Ecological Patterns in Prokaryotic Diversity and Community Composition in Tropical Soils of Southeast Asia

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 2. Jonathan Miles Adams.The dominant factors controlling soil microbial community composition and diversity variation within the tropics are poorly known. In this study, the extent of soil microbial diversity was investigated in tropical soils of Malaysia and the way microbial communities are affected by land use (forest to agriculture), spatial scaling (at local and regional scales) and biome (tropics vs. temperate), as well as the extent to which ecological processes or other environmental factors contribute to structuring the soil microbial communities. The effect of land use changes (i.e. conversation of forest to agriculture) on bacteria, archaea and ammonia oxidizing archaea (AOA) was studied in tropical soils of Malaysia. Acidobacteria and Proteobacteria were the most dominant bacterial phyla across all soil samples. Thaumarchaeota groups 1.1b and 1.1c appear to be the dominant archaeal lineages in tropical lowland soils, whereas, Nitrososphaera and Nitrosotalea clusters were recovered as the most dominant AOA taxa. It is found that land use in itself has a weak but significant effect on the microbial community composition. However, for all microbial taxa the community composition and diversity was strongly correlated with soil properties, especially soil pH. Soil pH was the best predictor of microbial community composition and diversity across the various land use types. Total bacterial and AOA diversity showed a hump-shaped correlation with soil pH, diversity peaking at ~ pH 6.0. However, total archaeal diversity showed a negative correlation with soil pH. In addition, variation in relative abundance of dominant lineages of bacteria, archaea and AOA was also significantly correlated with soil pH. Together, these results confirm the importance of soil pH in structuring soil microbial communities in Southeast Asia. How spatial scaling affects bacterial communities in tropical rainforest soils was further analyzed at two spatial scales: a local scale with samples spaced every 5 m over a 150 m transect, and a regional scale with samples 1 to 1,800 km apart. A weak correlation was found between spatial distance and whole bacterial community dissimilarity, but only at the local scale. In contrast, environmental distance was highly correlated with community dissimilarity at both spatial scales, stressing the greater role of environmental variables rather than spatial distance in determining bacterial community variation at different spatial scales. Soil pH was the only environmental parameter that significantly explained the variance in bacterial community at the local scale, whereas total nitrogen and elevation were additional important factors at the regional scale. Similar results were observed at both scales when only the most abundant OTUs were analyzed. A variance partitioning analysis showed that environmental variables contributed more to bacterial community variation than spatial distance at both scales. In total, these results support a strong influence of the environment, rather than neutral or dispersal effects, in determining bacterial community composition in the rainforests of Malaysia. It is possible that the remaining spatial distance effect is due to some of the myriad of other environmental factors which were not considered here, rather than dispersal limitation. The community of both total archaea and ammonia oxidizing archaea (AOA) was compared using 16S rRNA and amoA gene pyrosequencing respectively, on a geographical scale in two different biomes: tropical Malaysia and temperate Korea and Japan - to investigate to what extent archaeal and AOA diversity and community structure change between them. Total archaeal diversity showed a negative correlation with soil pH, and in contrast a hump-shaped curve for AOA diversity that peaked at ~ pH 6.0. Within each biome, soil pH also emerged as the dominant factor determining variation in community composition of both 16S rRNA and amoA genes. However, between biomes climate was also important in differentiating tropical from temperate archaea. For both 16S rRNA and amoA genes, significant phylogenetic signals were consistently detected across short phylogenetic distances. However, for 16S rRNA genes significant signals were detected also at intermediate phylogenetic distances. Quantifying phylogenetic turnover (as the deviation from a null expectation) suggested that deterministic factors imposed strong selection on total archaea and AOA communities, in both biomes. This study reveals that strong and predictable relationships exist between archaeal community structure and the environment, and that both climate and soil pH are major factors in archaeal community ecology.TABLE OF CONTENTS ABSTRACT ………………………………………………….………. i TABLE OF CONTENTS …………………………………………....iv ABBREVIATIONS ………………………………..……………….viii LIST OF FIGURES ….………………………………………….…..ix LIST OF TABLES …………………………………………….……xiv CHAPTER 1. Tropical Soil Microbial Diversity and Recent Trends in Microbial Ecology: An Introduction …………..………1 1.1. Tropical regions are exceptionally rich in biodiversity……......2 1.2. Metagenomics and soil microbial community analysis....…….3 1.2.1. Metagenomics approach………………………...……...3 1.2.2. The extent of prokaryotic diversity in soil………………5 1.2.3. The status of prokaryotic diversity in tropical soil………7 1.3. Microbial community analysis procedure……….………..10 1.3.1. Initial processing and sequence quality control..…........10 1.3.2. Sequence alignment, pre-clustering, chimera removal and taxonomic classification ...........................................................10 1.3.3. OTU based analysis and further analysis………………12 1.4. Objectives of this study.……………………..……..………...15 CHAPTER 2. Understanding the Effect of Land Use on Microbial Communities in Tropical Soils of Malaysia.….................17 2.1. Tropical soil bacterial communities in Malaysia: pH dominates in the equatorial tropics too……………………………………….18 2.1.1. Introduction …………………………...……………….18 2.1.2. Materials and Methods …………………..…………….20 Site description.……………………………………..….20 Soil collection and DNA extraction....……………..…...23 Amplification of 16S rRNA genes andpyrosequencing..............................................................................................23 Environmental variables...…………………………..….24 Processing of pyrosequencing data and taxonomic analysis…………………………………………………25 Phylogenetic analysis………………………………….26 Statistical analysis………………………………………26 2.1.3. Results.…………………………...…………………….28 General analysis of the pyrosequencing-derived dataset…………………………………………………..29 Land use and bacterial community......………...………29 Soil properties and bacterial community….……..……32 2.1.3. Discussion.……………………….…………………….42 2.2. pH dominates variation in tropical soil archaeal diversity and community structure.………………....………………………….……50 2.2.1. Introduction.…………………………...……………….50 2.2.2. Materials and Methods.…………………..…………….52 Collection site characterization, sampling and isolation of soil DNA………….…………………………..………52 PCR amplification and pyrosequencing..….………..….53 Processing of pyrosequencing data and taxonomic analysis……………….………………….………..…….54 Statistical processing and analysis of results………..….54 2.2.3. Results.…………………………...…………………….56 2.2.4. Discussion.……………………….…………………….64 Community composition, diversity, and soil pH……….64 Effect of land use……………………………………….66 CHAPTER 3. Spatial Scaling Effects on Bacterial Communities of Tropical Rainforest Soils.………………………….…......67 3.1. Spatial scaling effects on soil bacterial communities in Malaysian tropical forests………………………………..……….68 3.1.1. Introduction.…………………………...……………….68 3.1.2. Materials and Methods.…………………..…………….70 Sampling locality and field sampling strategy………….70 Environmental variables and soil DNA extraction……..71 PCR amplification, pyrosequencing, sequence processing, and taxonomic analysis….…………………………..….72 Statistical analysis…….……………….………………..72 Deposited 454 read accession numbers………………...76 3.1.3. Results.…………………………...…………………….78 3.1.4. Discussion.……………………….…………………….92 CHAPTER 4. Biome Effect on Community Composition and Diversity of Soil Archaea and Ammonia Oxidizing Archaea................................................................................98 4.1. Both climate and pH are key in determining soil archaeal community structure and diversity…………….….………………99 4.1.1. Introduction …………………………...……………….99 4.1.2. Materials and Methods.……………….………...…….101 Sample collection………………………...……………101 Soil chemical variables analyses……….……………..102 DNA extraction, PCR and pyrosequencing of 16S rRNA and amoA gene………………………….………....…102 Processing of pyrosequencing data and taxonomic analysis………………………………………………..103 Statistical procesing and analysis of results.…………104 4.1.3. Results.…………………………...…………………...107 4.1.4. Discussion ……………………….…………………...122 GENERAL CONCLUSIONS……………………………………..129 REFERENCES.…………………………………………………….132 APPENDIX.………………………………………………………...155Docto