Molecular profiling of bark beetle mycobiota

214 Pages·2016·24.85 MB·English

Molecular
profiling of bark
beetle mycobiota

Kirsten Eleanor Miller

A thesis submitted for the degree of Doctor of Philosophy

Department of Life Sciences
Imperial College London

September 2015
Abstract

No organism evolves in isolation; the symbiotic microorganisms associated with
insects are key determinates of their hosts ecology and evolution. This thesis
investigates the fungal communities found with bark and ambrosia beetles
(Curculionidae: Scolytinae & Platypodinae) in an attempt to better understand the
diversity and variability of this ecologically and economically important system. I
start by assessing the efficacy of high throughput sequencing (HTS) and
metabarcoding for the characterisation of fungal communities derived from whole
arthropod DNA extracts (Chapter 2). I then apply this approach to understanding
the biotic and abiotic determinants of beetle associated fungal taxa, and find that
both beetle species identity and environmental conditions significantly predict
community richness and composition (Chapter 3). In Chapter 4, I expand upon the
beetle species level differences in microbial communities to show that they are
driven by a relatively small subset of fungal taxa, predominantly belonging to the
beetle associated Microascales, but also to the relatively unexplored
Saccharomycetes. I further apply HTS to the recreation of beetle evolutionary
histories using mitochondrial genomics, and test for correlations between beetle
phylogenetic and mycobiotic similarities (Chapter 5). My investigations demonstrate
the utility of recently developed sequencing technologies to the study of microbial
symbioses and I conclude with a synthetic summary of my findings with suggestions
for further research.

2
Contents

List of Figures .......................................................................................................................... 5
List of Tables ............................................................................................................................ 7
Copyright Declaration ............................................................................................................ 7
Declaration of Originality ...................................................................................................... 8
Acknowledgements ................................................................................................................ 9

Chapter 1 : General Introduction ...................................................................................... 10

Chapter 2 : Metabarcoding of bark and ambrosia beetle associated fungal
communities
2.1 Abstract ............................................................................................................................. 28
2.2 Introduction ..................................................................................................................... 29
2.3 Methods ............................................................................................................................ 31
2.4 Results ............................................................................................................................... 40
2.5 Discussion ......................................................................................................................... 48

Chapter 3: Identifying the biotic and abiotic drivers of bark and ambrosia beetle
mycobiota
3.1 Abstract ............................................................................................................................. 54
3.2 Introduction ..................................................................................................................... 55
3.3 Methods ............................................................................................................................ 59
3.4 Results ............................................................................................................................... 67
3.5 Discussion ......................................................................................................................... 79

Chapter 4: Host specificity of bark and ambrosia beetle mycobiota
4.1 Abstract ............................................................................................................................. 86
4.2 Introduction ..................................................................................................................... 87
4.3 Methods ............................................................................................................................ 89
4.4 Results ............................................................................................................................... 93
4.5 Discussion ....................................................................................................................... 102

3
Chapter 5: Does phylogenetic distance between bark and ambrosia beetle species
predict mycobiotic similarity?
5.1 Abstract ........................................................................................................................... 110
5.2 Introduction ................................................................................................................... 111
5.3 Methods .......................................................................................................................... 114
5.4 Results ............................................................................................................................. 121
5.5 Discussion ....................................................................................................................... 130

Chapter 6: General Discussion
Thesis overview ................................................................................................................... 138

Bibliography ......................................................................................................................... 148

Appendices
Appendix I: Chapter 2 Supplementary Material ......................................................... 180
Appendix II: Chapter 3 Supplementary Material ........................................................ 186
Appendix III: Chapter 4 Supplementary Material ...................................................... 200
Appendix IIII: Chapter 5 Supplementary Material ..................................................... 206
Appendix V: Testing the effect of rarefaction and normalization methods on
Chapter 2 results…………………………………………………….....210

4
List of Figures
1.1 Scanning electron microscopy image of platypodine species……...……...14
1.2 Diagram of internal transcribed spacer region……………………………...25
2.1 Schematic flowchart of methodological stages.……..……………………...38
2.2 Percentage of ITS2 reads removed during bioinformatic processing steps 41
2.3 Taxonomic classification of pre-ITSx merged Illumina sequences ............. 43
2.4 Number of ITS2 sequences associated each fungal OTU ............................. 44
2.5. NMDS plots displaying fungal community dissimilarity between beetle
specimens ....................................................................................................... 45
2.6. Fungal OTU accumulation curves for replicate communities ................... 46
2.7. Overall OTU accumulation curves for each beetle specimen……………..47
2.6. Log fungal OTU richness differences for each beetle specimen ................ 48
3.1. Nestedness and turnover components of community dissimilarity ......... 57
3.2. Plantation pine forest collecting site locations throughout the UK .......... 59
3.3. Diagram of a Lindgren multiple funnel trap ................................................ 61
3.4. Proportion of Scolytinae species found at UK sampling sites ................... 71
3.5 Taxonomic composition of fungal OTU from 11 bark beetle species ......... 73
3.6. Partitioning of explained variance in fungal communities ........................ 76
3.7. NMDS plot displaying fungal community dissimilarity between beetle
species ............................................................................................................ 77
3.8. Pairwise nestedness and turnover resultant dissimilarities for intra and
interspecific comparisons ............................................................................ 78
4.1. Fungal OTU accumulation curves for each beetle species ......................... 93
4.2. Taxonomic composition of each bark beetle species mycobiota ............... 95
4.3 Fungal composition of beetle body parts ........................................................ 97
4.4 Distribution of specific OTUs across beetle species ..................................... 98
5
4.5 Proportion of specific and generalist OTUs for ten fungal classes .............. 99
4.6 The distribution of Ophiostomataceae OTUs across beetle species .......... 100
4.7. ML Ceratocystis phylogeny tree built from ITS2 .......................................... 101
5.1. Schematic flowchart showing principle methodological stages……….. 114
5.2. Barro Colorado Island and forest dynamics plot locations ....................... 115
5.3. GMYC analysis plots for Panamanian beetle specimens ........................... 124
5.4. Abundance of GMYC beetle species groupings .......................................... 121
5.5 ML bark beetle phylogeny based upon mitochondrial genes .................... 125
5.6. Dendrogram of beetle species based upon pairwise fungal
community dissimilarity .......................................................................... 128
5.7. Comparison of dendrograms based upon beetle phylogenetic similarity
and fungal community similarity ........................................................... 129
5.8. Relationship between beetle genetic divergence and fungal community
dissimilarity for beetle species .................................................................. 130

6
List of Tables

2.1. Beetle specimen species information ........................................................................... 32

2.2. Tag identity, specimen and library for each community replicate ......................... 32

2.3. Specimen sequence numbers and OTU richness ....................................................... 41

3.1. UK bark beetles associated with pine forests ............................................................. 58

3.2. Geographic coordinates and details for each UK collection site ............................. 60

3.3. List of models generated for each variance partitioning analysis ........................... 65

3.4. Collected Scolytine species with total number of individuals ................................. 69

3.5. Diversity metrics for beetles collected at each site .................................................... 70

3.6. Beetle specimens selected for mycobiome analysis ................................................... 70

3.7 PERMANOVA results for fungal community composition ..................................... 76

4.1 Diversity metrics for mycobiota of bark beetle species ............................................. 96

5.1. Summary of PCGs extracted from Scolytinae mitogenome contigs ..................... 126

Copyright Declaration

‘The copyright of this thesis rests with the author and is made available under a
Creative Commons Attribution Non-Commercial No Derivatives licence.
Researchers are free to copy, distribute or transmit the thesis on the condition that
they attribute it, that they do not use it for commercial purposes and that they do not
alter, transform or build upon it. For any reuse or redistribution, researchers must
make clear to others the licence terms of this work’

7
Declaration of Originality

I confirm that this thesis is my own work and that any assistance received is
acknowledged here and where appropriate throughout the thesis.

At the time of submission Chapter 2 has been submitted to Molecular Ecology
Resources. Co-authors are Daegan Inward, Kevin Hopkins and Alfried Vogler
reflecting their contribution to specimen collection, Illumina sequencing and
development of the final manuscript from my initial draft respectively.

UK fieldwork planning, data collection and morphological identification of
specimens was conducted in collaboration with Daegan Inward at Forest Research.
Panamanian fieldwork planning and execution was carried out by myself with the
help of a local field assistant. All molecular processing other than Illumina library
preparation and sequencing was carried out by myself. In Chapter 3 appropriate
analyses were selected in consultation with Andres Baselga and Carola Gómez-
Rodríguez. In Chapter 5 Alex Crampton-Platt aided with the assembly of beetle
mitochondrial genomes.

I acknowledge and thank Tom Bell and Clare Gray at Imperial College; Anne
Jungblut and Benjamin Linard at the NHM; Carola Gómez-Rodríguez and Andres
Baselga at the Universidad de Santiago de Compostela; James Probert at the
University of Liverpool and Stephen Miller at the University of Strathclyde for their
comments and suggestions on Chapters 3-5.

Kirsten Miller
September 2015

8
Acknowledgements

My gratitude goes to all past and present inhabitants of ‘The Beetle Lab’ but
particularly Alex Crampton-Platt, Benjamin Linard, Belen Arias, Carmelo Andújar,
Paula Arribas Blázquez, CT, Amrita Srivathsan and Martijn Timmermans, I really
appreciate all the advice and constructive criticism over the last few years. Alex I
know you don’t like overt affection but your friendship means a lot and you’ve
helped me more than you know. Benjamin, your mindless optimism has been a
constant source of amusement, and okay, I’ll admit it, comfort. Carola Gómez-
Rodríguez and Andres Baselga, you have both been incredibly supportive and so
generous with your time and advice – I’m so excited about working with you in the
future. Sandy Knapp, I’ve valued our conversations and your literary suggestions,
scientific and otherwise. And of course my enormous gratitude goes to Alfried
Vogler for giving me this opportunity in the first place.

This thesis wouldn’t have been possible without the help and support of many at the
NHM – Steve, Alex and Andie in the lab, Kevin, Lisa, Jackie et al. in the sequencing
facility, Peter Foster for bioinformatics support, Anne, Tiina and of course Eileen and
Anna in the postgrad office – thank you all.

Many, many thanks go to Daegan Inward at Forest Research, I don’t know what I
would have done without our collaboration and I learnt an awful lot during our
fieldwork. I hope my driving wasn’t too bad and that my Mum’s curry wasn’t too
hot. Thank also go to the Forest Research field operatives who helped to collect the
data.

Mum, Dad and Bean, thanks for indulging my passion for beetles and Mum in
particular - you have been so incredibly encouraging and supportive as you are with
everything. James, you’ve had to bear the brunt of my thinking-out-loud rambling
outbursts - thank you and I’m sure I’ll get an opportunity to reciprocate soon. Aly,
Aaron, Sean, Mike, Thea, Chris, Rajan and Pedro thanks to you all for being there.
Clare, you’ve really helped me out this year and its meant a lot.

Finally I’d like to thank NERC for the PhD scholarship that funded this research.
9
Chapter 1

General Introduction

10