Thesis submitted in fulfilment of the requirements for
Degree of Doctor of Philosophy
Antarctic biodiversity surveys using high throughput sequencing: understanding landscape and communities of the Prince
Charles Mountains
Paul Czechowski December 2015
School of Biological Sciences
Difficulties are just things to overcome, after all
- Ernest ShackletonContents
Abstract v
Publications, Presentations, Awards vii
Thesis Declaration ix
Acknowledgments x
Chapter Format xi
1. Antarctic terrestrial biodiversity and environmental metagenetics 3
1.1. Introduction . . . 4
1.2. Technical considerations . . . 6
1.2.1. Extraction of environmental samples . . . 6
1.2.2. High throughput platforms . . . 7
1.2.3. Marker choice . . . 8
1.2.4. Library generation . . . 8
1.2.5. Amplification . . . 9
1.2.6. Sequence analysis . . . 10
1.2.7. Recent improvements of metagenetic HTS approaches . . . 11
1.3. The potential of metagenetics for Antarctic biology . . . 15
1.3.1. Community structures . . . 15
1.3.2. Geographic distribution of Antarctic biota . . . 16
1.3.3. Supporting conservation efforts . . . 17
1.4. Summary and conclusions . . . 17
1.5. Acknowledgements . . . 18
1.6. Authors contributions . . . 18
2. Eukaryotic soil diversity of the Prince Charles Mountains 21 2.1. Introduction . . . 22
Contents
2.2. Methods and materials . . . 25
2.2.1. Fieldwork, soil storage and DNA extraction . . . 25
2.2.2. Amplification and library generation . . . 26
2.2.3. Read processing . . . 26
2.2.4. Eukaryotica and b diversity comparison . . . 27
2.2.5. Distribution of phylotypes across sites . . . 28
2.2.6. Species-level assignment of phylotypes . . . 30
2.3. Results . . . 31
2.3.1. Read processing . . . 31
2.3.2. Eukaryotica and b diversity comparison . . . 31
2.3.3. Distribution of phylotypes across sites . . . 33
2.3.4. Species-level assignment of phylotypes . . . 34
2.4. Discussion . . . 35
2.4.1. Technical considerations . . . 35
2.4.2. Differences in eukaryotic diversity among three locations . . . 36
2.4.3. Distribution of highly abundant phylotypes . . . 37
2.4.4. Validity of species-level taxonomic assignments . . . 38
2.5. Summary and conclusions . . . 39
2.6. Acknowledgements . . . 39
2.7. Supplemental data . . . 40
2.8. Supplemental information . . . 41
2.8.1. Methods and Materials . . . 41
2.8.2. Results and comments . . . 50
3. Phylotypes and morphotypes of Antarctic invertebrates 59 3.1. Introduction . . . 60
3.2. Methods . . . 63
3.2.1. Samples . . . 63
3.2.2. DNA extractions . . . 64
3.2.3. Primers . . . 65
3.2.4. Amplification and sequencing . . . 66
3.2.5. Reference data for taxonomic assignments . . . 66
3.2.6. Generation of phylotype observations . . . 67
3.2.7. Selection of processing parameters for 18S and COI phylotypes 68 3.2.8. Concordance between phylotypes and morphotypes . . . 69
Contents
3.3. Results . . . 71
3.3.1. Selection of analysis parameters . . . 71
3.3.2. Concordance between morphotypes and phylotypes . . . 71
3.4. Discussion . . . 72
3.4.1. Analysis parameters . . . 74
3.4.2. Detecting cryptic invertebrates . . . 75
3.4.3. Metagenetic marker choice for Antarctic invertebrates . . . 76
3.5. Conclusions . . . 77
3.6. Acknowledgements . . . 78
3.7. Supporting information . . . 78
3.8. Supplemental information . . . 79
3.8.1. Methods and Materials . . . 79
3.8.2. Tables and Figures . . . 83
3.8.3. Analysis code . . . 85
4. Salinity gradients determine invertebrate distribution 90 4.1. Introduction . . . 91
4.2. Methods . . . 94
4.2.1. Fieldwork . . . 94
4.2.2. Soil geochemical and mineral analysis . . . 94
4.2.3. Preparation and analysis of environmental observations . . . . 97
4.2.4. Preparation and analysis of biological observations . . . 98
4.2.5. Constrained ordination . . . 99
4.3. Results . . . 99
4.3.1. Environmental data . . . 99
4.3.2. Biological data . . . 100
4.3.3. Biological data in relation to environment . . . 100
4.4. Discussion . . . 101
4.5. Conclusion . . . 105
4.6. Data accessibility . . . 106
4.7. Authors contributions . . . 107
4.8. Acknowledgements . . . 107
4.9. Funding . . . 108
4.10. Supplemental information . . . 109
4.10.1. Phylotype data generation for 18S and COI . . . 109
Contents
4.10.2. Sequence tag selection and amplicon orientations . . . 110
4.10.3. Intermediate results of environmental data processing . . . 111
4.10.4. Intermediate results of biological data processing . . . 111
4.10.5. Intermediate results of biological data in relation to environment111 4.10.6. Data and analysis scripts, additional figures and tables . . . . 112
5. Synthesis 130 5.1. Summary . . . 130
5.1.1. Technical and computational methods . . . 130
5.1.2. Biodiversity information from the Prince Charles Mountains . 130 5.2. High throughput sequencing for Antarctica . . . 132
5.3. Implications and future improvements . . . 135
5.4. Conclusion . . . 137
5.5. Acknowledgements . . . 138
A. Phylotype information chapter 2 139
B. Analysis code chapter 3 162
C. Analysis code chapter 4 190
D. Molecular tagging of amplicons 236
Bibliography 244
Abstract
Antarctic soils are home to small, inconspicuous organisms including bacteria, uni- cellular eukaryotes, fungi, lichen, cryptogamic plants and invertebrates. Antarctic soil communities are distinct from other soil biota as a consequence of long-term persistence under harsh environmental conditions; furthermore their long history of isolation is responsible for a high degree of endemism. Of major concern is the establishment of non-indigenous species facilitated by human-mediated climate change and increased human activity, threatening the highly specialised endemic species. A lack of baseline information on terrestrial Antarctic biodiversity currently impairs efforts to conserve the unique but still largely unknown Antarctic biota.
In this thesis I apply metagenetic high throughput sequencing (MHTS) methods to address the deficiency of biological information from remote regions of continental Antarctica, and use the data generated to explore environmental constraints on Antarctic biodiversity.
In Chapter 1, I introduce current issues impeding the generation of baseline Antarctic biodiversity data and evaluate the application of using MHTS techniques. This review highlights the potential of using MHTS approaches using amplicon sequencing to retrieve Eukaryotic biodiversity information from terrestrial Antarctica. In Chapter 2, the eukaryotic diversity of three biologically unsurveyed regions in thePrince Charles Mountains, East Antarctica (PCMs) is explored. Total eukaryote biodiversity in the PCMs appears to follow an altitudinal or latitudinal trend, which is less obvious for terrestrial invertebrates. In order to apply MHTS to the study of Antarctic invertebrates, the comparative taxonomic assignment fidelities of metagenetic markers and morphological approaches are explored in Chapter 3. Fidelities of taxonomic assignments to four Antarctic invertebrate phyla differed depending on metagenetic marker, and only application of non-arbitrary sequence processing parameters resulted in these findings. In Chapter 4, I use MHTS-derived biodiversity information to explore the relationship between soil properties and invertebrate biodiversity in the PCMs. Across large spatial scales distribution of phyla Tardigrada and Arachnida and
classes Enoplea (Nematoda) and Bdelloidea (Rotifera) in inland areas are constrained by terrain-age-related accumulation of salts, while other Classes (Chromadorea, Nematoda and Monogonata, Bdelloidea) are better able to tolerate high salinity. In moister, nutrient-richer and more coastal areas, this effect was less pronounced and a higher invertebrate diversity was found.
The methods applied and developed in this thesis are a valuable starting point to advance the collection of biodiversity information across terrestrial Antarctica and other remote habitats. The work presented here provides examples for generation and usage of MHTS information from remote Antarctic habitats, demonstrates how biodiversity information retrieved using different metagenetic markers can be combined, developed methods for assessing the quality of MHTS markers and finally demonstrated the application of MHTS data to investigate the environmental determinants of invertebrate diversity in remote ice-free habitats. Future MHTS biodiversity studies of Antarctic terrestrial habitats should incorporate large sample numbers and use combined data from multiple genetic markers.
Publications, presentations and awards
Publications
2013 Laurence J. Clarke,Paul Czechowski, Julien Soubrier, Mark I. Stevens, Alan Cooper (2014). Modular tagging of amplicons using a single PCR for high-throughput sequencing. Molecular Ecology Resources. 14, 117–121.1
Conference presentations
2014 2014 SCAR Open Science Conference (SCAR, SkyCity, Auckland, New Zealand): Paul Czechowski, Duanne White, Laurence J. Clarke, Alan Cooper, Mark I. Stevens (April): High-throughput DNA sequencing reveals Antarctic soil biodiversity.
2014 Understanding Biodiversity Dynamics using diverse Data Sources (CBA, Australian National University, Canberra, Australia): Paul Czechowski, Duanne White, Laurence J. Clarke, Alan Cooper, Mark I. Stevens (April):
High-throughput DNA sequencing reveals Antarctic soil biodiversity.
2013 Biodiversity Genomics Conference (CBA, Australian National University, Canberra, Australia): Paul Czechowski, Laurence J. Clarke, Alan Cooper, Mark I. Stevens (April): Exploring Distribution and Evolution of Antarctic Invertebrates using High-Throughput Sequencing.
1See Appendix D
Awards
2014 Australian Government’s National Taxonomy Research Grant Program - Student Travel Grant, Australian Biological Resources Study, Government of Australia, $ 1 650- travel support for conference attendance.
2013 MiSeq Pilot the Possibilities Grant Program, Illumina Australia, One library reagents kit for experimental libraries.
2012 Small Research Grants Scheme, The Royal Society of South Australia,
$ 1 500,- for laboratory work.
2012 International Post-Graduate Research Scholarship, The University of Adelaide, $ 25 849,- p.a. livelihood.
Thesis declaration
I certify that this work contains no material which has been accepted for the award of any other degree or diploma in my name in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. In addition, I certify that no part of this work will, in the future, be used in a submission in my name for any other degree or diploma in any university or other tertiary institution without the prior approval of the University of Adelaide and where applicable, any partner institution responsible for the joint award of this degree.
I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968.
The author acknowledges that copyright of published works contained within this thesis resides with the copyright holder(s) of those works.
I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library Search and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.
Acknowledgments
I thank my supervisor Dr. Mark Stevens of the South Australian Museum for his long dedication towards this project and his efforts in supporting me in this work and in the years before. Mark has always been a kind mentor and supporter, both professionally and personally. I thank Chester Sands of the British Antarctic Survey, and Alejandro Velasco-Castrillon of the University of Adelaide. I would like to thank those working with me at Mount Menzies, in the Mawson Escarpment and Lake Terrasovoje, Fiona Shanhun, Tessa Williams, Josh Scarrow, Adrian Corvino and Nick Morgan. I appreciate comments and advice provided by several other persons, as further listed in individual chapters.
For this project the Australian Antarctic Division provided funding under science project 2355 to Mark Stevens. The Australian Research Council supported this work through funds from linkage grant LP0991985 to Alan Cooper and Mark Stevens.
The University of Adelaide supported this project through the International Post- Graduate Research Scholarship to Paul Czechowski.
Chapter format
This thesis is formatted according to guidelines provided by the University of Ad- elaide2. Reference formatting is uniform throughout the thesis. Sectioning of individual chapters follows the guidelines of currently targeted journals:
Chapter 1 Antarctic Science
Chapter 2 Soil Biology & Biochemistry Chapter 3 PLoS ONE
Chapter 4 Royal Society Open Science
As required by the University of Adelaide, Statements of Authorship are preceding individual chapters. Analysis source code is provided in the appendix as indicated within individual chapters, further supplemental material will be available online upon publishing of individual chapters.
2As provided by higher degree research rules for 2015