Cultured microalgae from shallow water and intertidal soft sediments in the Red Sea, Saudi Arabia Anders Wold Master of Science Thesis Department of Biosciences Section for Aquatic Biology and Toxicology (AQUA) UNIVERSITY OF OSLO 18.06.2015 © Anders Wold Year: 2015 Title: Cultured microalgae from shallow water and intertidal soft sediments in the Red Sea, Saudi Arabia Author: Anders Wold http://www.duo.uio.no Printed: Reprosentralen, University of Oslo II Abstract The tropical Red Sea coast of Saudi Arabia is dominated by intertidal soft sediments, seagrass meadows and mangrove beaches. These habitats have been under high pressure for decades partly due to anthropogenic activity. The water of the Red Sea is characterised by its high temperature and salinity throughout the year. The microalgae is a group of organisms characterised by their small size, unicellular or colonial organisation and ability or secondarily lost ability to photosynthesise including closely related non-photosynthetic species. The photoautotrophic microalgae are responsible for approximately 45% of the World's primary production and thus play a major role in many ecosystems. It is estimated that more than 40,000 algal species (including macroalgae) have been described, but recent advances in high throughput sequencing suggest there is a vast unknown diversity yet to be discovered. Little is known about the microalgal species diversity associated with the tropical habitats of the west coast of Saudi Arabia. Eleven strains were isolated in the shallow waters and beaches of Saudi Arabia. They were studied morphologically with light microscopy, scanning electron microscopy and transmission electron microscopy and molecularly with phylogenetic trees inferred from 18S and partial 28S (D1/D2) rDNA. Sixteen unique sequences and two previously recorded sequences were acquired and will be uploaded to NCBI GenBank. Four strains have been identified to species: Cafeteria roenbergensis Fenchel & Patterson, Lotharella reticulosa Ota, Cylindrotheca cf. closterium (Ehrenberg) Reimann & Lewin and Oxyrrhis marina Dujardin. The latter two have previously been recorded in the Red Sea. The first two have been recorded in similar habitats. Four strains have been identified to genus: genera Amphidinium Claparède & Lachmann, Chlamydomonas Ehrenberg, Nitzschia Hassall and Tetraselmis Stein, all of which are previously recorded in the Red Sea and similar habitats. Three potentially novel species with affinity for known species were partially described. They belong to genera Proteomonas Hill & Wetherbee, Pavlova Butcher and Rhizochromulina Hibberd & Chrétiennot-Dinet respectively. III Acknowledgement The work with this thesis was carried out mainly at the Section for Aquatic Biology and Toxicology (AQUA). Some of the laboratory work and the analyses were also carried out the Section for Genetics and Evolutionary Biology (EVOGENE), and the Centre for Ecological and Evolutionary Synthesis (CEES) and the Electron Microscopy Unit for Biosciences facilities at the University of Oslo (UiO) from September 2013 to June 2015 and at the Red Sea Research Center (RSRC) at the King Abdullah University of Science and Technology (KAUST) in Saudi Arabia in November 2012 and January 2014. First I would like to thank my main supervisor Bente Edvardsen for sharing your expertise on phylogeny, writing, providing relevant literature, proofreading of my thesis and your knowledge on ecology and the microalgae. Also, a big thanks to my co-supervisor Wenche Eikrem for helping me with preparation for EM, providing relevant literature, proofreading of my thesis and sharing some of your enormous knowledge of microalgal morphology with me. I would also like to thank my other two co-supervisors Stein Fredriksen and Stein Kaartvedt for providing relevant literature and proofreading. An extra big thanks goes to Sissel I. Brubak for all the help and assistance with cultivation and the molecular work. To the guys at the Electron Microscopy Unit: thanks for teaching me how to operate the different electron microscopes and preparation techniques. Furthermore, I owe a big thanks to Elianne S. Egge and Anders K. Krabberød for all the help with molecular work, analysis and very interesting discussions on microalgal topics. Finally I would like to thank Kristine Dobbe and Annie Evankow for proofreading, Randi F. Ose for helping me with the cloning, the guys at KAUST for giving me a pleasant stay in Saudi Arabia, to Siri R. Moy and the rest of the guys at my study room and all the people at UiO for making all these years unforgettable. IV Table of Contents 1 Introduction.......................................................................................................................................1 1.1 Red Sea......................................................................................................................................1 1.2 Mangrove, seagrass and intertidal soft sediment ecology.........................................................1 1.2.1 Mangrove ecology..............................................................................................................1 1.2.2 Seagrass ecology................................................................................................................2 1.2.3 Intertidal soft sediment ecology.........................................................................................2 1.3 Microalgae.................................................................................................................................3 1.4 Benthic microalgae....................................................................................................................4 1.5 Morphological and molecular phylogenetics.............................................................................4 1.6 Objectives..................................................................................................................................6 2 Materials and methods.......................................................................................................................7 2.1 Field work..................................................................................................................................7 2.1.1 Location description...........................................................................................................7 2.1.2 Sampling............................................................................................................................7 2.1.3 Raw cultures.......................................................................................................................7 2.2 Cultures......................................................................................................................................8 2.2.1 Culture medium..................................................................................................................8 2.2.2 Dilution series....................................................................................................................8 2.2.3 Agar dishes.........................................................................................................................9 2.2.4 Culture examination...........................................................................................................9 2.2.5 Other isolation techniques..................................................................................................9 2.3 Molecular work........................................................................................................................10 2.3.1 DNA isolation..................................................................................................................10 2.3.2 DNA amplification...........................................................................................................10 2.3.3 Gel electrophoresis...........................................................................................................13 2.3.4 Analysis of DNA concentration and DNA purity.............................................................13 2.3.5 PCR product purification and sequencing........................................................................14 2.4 Microscopy..............................................................................................................................14 2.4.1 Light microscopy.............................................................................................................14 2.4.2 Scanning electron microscopy (SEM).............................................................................15 2.4.3 Transmission electron microscopy (TEM).......................................................................15 2.4.4 Measurements..................................................................................................................16 2.5 Sequence processing, alignments and phylogeny....................................................................16 2.5.1 Sequence processing........................................................................................................16 2.5.2 Alignments.......................................................................................................................17 2.5.3 Phylogenetic trees............................................................................................................17 3 Results and species discussion........................................................................................................18