UNIVERSIDADE FEDERAL DO RIO DE JANEIRO INSTITUTO DE BIOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIODIVERSIDADE E BIOLOGIA EVOLUTIVA - PPGBBE TESE DE DOUTORADO Genômica estrutural e funcional de turfs e cianobactérias do Banco de Abrolhos (Bahia, Brasil) JULINE MARTA WALTER Rio de Janeiro 2016 Genômica estrutural e funcional de turfs e cianobactérias do Banco de Abrolhos (Bahia, Brasil) JULINE MARTA WALTER Tese apresentada como requerimento parcial ao título de Doutor pelo Programa de Pós Graduação em Biodiversidade e Biologia Evolutiva da Universidade Federal do Rio de Janeiro. Aprovada em: 19 de dezembro de 2016. Prof. Dr. Fabiano Lopes Thompson __________________________ Profª. Drª. Sigrid Neumann Leitão __________________________ Prof. Dr. Ronaldo Bastos Francini-Filho __________________________ Prof. Dr. Carlos Eduardo Guerra Scharago __________________________ Prof. Dr. Osmindo Rodrigues Pires Júnior __________________________ Profª. Drª. Laura Silvia Bahiense da Silva __________________________ Leite Rio de Janeiro 2016 iii Structural and functional genomics of turfs and cyanobacteria from the Abrolhos Bank (Bahia, Brazil) JULINE MARTA WALTER Thesis submitted in partial fulfillment of the requirements for the degree of Doctor in the Graduate Program of Biodiversity and Evolutionary Biology at Federal University of Rio de Janeiro. Adviser: Prof. Dr. Fabiano Lopes Thompson Doctoral Committee: Prof. Dr. Ronaldo Bastos Francini-Filho (UFPB) Prof. Drª. Sigrid Neumann Leitão (UFPE) Prof. Dr. Osmindo Rodrigues Pires Júnior (UnB) Prof. Dr. Carlos Eduardo Guerra Schrago (UFRJ) Prof. Drª. Laura Silvia Bahiense da Silva Leite (COPPE, UFRJ) Alternate Members: Profª. Drª. Cristiane Carneiro Thompson (UFRJ) Profª. Drª. Michelle Regina Lemos Klautau (UFRJ) Rio de Janeiro 2016 iv This thesis is dedicated to my parents v ACKNOWLEDGMENTS I want to thank the many people who contributed with this thesis, particularly my adviser Dr. Fabiano L. Thompson, who gives me this unique opportunity, the partnership and knowledgement into the coral microcosms. I also would like to thank you for the hours you spent meticulously correcting chapters and papers. I also thank the Phyto research group, particularly Dr. Paulo S. Salomon, for his continuous assistance and enthusiastic encouragements. Special thanks are offered to Dr. Rodrigo L. Moura for inspiration and introducing me to this fascinating ecosystem, helping me in the fieldtrip. I extend special thanks to the crewmembers behind the catamarã for their hard work: Zá, Maurício, and Van. Thank to undergraduate students who spend many hours assisting in the isolation of the clonal cultures. Thank to Paulo Iiboshi for the maintenance of the cultures. I thank Dra. Cristiane C. Thompson for being always there, with her supportive and pleasant atmosphere. Many thanks to all my friends and colleagues at the Micro Lab: Adriana Fróes (Dri), Ana Carolina Soares, Ana Paula Moreira, Angélica, Arthur Lima, Bruno Sérgio, Diogo Tschoeke, Felipe Coutinho, Gizele Garcia, Giselle Cavalcanti, Gabriela Calegario, Graciela Dias, Gustavo Pitta, Lívia Vidal, Louise Oliveira, Luciana Leomil, Luciana Reis, Mariana Campeão, Pedro Meirelles, Raphael Silva, Rosa Koko, and many others. Special thanks go to my darling Lílian J. K. Mayerhofer. I could not have completed this project without her constant love and encouragements. Love you! I would also like to thank my Brazilian, Dutch and Italian colleagues and friends from Utrecht University for their high quality discussion during the time I spend there. The fruitful discussions with Prof. Dr. Jean Swings were highly acknowledged. Suggestions vi from members of the Examining Evaluation greatly improved the manuscripts and thus, this Thesis. Thank to the contribution of Rede Abrolhos (Abrolhos Network), funded by the National Biodiversity System (SISBIOTA). I would like to thank the Abrolhos Marine National Park to facilitate the access to the reefs. Thank to the Biodiversity and Evolutionary Biology Graduate Program at Federal University of Rio de Janeiro. Thank to all PPGBBE Committee especially during the year (2014) that I was Vice-Representative of student body. Financial support was provided in part by the CAPES, and from the CNPq through grant 207751. Grant from the CNPq enabled me to spend one year (2015-2016) visiting a Bioinformatics laboratory in The Netherlands. Special thanks are offered to Dr. Bas Dutilh for their time, patience and advices while I was pretty new in the bioinformatics field. Last but not least, thanks to my whole family, been always the biggest support. Rio de Janeiro, November 2016. vii “With every drop of water you drink, Every breath you take, You're connected to the sea. No matter where on Earth you live.” Sylvia Earle - Marine biologist. She was the first woman chief scientist of the U.S. National Oceanic and Atmospheric Administration. - viii LIST OF FIGURES Chapter I Figure 1. Global distribution of coral reefs ……….. 2 ……….. 5 Figure 2. Study site: the South Atlantic’s largest coral reefs Figure 3. Turf assemblages samples ……….. 8 Figure 4. Microbialization process of coral reefs ……….. 9 Figure 5. Abundance of benthic organisms in the Abrolhos Bank ……….. 10 Figure 6. Cost per raw Megabase of DNA sequence ……….. 11 Figure 7. Phylogenetic tree from Woese (1987) based on 16S rRNA sequences. ……….. 14 Figure 8. Phylogenetic reconstruction based on 31 conserved proteins sequences ……….. 15 Chapter II ……….. 41 Figure 1. Turf system taxonomic and functional profiles Figure 2. Most abundant genes from the different lifestyles co- occurring in Abrolhos Bank turfs ……….. 44 Figure 3. Principal Component analysis of the 41 metagenomes using the top eleven variables identified from the Random Forest analysis ……….. 47 ……….. 59 Figure S1. Underwater pictures of turfs ……….. 60 Figure S2. Scheme of turf system and major metabolisms Chapter III Figure 1. Absorption spectra for Acrophormium turfae strains ……….. 77 ……….. 79 Figure 2. Whole genome comparison ……….. 80 Figure 3. Phylogenetic position of Acrophormium turfae strains Figure 4. Distribution of the secondary metabolites across the Acrophormium turfae strains ……….. 83 ix Figure 5. Abundance and distribution profiles of the novel cyanobacterial strains across turfs metagenomes from Abrolhos coral ……….. 84 reefs Figure S1. Overview of turfs and Acrophormium turfae culture isolated from them ……….. 95 Figure S2. Growth rates and optical characteristics of strains CCMR0081T and CCMR0082 ……….. 96 Figure S3. Binning and G+C skew ……….. 96 Figure S4. Distribution of Acrophormium turfae strains ……….. 97 Figure S5. Distribution of genes involved in photosynthesis apparatus ……….. 97 Chapter IV Figure 1. Multilocus sequence analysis (MLSA) phylogenetic tree of the Cyanobacteria phylum with the proposed new names ……….. 129 Figure 2. Heatmap based on similarity matrix of AAI between 100 genomes. ……….. 132 Figure 3. Environmental correlations profile used to define ecogenomic groups ……….. 135 ……….. 138 Figure 4. Ecogenomic of Cyanobacteria across the Earth Supplementary Figure S1. Ribosomal phylogenetic reconstruction of the Cyanobacteria phylum ……….. 151 Supplementary Figure S2. Heatmaps based on GGD metrics of especific cases ……….. 152 Supplementary Figure S3. Abundance and distribution of ecogenomic clusters across freshwater metagenomes ……….. 152 x LIST OF TABLES Chapter II Table 1. General features of each metagenomic sample of the benthic organisms (turf, coral, and rhodolith) and seawater ……………… 34 collected from the Abrolhos Bank ……………… 47 Table 2. Genes abundance ……………… 61 Table S1. Turf pigment profile ……………… 62 Table S2. General features of the turf assemblage metagenomes Table S3. The taxonomic composition of turf metagenomes are statistically indistinguishable in different locations and seasons ……………… 63 (H1) [Online Material] Table S4. Taxonomic contribution to the turf composition from the Abrolhos Bank, indicated by collection period and reef ……………… 63 location [Online Material] Table S5. The functional composition of turf metagenomes are statistically indistinguishable in different locations and seasons ……………… 63 (H2) [Online Material] Table S6. Variable importance (taxonomic Order level) determined by the unsupervised Random Forest analysis ranking of Taxonomy [Online Material] ……………… 63 Table S7. Variable importance (level 1 Subsystems) determined by the unsupervised Random Forest analysis ranking of Function [Online Material] ……………… 63 Table S8. Comparison of the different types of metabolism in the turf, coral, rhodolith, and seawater samples from the Abrolhos ……………… 63 Bank [Online Material] Table S9. Turkey Honest Significant Differences (HSD) pos hoc test results of H3 ANOVA [Online Material] ……………… 63 Chapter III ……………… 74 Table 1. Features of Acrophormium strains included in this study Table S1. Prediction of RNAs in cyanobacterial genomes. ……………… 98 Table S2. The 30 more abundant proteins of the core genome of ……………… 99 CCRM0081T, CCRM0082, and PCC7375 strains. xi Table S3. Distribution of the nonribosomal encoded peptide and polyketide biosynthetic pathways in Cyanobacteria ……………… 101 Chapter IV Table 1. Details of all cyanobacterial genomes included in this ……………… 111 study Table 2. Conserved marker genes used in MLST phylogenetic ……………… 124 reconstruction ……………… 153 Supplementary Table S1. Estimates of genome relatedness of cyanobacterium strains [Online Material] Supplementary Material. Formal description of new genera and ……………… 153 species xii ABBREVIATIONS AND SIMBOLS ºC degrees Celsius