Fingerprinting Marine Macrophytes in Blue Carbon Habitats Thesis by Alejandra Ortega In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Bioscience King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia November, 2019 2 EXAMINATION COMMITTEE PAGE The thesis of Alejandra Ortega is approved by the examination committee. Committee Chairperson and Thesis Supervisor: Prof. Carlos M. Duarte Committee Members: Prof. Mark Tester, Prof. Takashi Gojobori, and Prof. Hugo de Boer [External] 3 © November, 2019 Alejandra Ortega All Rights Reserved 4 ABSTRACT Fingerprinting Marine Macrophytes in Blue Carbon Habitats Alejandra Ortega Seagrass, mangrove, saltmarshes and macroalgae - the coastal vegetated habitats, offer a promising nature-based solution to climate change mitigation, as they sequester carbon in their living biomass and in marine sediments. Estimation of the macrophyte organic carbon contribution to coastal sediments is key for understanding the sources of blue carbon sequestration, and for establishing adequate conservation strategies. Nevertheless, identification of marine macrophytes has been challenging and current estimations are uncertain. In this dissertation, time- and cost-efficient DNA-based methods were used to fingerprint marine macrophytes and estimate their contribution to the organic pool accumulated in blue carbon habitats. First, a suitable short-length DNA barcode from the universal 18S gene was chosen among six barcoding regions tested, as it successfully recovered degraded DNA from sediment samples and fingerprinted marine macrophyte taxa. Second, an experiment was performed to test whether the abundance of eDNA represents the content of organic carbon within the macrophytes; results supported this notion, indicating a positive correlation (R2 = 0.85) between eDNA and organic carbon. Third, using the chosen barcode, eDNA of marine macrophyte was identified from sediments of seagrass meadows and mangrove forests in the Arabian Red Sea, to further estimate contributions to the organic carbon pools. Estimations based on eDNA were compared against estimations of organic carbon based on stable isotope analyses from the same sediments; results from both methods were similar. In addition, this research 5 provided the first quantitative evidence of the contribution of macroalgae to coastal and oceanic carbon pools. Hitherto, macroalgae have been ignored in blue carbon assessments because their fingerprinting was challenging and there was no evidence of their carbon export. The results of this dissertation demonstrate that eDNA offers an unprecedent taxonomic discrimination, and resolve the contribution of marine macrophytes to the organic pools in blue carbon sediments. 6 ACKNOWLEDGEMENTS This doctoral research is a collaborative effort between KAUST and other institutions; I would like to thank my co-authors for their contribution to this work. Especially, to Sarah Bachmann Øberg, Marlene Wesselmann, Intikhab Alam and Allan A. Kamau. I extent this gratitude to lab support specialists Jasmine Raja Mohamed Sait and Mongi Ennasri. My profound appreciation goes to Nathan R. Geraldi and Rubén Rua Díaz; their guidance was limitless. This appreciation also goes to Daniel Binham for his unconditional friendship and editing skills. Finally, I thank Prof. Carlos M. Duarte for offering and advising me on this research. 7 TABLE OF CONTENTS Page EXAMINATION COMMITTEE PAGE.................................................... 2 COPYRIGHT PAGE.................................................................................... 3 ABSTRACT................................................................................................... 4 ACKNOWLEDGEMENTS......................................................................... 6 TABLE OF CONTENTS............................................................................. 7 LIST OF ABBREVIATIONS...................................................................... 9 TABLE OF FIGURES.................................................................................. 11 LIST OF TABLES........................................................................................ 12 Dissertation synopsis.................................................................................... 13 Introduction........................................................................................... 13 Dissertation Outcome............................................................................ 16 Publications........................................................................................... 17 References............................................................................................. 18 Part I: Fingerprinting blue carbon in coastal ecosystems......................... 21 Chapter 1: A DNA mini-barcode for marine macrophytes....................... 22 1.1 Abstract............................................................................................ 23 1.2 Introduction...................................................................................... 24 1.3 Methods............................................................................................ 27 1.4 Results.............................................................................................. 37 1.5 Discussion........................................................................................ 49 1.6 Acknowledgements.......................................................................... 52 1.7 Supplementary Information............................................................. 53 1.8 eDNA extraction protocol................................................................ 55 1.9 References........................................................................................ 61 Chapter 2: Environmental DNA estimates marine macrophyte contribution to blue carbon sediments.............................................................................. 66 2.1 Abstract............................................................................................ 67 2.2 Introduction...................................................................................... 67 2.3 Methods............................................................................................ 69 2.4 Results and discussion...................................................................... 76 2.5 Acknowledgements.......................................................................... 90 2.6 Supplementary Information............................................................. 91 2.7 Supplementary Results.................................................................... 100 2.8 References....................................................................................... 102 8 Part II: Fingerprinting blue carbon in the open ocean............................. 105 Chapter 3: Important contribution of macroalgae to oceanic carbon Sequenstration............................................................................................... 106 3.1 Abstract.................................................................................................. 107 3.2 Main....................................................................................................... 107 3.3 Tracing macroalgae............................................................................... 110 3.4 Macroalgae diversity in the ocean......................................................... 115 3.5 Export of macroalgae throughout the water column............................. 117 3.6 Implications for blue carbon assessments............................................. 122 3.7 Methods.................................................................................................. 123 3.8 Acknowledgements................................................................................ 131 3.9 Supplementary Information................................................................... 132 3.10 References.............................................................................................. 138 9 LIST OF ABBREVIATIONS 18S rDNA 18S ribosomal DNA δ13C delta Carbon 13 δ15N delta 15 Nitrogen l microliter μm micrometer mol micromolar ANOVA Analysis of variance BLASTp Basic local alignment search tool for proteins BOLD Barcode of life database bp Base pair CBOL Consortium for the barcode of life CI Chloroform-isoamyl alcohol CO2 Carbon dioxide COI Cytochrome C oxidase subunit I gene Corg Organic carbon CTD Conductivity, temperature, and depth instrument DADA2 Divisive amplicon denoising algorithm 2 df Degree of freedom DMAP Dragon metagenomics analysis platform DOC Dissolved organic carbon eDNA environmental DNA F Pseudo-F statistic IO Indian Ocean ITS2 Internal transcribed spacer 2 gene KEGG Kyoto encyclopedia of genes and genomes LPA Linear polyacrylamide matK Maturase K plastid gene Mg C Megagram of carbon MS Mediterranean Sea NaCl Sodium Chloride NAO North Atlantic Ocean NaOH Sodium hydroxide nMDS Non-metric multidimensional scaling ordination NPO North Pacific Ocean OTU Operational taxonomic units PCR Polymerase chain reaction PEG Polyethylene glycol PERMANOVA Permutational multivariate analysis of variance PERMDISP Analysis of homogeneity of multivariate dispersion Pg C Petagram of carbon PO4 Phosphate POC Particulate organic carbon POM Particulate organic matter rbcL Ribulose bisphophate carboxylase large subunit gene 10 RPM Read per million RS Red Sea SAO South Atlantic Ocean SCG