DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES PHENOTYPIC AND GENOTYPIC ADAPTATIONS IN A RIVERINE GREEN ALGA (CHLOROPYCEAE, SELENASTRACEAE) AS A RESPONSE TO LONG-TERM EXPOSURE TO CHEMICAL STRESS Max Karlsson Degree project for Master of Science with a major in Ecotoxicology ES2520, Examination course in ecotoxicology, 60 ECTS Second cycle Semester/year: Spring 2019 Supervisor: Natàlia Corcoll Cornet, Department of Biological and Environmental Sciences Examiner: Thomas Backhaus, Department of Biological and Environmental Sciences Acknowledgements There have been a lot of people involved in this study without whom it would not have been possible to see it through to the end. Your help has been of great value and appreciated beyond words. First and foremost, I would like to thank Oskar Johansson at the department of Biology and Environmental Sciences for his “never give-up” attitude and his pedagogic tutoring throughout the genetic part of the study. Without your help, I would never have gotten past the first step in the process, let alone able to make it all the way through to the end. Secondly, I would like to thank Åsa Arrhenius and Britt Wassmur of the research group of ecotoxicology at the department of Biology and Environmental Sciences for your daily help in finding materials in the laboratory and valuable tips on how to proceed with certain issues. You have given me many quick fixes to problems that otherwise would have taken hours of my time to resolve. I would also want to express my gratitude towards Björn Andersson and Mats Töpel for your genuine interest in the study and for contributing with your specialist knowledge regarding subjects related to your own studies. Lastly, I would like to issue my sincerest gratitude to my supervisor Natàlia Corcoll Cornet, for her incredible patience, encouragement and kindness in her supervision of this thesis. Nothing of this would have been possible without your support and will to succeed. Another testament to your character is that you managed to do all this while simultaneously supervise another student as well as an additional human being. There are no limits to what you can accomplish. I wish you all the best in the future! Sincerely, Max Karlsson. 2 Table of contents Abbreviations ............................................................................................................................. 5 Abstract ...................................................................................................................................... 6 1. Introduction ......................................................................................................................... 7 1.1 General introduction ......................................................................................................... 7 1.2. Carotenoids: Background, synthesis and their biological function ........................... 10 1.2. Herbicide resistance in microalgae ................................................................................ 11 1.3. Project background .................................................................................................... 11 1.4. Aims and hypotheses ................................................................................................. 12 2. Materials and methods ......................................................................................................... 12 2.1 Phenotypic parameters .................................................................................................... 12 2.1.1 Growth conditions .................................................................................................... 12 2.1.2 Growth tests ............................................................................................................. 13 2.1.3 Calibration curves .................................................................................................... 13 2.1.4 Size determination ................................................................................................... 14 2.1.5 Pigment extraction ................................................................................................... 15 2.1.6 Photosynthetic activity ............................................................................................. 16 2.1.7 Tolerance tests ......................................................................................................... 16 2.2 Genotypic parameters ..................................................................................................... 17 2.2.1 DNA extraction ........................................................................................................ 17 2.2.2 PCR-amplification ................................................................................................... 18 2.2.3 Gel electrophoresis ................................................................................................... 18 2.2.4 Product cleaning ....................................................................................................... 19 2.2.5 Cloning ..................................................................................................................... 19 2.2.6 Extraction of fragments from clones ........................................................................ 20 2.2.7 Sequenceing of fragments ........................................................................................ 20 2.3. Statistics ......................................................................................................................... 20 3. Results .................................................................................................................................. 21 3.1 Phenotypic parameters .................................................................................................... 21 3.1.1 Growth and cell size ................................................................................................. 21 3.1.2. Pigment composition .............................................................................................. 23 ........................................................................................................................................... 24 3.1.3. Photosynthetic activity ............................................................................................ 24 3.1.4. Dose-response tests: Diflufenican sensitivity ......................................................... 27 3.2. Genotypic parameters .................................................................................................... 29 3.2.1 DNA extraction, PCR-fragments and sequencing ................................................... 29 3 4. Discussion ............................................................................................................................ 31 4.1 Phenotypic differences ................................................................................................... 31 4.2 Genotypic differences ..................................................................................................... 33 4.3 General remarks .............................................................................................................. 34 5. Conclusion ............................................................................................................................ 34 6. Bibliography ......................................................................................................................... 36 Appendix I .................................................................................................................................. 1 Appendix II ................................................................................................................................ 2 4 Abbreviations DFF – Diflufenican, an herbicide that inhibits carotenoid-synthesis in plants and algae DMAPP - Dimethylallyl diphosphate, one of two important substrates participating in the initial steps of the carotenoid synthesis EFSA – European Food Safety Authority EQY – Effective Quantum Yield, photosynthetic measurement describing number of functional complexes of Photosystem II FU – Fluorescence Units MQY – Maximum Quantum Yield, photosynthetic measurement describing the proportion of absorbed light that participate in photosynthesis NPQ – Non-Photochemical Quenching, photosynthetic process that takes place when light absorption exceeds photosynthetic capacity IPP - Isopentenyl diphosphate, one of two important substrates participating in the initial steps of the carotenoid synthesis pds – gene encoding for the enzyme phytoene desaturase PICT – Pollution Induced Community Tolerance, a concept where sensitive species and/or genotypes are replaced by more tolerant ones in a community as a result of chemical pollution PSII – Photosystem II 5 Abstract Herbicide pollution is a common problem in agricultural streams due to their extensive use and their unwanted effects on non-target species. Selanastraceae is a family of green algae that are particularly dominant in freshwater, making them a key component in the base of the food chain and an important part of the oxygen production. Therefore, it is crucial from a conservation point of view to accurately monitor the ecotoxicological impact of herbicides on this group of algae. In this work, phenotypic and genotypic differences are studied between different populations of Kirchneriella aperta (Chlorophyceae, Selenastraceae). Three