ontogeny; therefore the fertilized
embryo and the adult are exposed to
independent selective pressures resulting in distinct niche
specialization and increasing modularity
of “larval” forms (Figure 1.4B). In this scenario, they predict
that the LCA of metazoans had already
developed a biphasic life cycle prior to metazoan cladogenesis.
Thus, there is only one evolution of
biphasy, and larvae are a pleisiomorphic trait among
metazoans.
Adult-like ancestor
Phyla 1 Larva Phyla 2 Larva Phyla 3 Larva
Phyla 1 Adult Phyla 2 Adult Phyla 3 Adult
A B
Figure 1.4 Implications of the intercalation hypothesis. (A)
Schematic representation of the intercalation of larval features
into adult ontogenies. Blue lines represent ‘adult-like’ ontogeny,
while red indicates larval ontogeny. Larval ontogeny arises in an
‘adult-like’, direct devel-oping ancestor as a facultative pathway
free of evolutionary constraint, while adult specific gene pathways
remain intact. Subsequently, adult pathways are discarded, as
facultative genes become an adaptive advan-tage, resulting in a
truly biphasic species (B) Intercalation hypothesis asserts a
direct developing, ‘adult-like’ ancestor with complex genetic
structure. Evolution of larvae occurs from co-option of previously
acquired genes, resulting in homologous components of both larval
and adult forms. Both figures redrawn from Sly et al. 2003.
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1.2.5 Ancient synapomorphy and the adaptive decoupling
hypothesis
Three of the most recent reviews on larval evolution (Degnan and
Degnan 2006, Mikhailov et al.
2009, Nielsen 2013), including proponents of both intercalation
and terminal addition theories, agree
that the last common ancestor of the Metazoa was likely to
display some degree of biphasy; therefore,
the pelagobenthic life cycle is hypothesized to be an ancient
synapomorphic trait of extant animal
lineages. These theories (particularly that of Degnan and Degnan
2006) are largely consistent with ideas
proposed by Balfour and Garstang, specifically that different
life cycle stages evolve independently
of one another. This is in accordance with other hypotheses for
the evolution of complex life cycles,
such as Nancy Moran’s adaptive decoupling hypothesis (Moran
1994), which proposes an alternative
scenario, whereby larvae and adult body plans evolve in parallel
to one another due to ecologically and
developmentally phase-specific selective pressures. Thus, if
life cycle stages are decoupled, genetic
traits of different phases (such as in larvae and adults) will
be less correlated than genetic traits of the
same ecological niche (Ebenman 1992), resulting in a distinct
genetic barrier between pelagic and
benthic phases.
Other studies suggest that larval traits are equally important
to the reproductive success of the juvenile,
therefore it is unlikely that larval and adult phases could be
entirely decoupled (Pechenik 2006,
Crean et al. 2011). This implies that larval traits are likely
to be constrained due to intense selection
pressures associated with larval settlement (Crean et al. 2011).
Interestingly, this hypothesis echoes
Garstang’s early observation that larvae play a crucial role in
the life cycle evolution (Garstang 1922).
In addition to these selective pressures based on larval ecology
in biphasic species, the constraint of
earlier developmental stages is consistent with hypotheses for
the phylotypic stage (Duboule 1994).
It is hypothesized that the similarities between animal body
plans during this stage are the result of
stabilizing selection due to the increased connectivity of genes
expressed during that developmental
period (Raff 1992, 1994). This hypothesis has been supported by
genetic approaches (Galis and Metz
2001), suggesting that novel genes are more prevalent during
early and late development, while older
genes are expressed during the phylotypic stage (Domazet-Lošo
and Tautz 2010, Akhshabi et al. 2014).
Given these findings, many contemporary theoretical frameworks
(i.e. phylotypic stage, hourglass
model of evolution, and adaptive decoupling hypothesis) for life
cycle evolution are in accordance
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Ch a p t e r 1: In t r o d u C t I o n
with one another. However, many critical questions remain
unsubstantiated by empirical evidence –
particularly if biphasic life cycles are homologous across crown
metazoans.
1.3 aims of This sTudyThe more current hypotheses for larval
evolution (Intercalation theory: Degnan and Degnan 2006;
and Terminal addition: Nielsen 2010, 2013) assert a complex
biphasic metazoan ancestor, suggesting
that indirect development is a synapomorphic trait of all animal
phyla. If this is true, then the close
examination of larval homology should uncover deeply conserved
evolutionary signatures. However,
it is also possible that larval traits are only shared between
certain lineages, which would result in four
primary evolutionary scenarios (Figure 1.5). The first scenario
is the ancient metazoan synapomorphy
of biphasic life cycles, as proposed by Degnan and Degnan 2006,
Nielsen 2013, and Mikhailov et al.
2009 (Figure 1.5A). The second scenario suggests that poriferan
larvae evolved independently from the
planula-like larvae, which is the ancestral larval-type to
Cnidarian-Bilaterian lineages. This hypothesis
would be consistent with examinations of apical morphology as
suggested by Marlow et al. 2014 (Figure
1.5B). The third scenario describes the independent evolution of
poriferan, cnidarian, and bilaterian
lineages. This scenario is consistent with the
planuloid–acoeloid hypothesis, which postulates that
biphasic species in bilaterians evolved from a planula-like last
common ancestor (reviewed in Baguñà
and Riutort 2004). This hypothesis is also consistent with the
deeply conserved gut patterning shown
between protostome and deuterostome larvae (Arendt et al. 2001;
Figure 1.5C). Lastly, it is possible
that biphasy has evolved convergently (Hadfield 2000) in each of
these animal lineages, consistent
with early Trochaea theories (e.g. Nielsen and Nørrevang 1985;
Figure 1.5D).
Poriferans
Cnidarians
Protostomes
Deuterostomes
Poriferans
Cnidarians
Protostomes
Deuterostomes
Poriferans
Cnidarians
Protostomes
Deuterostomes
Poriferans
Cnidarians
Protostomes
Deuterostomes
A B C D
Figure 1.5 Possible scenarios for the evolution of biphasic life
cycles. Different evolutionary scenarios can be inferred through
homology. Colored blocks represent shared traits between clades and
black circles represent a distinct evolutionary event leading to
biphasy, following the assumption that homologous traits were also
present in the LCA.
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In light of these theories, fundamental unanswered questions
emerge: can modern genomics provide
novel insight into the evolution of the pelagobenthic life
cycle, and how can this new information be
incorporated into the existing theoretical frameworks for life
cycle evolution? More specifically, (1)
under what mechanism can a single genome pattern two different
body plans, and (2) what are the
implications of these findings for the evolution of animal life
cycles? In this thesis, I aim to answer these
fundamental questions using the sponge, Amphimedon
queenslandica, as a case study. Furthermore, I
aim to use comparative techniques to identify and compare
commonalities between A. queenslandica
biphasy with that of other metazoans.
1.3.1 The Amphimedon queenslandica model system: a foundational
case study
Sponges are widely considered to be the earliest branching
metazoan phyletic lineage (Edgecombe
et al. 2011), due to their simple adult body plan, which lacks
neurons or a central gut. However,
recent phylogenomic studies report that ctenophores may have
branched off from the metazoan stem
before sponges (Ryan et al. 2013, Moroz et al. 2014). This would
suggest that the simplified adult
body plan of the sponge represents a highly derived state, which
resulted from the loss of many tissue
types previously assumed to be conserved cnidarian-bilaterian
synapomorphies (Ryan and Chiodin
2015). However, despite this phylogenetic ambiguity, the
majority of sponges possess a complex
pelagobenthic life cycle (Wörheide et al. 2012), which is
arguably the earliest branching example of
a primary biphasic life cycle.
A. queenslandica is a brooding haplosclerid demosponge with a
biphasic life cycle consisting of a
ciliated parenchymella larva and a sessile, filter-feeding adult
(Degnan et al. 2015; Figure 1.6). Despite
a lack of any neuronal signaling, A. queenslandica larvae are
photosensitive (Leys and Degnan 2001),
and display highly coordinated behaviors, including phototaxis
and settlement selection (Jackson et al.
2002, Degnan and Degnan 2010). Additionally, like most biphasic
organisms, A. queenslandica larvae
develop competence before settlement upon exposure to a specific
settlement cue, such as crustose
coralline algae rubble (Degnan and Degnan 2010).
A. queenslandica provides important insight into the early
evolution of animal ontogenies, particularly
since hallmarks of early embryogenesis and the patterning of
larval body plans are largely conserved
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Ch a p t e r 1: In t r o d u C t I o n
with other eumetazoan species (reviewed in Degnan et al. 2015).
For instance, like eumetazoan species,
A. queenslandica displays characteristic features of animal
embryogenesis, including asymmetric cell
division and cell fate determination by well conserved
transcription factors and signaling pathways
(reviewed in Degnan et al. 2015; Adamska et al. 2007).
Furthermore, A. queenslandica offers a unique
opportunity for genomic studies, as it is one of the few basal
organisms with a fully sequenced and
annotated genome (Srivastava et al. 2010) and reasonably-well
annotated developmental transcriptome
(Fernandez-Valverde et al. 2015).
1.3.2 Aim 1: Gleaning biological meaning from transcriptomes of
non-model organisms
Despite the abundant genomic resources available for A.
queenslandica, like many other early branching,
non-model organisms, only a small portion of the putative gene
models have been functionally tested
in the lab. Hence, genome wide studies are largely restricted to
bioinformatically-derived functional
Embryogenesis
adult brood chamber
Acquisition of competence
Larv
al rel
ease
Settlement
MetamorphosisMaturation
Figure 1.6 Life cycle of the demosponge, Amphimedon
queenslandica. A. queenslandica displays a typically biphasic life
cycle with motile pelagic larva, and a benthic adult form. Pelagic
phase is separated by metamorphosis, followed by the settlement of
a competent larva in response to a specific chemical cue.
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annotations (such as gene ontology, KEGG, and Pfam) based on
sequence similarity to better-
characterized model systems such as Drosophila, C. elegans, and
mice. Therefore, the first aim of my
dissertation (Chapter 2) is to substantiate ubiquitously used
computational tools in the A. queenslandica
system against experimentally validated biological function.
Based on these results, I develop a strategy
for confidently mining existing A. queenslandica data sets to
uncover how the biphasic life cycle is
orchestrated in this species of sponge.
1.3.3 Aim 2: Analysis of the genomic orchestration of biphasy in
A. queenslandica
Little is currently known about how a single genome is
partitioned to pattern morphologically and
ecologically distinct larval and adult body plans, and whether
each phase expresses genes that are
unique or shared between body plans. Therefore, the second aim
of my dissertation (Chapter 3) is to
use the A. queenslandica developmental transcriptome to
investigate how biphasy is orchestrated in an
extant species of sponge. Furthermore, I use computational
methods of gene age to infer how biphasy
in A. queenslandica may have evolved.
1.3.4 Aim 3: Is metamorphosis conserved across the metazoan
phyla?
Given the hypothesis that the biphasic life cycle is an ancient
synapomorphic trait of the Metazoa, my
final chapter (Chapter 4) aims to use a cross-phyla comparative
transcriptome approach to substantiate
theoretical claims for the homology of marine life cycles. While
larval traits can inform questions of
larval homology, it is difficult to infer evidence for the
mechanism of life cycle evolution (i.e. larval-
first vs. adult-first hypotheses) from the larva alone.
Therefore, to do this I examined developmental
transcriptomes across metamorphosis (precompetent larval,
competent larval, and feeding juvenile
stages) from six indirectly developing species from different
phyla, including the primary biphasic
species – the cnidarian Acropora sp., the gastropod Haliotis
asinina, the hemichordate Balanoglossus
misakiensis, and the echinoderm Strongylocentrotus purpuratus -
and one secondary biphasic species
– the urochordate Herdmania momus.
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41
chapTer 2 - gleaning biological meaning from TranscripTomes of
non-model organisms: assessmenT of cel-seq developmenTal expression
profiles in The sponge Amphimedon queenslAndicA
2.1 absTracTBioinformatic methods that characterize genome and
transcriptome content, such as gene ontology
(GO) and KEGG, are widely used to infer biological function.
However, there have been few studies
comparing the reliability of these computational tools in
non-model organisms. Here, I use the marine
sponge, Amphimedon queenslandica – a species with an annotated
genome and relatively well-described
development – to compare and test these annotation techniques
against experimentally validated results
from the literature. BLIND clustering and correlation analysis
of 82 A. queenslandica developmental
CEL-Seq transcriptomes divided embryogenesis, larval
development, metamorphosis, juveniles and
adult stages into six distinct transcriptional blocks. Through
the pairwise comparisons of each sequential
block, I identified 13,580 significantly differentially
expressed genes (DEGs). I then analyzed the A.
queenslandica genome and developmental DEGs using three GO
annotation methods, ‘Blast2GO/
InterPro’, ‘Trinotate – BLAST’, and ‘Trinotate – Pfam’, and
found that each yields different, often
non-overlapping sets of annotations. GO enrichment analyses
accentuate annotation bias, resulting
in more uniquely enriched ontologies than shared ones between
these three different annotations.
However, GOSlim enrichments showed a higher degree of
consistency than enrichment analyses using
the entire GO hierarchy. Furthermore, I illustrated that Pfam
and InterPro methods are more effective
at identifying particular candidate genes in the A.
queenslandica genome, such as G-protein coupled
receptors, than BLAST-based methods. However, other candidate
genes, such as putative transcription
factors, are not effectively recovered by any GO annotation
methods. Nonetheless, GO enrichment
of DEG lists still largely correlated with known biological
function. GOSlim appeared to be the most
appropriate means to assess biological function, and when used
in conjunction with KEGG analyses,
can infer broad-scale biological trends across the A.
queenslandica life cycle. In light of highlighted
caveats of GO analyses, I provide a combined workflow that
utilizes BLIND clustering, GOSlim, and
KEGG to provide an accurate picture of gene function across the
ontogeny of a non-model species.
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2.2 inTroducTionOver the last decade, large-scale sequencing
techniques have become cheaper and more accessible,
allowing previously understudied species to be examined for the
first time on a genome- and
transcriptome-wide scale (e.g. Putnam et al. 2007, Srivastava et
al. 2010b, Simakov et al. 2013). When
combined with new computational approaches (Anavy et al. 2014,
Stegle et al. 2015), large RNA-Seq
data sets, including single-cell transcriptomes (Shapiro et al.
2013, Schwartzman and Tanay 2015,
Stegle et al. 2015) such as those generated by CEL-Seq
(Hashimshony et al. 2012), can be assessed in
novel ways with little prior knowledge of the underlying biology
(Levin et al. 2012, 2016). However,
this rapid influx of sequencing data has also created multiple
challenges, including the functional
characterization of thousands of newly sequenced and previously
unidentified genes.
Bioinformatic tools, such as gene ontology (GO) (Blake et al.
2015), Pfam (Finn et al. 2015), and
KEGG (Ogata et al. 1999, Kanehisa et al. 2016a), are often
employed to computationally characterize
and annotate new genomes and transcriptomes, inferring gene
function through sequence similarity to
known orthologs in other species. Given its ease of use and
customizability, GO is the most commonly
employed method of in silico gene annotation. GO has been widely
applied to a variety of studies
focusing on previously uncharacterized species (e.g. Glöckner et
al. 2016, Ricci et al. 2016), including
developmental RNA-Seq studies seeking to understand the role of
genes expressed at a particular life
cycle stage or transition (Reyes-Bermudez et al. 2009a, 2016,
Fiedler et al. 2010, Heyland et al. 2011,
Conaco et al. 2012, Du et al. 2012, Vaughn et al. 2012, Qiu et
al. 2015).
The GO database consists of a hierarchical network of
increasingly specific functional ontologies,
generated from direct experimental assays, mutant phenotypes,
and computationally-derived sequence
or structural similarity (reviewed in Rhee et al. 2008). GO
annotations can be assigned to new sequences
though various methodologies, such as Blast2GO (Conesa et al.
2005, Götz et al. 2008) or Trinotate
(Haas et al. 2013). These in turn can be further subdivided into
methods that rely on whole-gene
sequence similarity (i.e. BLAST-based inferences of gene
orthology), the identification of known
functional protein domains (i.e. Pfam and InterPro), or a
combination of both. However, because the
GO database is largely derived from a handful of
well-characterized model bilaterians, these in silico
methods of gene annotation are potentially unreliable in
non-model species (e.g. Reyes-Bermudez et
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Ch a p t e r 2: Gl e a n i n G b i o l o G i C a l m e a n i n G
f r o m t r a n s C r i p to m e s
al. 2009b, 2016, Fiedler et al. 2010, Heyland et al. 2011,
Conaco et al. 2012b, Du et al. 2012, Vaughn
et al. 2012, Ventura et al. 2013, Qiu et al. 2015). This may be
particularly pronounced in more distantly
related taxa, such as non-bilaterian animals (i.e. sponges,
ctenophores, placozoans and cnidarians; e.g.
Putnam et al. 2007, Srivastava et al. 2008, 2010b, Shinzato et
al. 2011, Conaco et al. 2012b, Fortunato
et al. 2012, Ryan et al. 2013, Moroz et al. 2014, Qiu et al.
2015) and non-metazoan eukaryotes (e.g.
choanoflagellates, plants, and fungi - excluding model systems
such as Arabidopsis and yeast; Galagan
et al. 2003, Dean et al. 2005, King et al. 2008, Martinez et al.
2009, Collén et al. 2013, Suga et al.
2013, Fairclough et al. 2013).
To test the reliability of GO in a non-model species, I examine
gene expression during the ontogeny of
a marine sponge, Amphimedon queenslandica. As an early branching
animal species with an annotated
genome (Srivastava et al. 2010b), published developmental
transcriptomes (Conaco et al. 2012,
Anavy et al. 2014, Fernandez-Valverde et al. 2015; Levin et al.
2016), and well-described life cycle
(reviewed in Degnan et al. 2015), A. queenslandica is a good
non-bilaterian case study for in silico
analysis of biological function across development. Here, I use
BLIND ordering (Anavy et al. 2014)
of an 82-sample replicated CEL-Seq data set (Levin et al. 2016)
to divide the entire A. queenslandica
ontogeny into six distinct transcriptional blocks that delineate
different developmental stages. I test
the validity of GO and KEGG methods by comparing the annotations
of genes differentially expressed
across key life cycle transitions with known cellular and
developmental processes occurring during
these periods. I find that each GO annotation method yields
different functional enrichments. However,
GOSlim can be used to conservatively represent A. queenslandica
development in accordance with
observed, and in some cases, experimentally tested biological
function. Using this information I
propose a streamlined workflow for future bioinformatic
investigations in non-model organisms that
minimizes spurious results.
2.3 resulTs The life cycle of the sponge Amphimedon
queenslandica (Figure 2.1A; reviewed in Degnan et al. 2015)
begins in the maternal brood chamber, where the early cleaving
embryos (cleavage stage) undergo
development into a free-swimming larva. Embryogenesis is
complete when the larval body plan is
fully established, at which time the brood chamber-bound
late-ring embryos are morphologically
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indistinguishable from larva. Upon emergence from the maternal
sponge, the ciliated larva swims in
the water column until it is competent to respond to ecological
and chemical sensory stimuli (Jackson
et al. 2002, Degnan and Degnan 2010). Once competence is
attained, the larva settles and initiates
metamorphosis on the benthos. Metamorphosis from a pelagic larva
to a benthic feeding juvenile
(oscula stage) takes three to four days at 25oC, before the
established juvenile (oscula) grows and
matures over an undetermined period of time into a
sexually-mature adult.
2.3.1 BLIND clustering largely places transcriptomes in expected
temporal order based on morphology
Because the maternal brood chamber contains mixed embryos from
all stages of embryogenesis,
identification of particular developmental stages is limited to
manual morphological characterization.
However, a computationally-based method called BLIND ordering
(Anavy et al. 2014) has been
developed to computationally sort individual RNA-Seq data sets
into the correct temporal order. This
method has been used to compare and contrast developmental
transcriptomes between species (Levin
et al. 2016), including a comprehensive timecourse of A.
queenslandica embryogenesis consisting of
59 individual transcriptomes from early cleavage to late-larvae
(Anavy et al. 2014, Levin et al. 2016).
However, these previous studies lack representa