MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Wei Li Candidate for the Degree: Doctor of Philosophy Dr. Rachael Morgan-Kiss, Director Dr. Annette Bollmann, Reader Dr. Thomas Crist, Reader Dr. Michael Vanni, Reader Dr. Richard Edelmann, Graduate School Representative Dr. Rebecca Gast, Distinguished Off Campus Scholar ABSTRACT INFLUENCE OF ENVIRONMENTAL DRIVERS AND INTERACTIONS ON THE MICROBIAL COMMUNITY STRUCTURE IN PERMANENTLY STRATIFIED MEROMICTIC ANTARCTIC LAKES by Wei Li The microbial loop plays important roles in the cycling of energy, carbon and elements in aquatic ecosystems. Viruses, bacteria, Archaea and microbial eukaryotes are key players in global carbon cycle and biogeochemical cycles. Investigating microbial diversity and community structure is crucial first step for understanding the ecological functioning in aquatic environment. Meromictic lakes are bodies of water and exhibit permanent stratification of major physical and chemical environmental factors. Microbial consortia residing in permanently stratified lakes exhibit relatively constant spatial stratification throughout the water column and are adapted to vastly different habitats within the same water. Pristine perennially-ice-covered lakes (Lake Bonney, Lake Fryxell and Lake Vanda) are meromictic lakes located in the McMurdo Dry Valleys (MDV) of Southern Victoria Land, Antarctica. The lakes have isolated water bodies and extremely stable strata that vary physically, chemically, and biologically within and between the water columns. The unique characteristics support microbially dominated food webs in these lakes. In the research presented here, we gathered new understanding of how environmental drivers influence microbial community structure in these aquatic ecosystems. We explored the lake microbial ecology from three major approaches: 1). Assess trophic activities in the natural environment and identify potential environmental drivers impacting heterotrophic (β Glucosaminidase) and autotrophic (Ribulose 1,5 bisphosphate carboxylase) enzyme activities; 2). Resolve the protist community composition (i.e. autotrophic, heterotrophic and mixotrophic groups) based on high throughput sequencing and bioinformatics. Identify how the community structures correlate with specific environmental and biological factors; 3). Reveal the diversity of potential microbial interactions between the microorganisms in the MDV lakes at individual cell level, and investigate how the interactions vary between organisms with different nutritional strategies. Studies of polar microbial communities on the cusp of environmental change will be important for predicting how microbial communities in low latitude aquatic systems will respond. This study expands the understanding of how environmental drivers interact with microbial communities in the Antarctica lakes, and provide new information to predict how the community structure will alter as response to climate changes. INFLUENCE OF ENVIRONMENTAL DRIVERS AND INTERACTIONS ON THE MICROBIAL COMMUNITY STRUCTURES IN PERMANENTLY STRATIFIED MEROMICTIC ANTARCTIC LAKES A DISSERTATION Submitted to the faculty of Miami University In partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology Ecology, Evolution and Environmental Biology Program by Wei Li Miami University Oxford, OH 2016 Advisor: Dr. Rachael Morgan-Kiss TABLE OF CONTENTS PAGE List of Tables v List of Figures vi Acknowledgments vii Chapter I: Introduction 1.1. introduction 2 Chapter II: Spatial heterogeneity and the impact of biotic and abiotic drivers on microbial autotrophic and heterotrophic activities in three chemically stratified Antarctic lakes 2.1. Introduction 16 2.2. Materials and methods 20 2.2.1. Field sampling and limnological parameters 20 2.2.2. Rubisco carboxylase activity assay 21 2.2.3. Glucosaminidase activity assay 21 2.2.4. Protein concentration determination 22 2.2.5. Bacterial enumeration 22 2.2.6. Statistical analyses 23 2.3. Results 24 2.3.1. Lake Bonney 24 2.3.2. Lake Fryxell 25 2.3.3. Lake Vanda 25 2.3.4. Cluster analyses of lake physicochemical parameters 26 2.3.5. Correlation of autotrophic and heterotrophic activities with physicochemical parameters 26 2.4. Discussion 28 2.5. Acknowledgements. 32 2.6. References 41 Chapter III: Influence of environmental drivers and potential interactions on the distribution of microbial communities from three permanently stratified Antarctic lakes 3.1. Introduction 45 3.2. Materials and methods 48 3.2.1. Site description and sample collection 48 3.2.2. Environmental parameter measurements 48 3.2.3. DNA library preparation and Illumina MiSeq Sequencing 49 3.2.4. Sequence analysis 49 3.2.5. Diversity assessment 50 3.2.6. Network analysis 50 3.3. Results 52 3.3.1. Environmental parameters 52 3.3.2. Ilumina MiSeq sequencing summary 52 iii 3.3.3. Bacterial community composition 53 3.3.4. Eukaryotic community composition 54 3.3.5. Co-occurrence microbial network and associated environmental factors 55 3.4. Discussion 57 3.5. Acknowledgements. 61 3.6. References 76 Chapter IV: Ultrastructural and single-cell level characterization reveals metabolic versatility in a microbial eukaryote community from an ice-covered Antarctic lake 4.1. Introduction 87 4.2. Materials and methods 90 4.2.1. Site description, sample collection and enrichment cultures 90 4.2.2. Single cell sorting 91 4.2.3. DNA template preparation for PCR 91 4.2.4. Sanger sequencing 92 4.2.5. Illumina sequencing 92 4.2.6. Analysis of the sequences 93 4.2.7. Confocal Laser Scanning Microscopy 93 4.2.8. Scanning Electron Microscopy 94 4.3. Results and discussion 95 4.3.1. Identities and trophic modes of sorted eukaryotes 95 4.3.2. Isolation and description of two key photosynthetic protists 98 4.3.3. Community composition of organisms co-sorted with Lake Bonney eukaryotes 99 4.3.4. Potential interactions between Dry Valley protists and bacteria 100 4.4. Conclusions 105 4.5. Acknowledgements 105 4.6. References 112 Chapter V: Conclusions 5.1 Conclusions 127 5.2 References 133 iv LIST OF TABLES PAGE 1. Pearson correlation coefficient values (R) for average RubisCO and βGAM activity with lake physical, chemical, and biological parameters for all samples 39 2. Linear regression models explaining the enzyme activities in different portions of water columns 40 3. Summary of major physicochemical parameters in the studied lakes 68 4. Pairwise comparison of species richness between lakes using Tukey’s HSD test 69 5. ANOSIM of communities from different lakes and layers 70 6. List of nodes in co-occurrence network 72 7. Basic growth physiology of Chlamydomonas sp. ICE-MDV and Isochrysis sp. MDV isolates 121 8. Diversity of major algal classes in Lake Bonney enrichment cultures 122 v LIST OF FIGURES PAGE 1. Schematic of Microbial loop and energy flows in aquatic environment 7 2. Map showing locations of study sites within the McMurdo Dry Valleys, Antarctica 8 3. Depth profiles of physical and chemical characteristics for the MDV lakes in this study 9 4. Map showing locations of study sites within the McMurdo Dry Valleys, Antarctica 33 5. Salinity profiles for Lake Bonney (east lobe, ELB; west lobe, WLB), Lake Fryxell (FRX) and Lake Vanda (VAN) 34 6. Depth profiles of physical and chemical characteristics for the three lakes in this study 35 7. Depth profiles for phytoplankton biomass (Chl a) and bacterial abundance for study sites 36 8. Spatial variability in enzyme activity of RubisCO and β-Glucosaminidase (B-GAM) in study sites 37 9. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) 38 10. Observed and estimated alpha diversity of 16S and 18S OTUs. 62 11. 16S OTUs relative abundance at phylum level 63 12. 18S OTUs relative abundance at phylum level 64 13. NMDS plot of bacterial communities 65 14. NMDS plot of eukaryal communities 66 15. Association network of concurrent of bacteria, eukaryotes and correlation with environmental parameters 67 16. Rarefaction curves of 16S and 18S OTUs 84 17. Representive SEM micrographs of protists found in studied lakes 85 18. Sample cytogram showing flow cytometric sort regions for microbial eukaryotes from an enrichment culture (MDV87) generated from the Antarctic Dry Valley Lake Bonney. 106 19. Maximum-likelihood tree (1000 bootstrap) showing identity of Lake Bonney microbial eukaryote single amplified genomes (EUK-SAGS) recovered from enrichment culture MDV87 107 20. Confocal microscopic images of Chlamydomonas sp. ICE-MDV and Isochrysis sp. MDV isolates 108 21. Principle Coordinates Analysis (PCoA) 109 22. SEM micrographs and the diversity of microbial partners associated with the heterotrophic nanoflagellate Pteridomonas 110 23. Evidence of parasite-host interactions between Lake Bonney microbial eukaryotes. a, Diversity of 16S rRNA gene OTUs recovered from Pirsonia EUK-SAGs 111 24. Confocal microscopic images of Isochrysis sp. MDV isolates without LysoTracker Green staining 123 25. Confocal microscopic images of Chlamydomonas sp. ICE-MDV isolates without LysoTracker Green staining 124 26. Heat map of communities associated with sorted eukaryotic organisms based on 16s rRNA sequence abundance 125 27. Working models of our current understanding of major carbon and energy cycles in Lake Bonney (west) vs. Lake Fryxell 132 vi Acknowledgements I would like to express my sincere appreciation and thanks to my mentor and Team Protist leader, Dr. Rachael Morgan-Kiss, who has been extremely