Modelling Microbial Diversity in Antarctic Soils Victoria. J. Ord A thesis submitted in accordance with the requirements of Newcastle University for the degree of Doctor of Philosophy School of Biology Newcastle University Newcastle Upon Tyne, United Kingdom June 2014 1 Memorandum Except where acknowledgement is given this thesis is the unaided work of the author. Material presented has never been submitted to Newcastle University or to any other educational establishment for the purposes of obtaining a higher degree. 2 Abstract Microorganisms play a crucial role in supporting biodiversity, maintaining marine and terrestrial ecosystems at the crux of the nutrient cycle. They are the most diverse and abundant of all living creatures, yet little is understood about their distribution or their intimate relationship with the environment. Antarctic ecosystems are among the most simple on Earth; with basic trophic structuring and the absence of many taxonomic groups, they are also isolated geographically with small patchy areas of nutrient inputs. In this instance, Antarctica becomes a pristine laboratory to examine the ecological paradigms already applied to macro-organisms, to determine if common biological laws govern the distribution of biology globally. The decline of biodiversity with increasing latitude is one such observation in the distribution of macro-organisms. In this study, soil microbial community samples were retrieved over a latitude of 56 to 72 °S across the Antarctic Peninsula region. This is a region of special interest due to a rapidly warming climate with mean temperatures increasing at several times the rate of mean global warming. Sites were biologically and environmentally profiled and data used in a variety of multivariate analysis in order to identify spatial trends and infer mechanisms that may be driving Antarctic terrestrial food webs; or where this was not possible, the areas where focus was needed to increase the information profile to allow this. Results indicate a lack of linear latitudinal gradient in microbial diversity, but do show a correlation with environmental heterogeneity; analysis of site diversity identified a gradient between warmer wetter areas, and areas synonymous with cold desert environment at 66⁰S, supported by both phylum composition and indicative soil chemistry. This was confirmed through principal co-ordinates of neighbours’ matrices analysis (PCNM), with distinct regions of community composition being identified when viewed with respect to environmental variables. Considering an overview of diversity with respect to environmental variables provided additional structure to test hypotheses about nutrient webs through structural equation modelling (SEM), and inferred that areas of patchy nutrient input exist and by means of ornithogenic guano additions promote higher C and N availability, increasing microbial abundance and richness. 3 Acknowledgements I would like to acknowledge NERC for funding provided to the Project AFI7/05 and to British Antarctic Survey for making it all logistically possible. Many people at BAS provided support to the PI (Professor David Hopkins) and RA (Dr Paul Dennis) in accessing difficult locations, collecting and transporting samples and also in the data collection itself, In particular Mr John Loines and Mr Matt Von Tersch; the old and new Bonner laboratory managers, who helped in the packaging and organisation of the samples. Numerous field general assistants, who aided in collection and made life easier in the field and other people who provided a helping hand here or there. Also, thanks to the Scottish Crop Research Institute for supporting Laboratory analyses where most of the community and chemical analyses were conducted by Dr Paul Dennis, supervised by Professor David Hopkins. Special thanks to Dr Benhua Sun and particularly, Dr Armando Laudicina and for their help with these analyses. I would like to thank Professor Steve Rushton and Professor David Hopkins for their guidance and support. Professor Tony O’Donnell for early guidance, Dr Sasha Jenkins and Mr Ian Waite for always having time to answer questions and to all the Life Sciences Modelling Group at Newcastle University for all their help and advice over the years. Thanks to my parents for their endless support and love. Last, but most certainly not least, thanks to Michael Chester, who turned out to be so much more than a guide on the glacier. 4 Common Terms and Abbreviations AIC Akaike Information Criterion BIC Bayesian Information Criterion CA Correspondence Analysis CCA Canonical Correspondence Analysis CFI Comparative Fit Index DOC Dissolved Organic Carbon EC Electrical Conductivity ELFA Ester Linked Fatty Acid LGM Last Glacial Maximum MDR Mean Daily Range MEM Moran’s Eigenvector Map MLE Maximum Likelihood Estimation MSPA Multi-Scale Pattern Analysis + NH4 Ammonium - - NO3 /NO2 Nitrate/Nitrite OTU Operational Taxonomic Unit PCR Polymerase Chain Reaction T-RFLP Terminal Restriction Fragment Length Polymorphism PCA Principle Components Analysis RDA Redundancy Analysis RDP-II Ribosomal Database Project PCNM Principle Components of Neighbours Matrices RMSEA Root Mean Square Error of Approximation SEM Structural Equation Modelling VIF Variable Inflation Factor Analysis 5 Contents Modelling Microbial Diversity in Antarctic Soils .....................................................................1 Memorandum.............................................................................................................................2 Abstract......................................................................................................................................3 Acknowledgements....................................................................................................................4 Common Terms and Abbreviations...........................................................................................5 Contents .....................................................................................................................................6 Equation boxes, Figures and Tables ..........................................................................................9 Thesis Introduction ..............................................................................................................12 1.1. The Antarctic Environment.......................................................................................12 1.1.1. Continental History............................................................................................17 1.1.2. Modern climate and environment ......................................................................18 1.1.3. Impacts of climate change ................................................................................22 1.1.4. Humans in Antarctica .......................................................................................24 1.1.5 Antarctic soils ....................................................................................................25 1.1.6 Carbon cycle ......................................................................................................27 1.1.7 Nitrogen cycle....................................................................................................27 1.2. Antarctic Biology ......................................................................................................29 1.2.1. Flora and fauna ..................................................................................................30 1.2.2. Bacteria ..............................................................................................................31 1.2.3. Fungi ..................................................................................................................33 1.3. Biodiversity and Biogeography.................................................................................34 1.3.1. Microbial diversity: considerations and analysis...............................................35 1.3.2. Trends in Antarctic biodiversity ........................................................................38 1.3.3. Functional diversity ...........................................................................................41 1.4 References .................................................................................................................43 Project Data and Thesis Methods ........................................................................................64 2.1. Project Data...............................................................................................................64 2.1.1. Soil collection Sites............................................................................................65 2.1.2. Physical Observation Data.................................................................................68 2.1.3. Major Soil Characteristics Data.........................................................................69 2.1.4. Microbial Community Data; Fatty Acid Analysis .............................................71 2.1.5. Microbial Species Taxonomic Diversity Data; Pyrosequencing Analysis ............74 2.2. Thesis Methodologies ...............................................................................................76 2.2.1. Ordination ..........................................................................................................78 2.2.2. Spatial eigenvector analyses ..............................................................................80