Freie Universität Berlin • Institute of Chemistry and Biochemistry Genetic diversity in green algae (Hydrodictyaceae) obtained from modern and ancient sedimentary DNA of Siberian lakes Bachelor thesis Submitted by Jan Patrick Lütje September 2014 Supervisor: Prof. Dr. Ulrike Herzschuh, Second Referee: Dr. Jens Peter Fürste, Alfred Wegener Institute for Polar and Institute of Chemistry and Marine Research, Potsdam Biochemistry, Freie Universität Berlin The present work was written between the 4th of August and 29th of September 2014. By submitting this work, I declare that it was solely undertaken by me and that no help was provided from other sources as those allowed. All source material that was used is listed in part 6 “References”. Furthermore, I declare no competing financial interests or any other conflict of interest. Potsdam, 29th of September 2014 Jan Lütje Bachelor thesis Note of thanks I wish to thank Prof. Dr. Ulrike Herzschuh (Alfred Wegener Institute for Polar and Marine Research, Potsdam) and Dr. Jens Peter Fürste (Freie Universität Berlin) for their willingness to review my work as supervisor and second referee, and Bastian Niemeyer (AWI) for cartographic and algae pictures. In particular, my gratitude goes to Dr. Kathleen Stoof-Leichsenring (AWI) for her supervision of my lab work and her constant help and support during the process of data analysis, writing and review of my thesis. Thank you also to the whole lab group at the AWI; your help and the pleasant working atmosphere is greatly appreciated. 3 Bachelor thesis Index Abstract ............................................................................................................................. 6 Zusammenfassung ............................................................................................................ 7 1. Introduction ............................................................................................................... 9 1.1. Hydrodictyaceae ................................................................................................. 9 1.2. Pediastrum as bioindicator ............................................................................... 10 1.3. RbcL as genetic marker .................................................................................... 11 1.4. Environmental DNA and metabarcoding ......................................................... 11 1.5. Study area ......................................................................................................... 12 2. Objective .................................................................................................................. 14 3. Materials and Methods ............................................................................................ 15 3.1. Materials ........................................................................................................... 15 3.1.1. Chemicals and buffers .............................................................................. 15 3.1.2. Kits and other materials ............................................................................ 15 3.1.3. Laboratory equipment ............................................................................... 16 3.1.4. Samples ..................................................................................................... 17 3.2. Methods ............................................................................................................ 19 3.2.1. Security and decontamination measures ................................................... 19 3.2.2. DNA isolation ........................................................................................... 19 3.2.3. Preliminary experiment: primer test and PCR .......................................... 21 3.2.4. PCR ........................................................................................................... 22 3.2.5. Gel electrophoresis ................................................................................... 23 3.2.6. Purification ................................................................................................ 24 3.2.7. Cloning ...................................................................................................... 24 3.2.8. Sanger sequencing .................................................................................... 26 3.2.9. Sequence alignment and verification ........................................................ 27 3.2.10. Phylogenetic analyses ............................................................................... 28 4. Results ..................................................................................................................... 29 4.1. DNA isolation and quantification .................................................................... 29 4.2. Preliminary experiment: primer test ................................................................. 29 4.3. Sequence verification ....................................................................................... 32 4 Bachelor thesis 4.4. Diversity, distribution and classification of Hydrodictyaceae lineages ........... 32 4.4.1. Diversity and distribution of Hydrodictyaceae lineages ........................... 32 4.4.2. Taxonomic assignment (NCBI BLAST) .................................................. 36 4.5. Phylogenetic analyses ...................................................................................... 37 4.5.1. Bayesian phylogenetic tree based on the 82 bp rbcL fragment ................ 37 4.5.2. Bayesian phylogenetic tree based on a 1052 bp rbcL fragment ............... 37 5. Discussion ................................................................................................................ 40 5.1. Specificity and reliability of tested primers ..................................................... 40 5.2. Distribution of lineages obtained from surface and core sediments ................ 40 5.3. Phylogenetic analyses and comparison of 82 bp and 1052 bp fragments ........ 42 5.4. Indications for the use of sedDNA analyses in paleoecological studies .......... 43 6. References ............................................................................................................... 45 7. List of figures and tables ......................................................................................... 47 8. List of symbols and abbreviations ........................................................................... 48 9. Appendix ................................................................................................................. 49 5 Bachelor thesis Abstract Abstract Pediastrum and other representatives of the green algae family Hydrodictyaceae (Chlorophyta), commonly found in freshwater environments, are potential bioindicators for paleolimnological studies as they are preserved in sediments and morphologically classified as non-pollen palynomorphs obtained along with pollen spectra. The aim of this study was to examine the diversity in Hydrodictyaceae obtained from modern and ancient Siberian lake sediments following a molecular genetic approach. Environmental samples were obtained from lakes across the arctic-boreal tree line in Siberia. Modern sedimentary DNA (sedDNA) was isolated from the samples and a selected fragment of the rbcL gene, encoding the large subunit of the enzyme RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), was amplified via polymerase chain reaction (PCR). The respective primer pairs were specifically developed beforehand and optimized for the desired target fragment in Hydrodictyaceae. Four primer combinations were tested on two modern sediment samples and the most suitable primer combination was selected and applied to additional modern and core samples. The PCR products were cloned and sequenced by Sanger sequencing; the sequences were aligned and verified and taxonomic identification was conducted based on BLAST nucleotide search. 71.2 % of the obtained sequences were assigned to Hydrodictyaceae in ten out of eleven samples and 28.8 % to unknown algae strains in nine out of eleven samples. In total, thirteen different rbcL lineages were detected, among them twelve lineages of Pediastrum and Pseudopediastrum in modern sediments and six in ancient core sediments, including a single Stauridium lineage in one core sample. Five different lineages were detected in both sample types. One sample did not yield any Hydrodictyaceae lineage. Although the preliminary results of this study indicated that the distribution of lineages was fairly heterogeneous, we recognized a general tendency in terms of sample type (surface/core) and vegetation type (tundra/forest tundra/forest), but could not identify distinct preferences of single lineages. Phylogenetic inferences of the lineages indicated that a resolution down to species and strain level is not possible for most of the examined lineages. 6 Bachelor thesis Zusammenfassung However, this study showed that Pediastrum DNA was reliably amplified from modern lake sediments and from core depths of up to 62 cm, the latter of which yielded ancient DNA (aDNA) with an age of approximately 3000 years. Possibly further studies covering larger datasets and additional genetic markers will give better resolution in terms of quantification, taxonomic coverage and identification. Zusammenfassung Pediastrum und andere in Süßwasser-Ökosystemen verbreitete Vertreter der Familie Hydrodictyaceae (Grünalgen, Chlorophyta) sind potentielle Bioindikatoren