Antarctic biodiversity surveys using high throughput sequencing

277 Pages·2016·11.35 MB·English

by  Paul Czechowski

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
Di�culties are just things to overcome, after all - Ernest Shackleton
Contents
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 e�orts . . . . . . . . . . . . . . . . . 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
i
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. Eukaryotic a 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. Eukaryotic a 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. Di�erences 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
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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
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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
iv
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 e�orts 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
biodiversitydataandevaluatetheapplicationofusingMHTStechniques. Thisreview
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 the Prince 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, thecomparativetaxonomicassignmentfidelitiesofmetageneticmarkers
and morphological approaches are explored in Chapter 3. Fidelities of taxonomic
assignments to four Antarctic invertebrate phyla di�ered depending on metagenetic
marker,andonlyapplicationofnon-arbitrarysequenceprocessingparametersresulted
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. AcrosslargespatialscalesdistributionofphylaTardigradaandArachnidaand
v
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 e�ect 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 di�erent 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.
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Publications, presentations and
awards
Publications
2013 Laurence J. Clarke, Paul Czechowski, Julien Soubrier, Mark I. Stevens,
AlanCooper(2014). ModulartaggingofampliconsusingasinglePCRfor
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,
AustralianNationalUniversity,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
vii
Awards
2014 Australian Government’s National Taxonomy Research Grant Program -
StudentTravelGrant,AustralianBiologicalResourcesStudy,Government
of Australia, $ 1650- 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,
$ 1500,- for laboratory work.
2012 International Post-Graduate Research Scholarship, The University of
Adelaide, $ 25849,- p.a. livelihood.
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