Estimation of genetic diversity in the colony forming polar prymnesiophyte species Phaeocystis antarctica

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(3) . . DISSERTATION zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. nat. am Fachbereich 2 (Biologie/Chemie) der Universität Bremen. Vorgelegt von. Steffi Gäbler-Schwarz Bremerhaven 23. Oktober 2009.

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(5) 1. Gutachter:. Prof. Dr. Ulrich Bathmann, Alfred-Wegener-Institut für Polar- und Meeresforschung, Bremerhaven. 2. Gutachter:. Prof. Dr. Gunter-Otto Kirst, Universität Bremen.

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(7) Hiermit erkläre ich, dass ich die vorliegende Dissertation selbstständig verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe. Die entnommenen Stellen aus benutzten Werken wurden wörtlich oder inhaltlich als solche kenntlich gemacht.. Steffi Gäbler-Schwarz.

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(12) Table of contents Page List of Tables. ii. List of Figures. iv. Summary. x. Zusammenfassung. xii. Chapter 1 General introduction. 1. Southern Ocean. 3. Sea ice, temperature and light Southern ocean regimes The genus Phaeocystis. 6 7 7. Molecular methods Aim and outline of the thesis. 10 17. References. 23. Chapter 2 A new cell stage in the haploid-diploid life cycle of the colony-forming haptophyte Phaeocystis antarctica and its ecological implications. 31. Chapter 3 Methods used to reveal genetic diversity in the colony forming prymnesiophytes Phaeocystis antarctica, P. globosa and P. pouchetii – preliminary results (short communication). 65. Chapter 4 STAMP: Extensions to the STADEN sequence analysis package for high throughput interactive microsatellite marker design. 87. Chapter 5 PRIMER NOTE: Microsatellite markers for the polar prymnesiophyte species Phaeocystis antarctica KARSTEN. 115. Chapter 6 Genetic structure and diversity of Phaeocystis antarctica assessed by fast-evolving microsatellite markers. 125. Chapter 7 Responses of Different Antarctic Genotypes of Phaeocystis antarctica to three salinities: Evidence for Ecosystem Resilience. 155. Chapter 8 Synthesis and further perspectives References. 191 203. Acknowledgements. xiii.

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(14) List of Tables Table 2.1. page AAD strains used in this manuscript, FC = flow cytometry; ITS = molecular 48 identification ITS1 region. 2.2. Flow cytometry: cellular optical properties of AAD strains (arbitrary units: 49 geometric mean of cells normalized to the geometric mean of calibration beads). For details see text.. 3.1. Selective nucleotides (SN) used for selective amplification. X= these primer sets 74 have been used.. 3.2. Algal species used to test best primer set and to create microsatellite enriched librariesį. 74. 3.3. Microsatellite (MS) motifs of the investigated Phaeocystis strains. Some clones contained multiple motifs.. 75. 4.1. Summary and comparison of the results of the four laboratory tests. The 101 "percentage yield" is the ratio of succesful amplifications over the number of loci with tested primers.. 5.1. Microsatellites reaction Mastermix. 5.2. Attributes of eight microsatellites loci isolated from P. antarctica strain SK23 122 tested on 89 P. antarctica isolates. Ta=annealing temperature; allele size range to (bp), NA=number of alleles, and N0 number of non-amplifying samples or null 124 alleles. Isolates tested are from the following regions: PB = Prydz Bay, ACC = Antarctic Circumpolar Current, AM & MS = Amundsen Sea & McMurdo Sound, WS = Weddell Sea.. 6.1. Sampling size details and Cruise information for the populations of P. antarctica 139. 6.2. Microsatellites reaction Mastermix. *labelled fluorescently with FAM/HEX. 140. 6.3. Microsatellite (MS) data sets used for calculations. 140. 6.4. Total number of isolates scored for each population and locus (N), number of 141 alleles (NA), number of effective alleles (NE), Information Index (I), observed heterozygosity (HO), expected heterozygosity (HE, genetic diversity), unbiased expected heterozygosity (UHE) and fixation index (F).. 6.5. Pairwise FST/RST values among the five P. antarctica populations for MS data 143 set a).. 6.6. Pairwise FST/RST values between the five P. antarctica populations for MS data 143 set f).. 7.1. Phaeocystis antarctica isolates used in salinity treatments.. 121. ii. 175.

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(16) List of Figures Fig. 1.1. page a) Overview showing the main ocean currents in the Southern Ocean: ACC 5 (Antarctic Circumpolar Current), thin white arrows close to the coast line show the Antarctic Coastal Current, Map modified after Rintoul et al. (2001) b) Scheme on Currents, zones & fronts in the Southern Ocean. Sea surface temperature (SST) is given (Smith et al. 2005).. 1.2. Southern Hemisphere 28-year average ice concentration maps for February 6 and September, the months of average minimum and maximum sea ice extents, respectively (after Cavalieri & Parkinson 2008). 1.3. Different P. antarctica morphotypes (developmental stages) a) flagellates, b) 9 new cell stage, c) overview of various colony stages, d) young colony, e) middle-sized colony and f) large colony. (Scale bar for a, b, d = 20 μm; c = 50 μm, e, f = 100 μm). 1.4. a) Maximum-likelihood phylogeny (fastDNAml) of 17 Phaeocystis 11 species/strains and other prymnesiophytes inferred from 18S rDNA. The class Pavlovophyceae was used as an outgroup. Bootstrap values are placed on the nodes that are identical from ML/NJ/MP analyses. The scale bar corresponds to two base changes per 100 nucleotides. b) Maximum-likelihood phylogeny of the ITS regions showing the multiple sequences from a single strain of P. globosa and P. pouchetii. Each different sequence comes from a different bacterial vector clone. All haplotypes from one strain of Phaeocystis are connected by the arrows. P. antarctica exhibited a single sequence per strain and these are collapsed into a triangle. Both redrawn from Lange et al. (2002) and Medlin & Zingone (2007).. 1.5. Workflow scheme showing how to generate AFLP markers (Vos et al. 1995, 14 Müller & Wolfenbarger 1999, Müller 2005). Description Box 1.. 1.6. Scheme on how to design a microsatellite marker. Clones from that library are 16 picked, sequenced, and primer pairs (red) spanning the microsatellites are designed.. 1.7. Overview of the Southern Ocean showing the sampling locations (light blue) 18 where P. antarctica isolates were obtained for this thesis. The ACC (grey area) and its current flow pattern (red arrows) are shown highly schematically.. 2.1. AA´s observed in field material from EIFEX (a-f), from ANT XXII/4 (g+h). 51 Light micrographs a-f were taken by P. Assmy and by g+h S. Gäbler-Schwarz.. 2.2. Temporal development of AAs on diatoms (epiphytic stage), flagellates and free-floating colonies of Phaeocystis antarctica inside and outside the fertilized patch during EIFEX.. 53. 2.3. AA´s observed in culture material. a-f: different developmental stages of AA´s. photos: a, e-i A. Davidson; b-d S. Gaebler-Schwarz. 55. iv.

(17) 2.4. Event frequency distribution of green DNA fluorescence of the three AAD strains. [DE12.2 (dashed line), KACTAS_A (solid line), T4.3 (dotted)].. 57. 2.5. Photo-sequence of an AA releasing flagellates (Four of eight shown in this graph).. 59. 2.6. Tree inferred by maximum likelihood analysis of combined ITS1 data of P. antarctica.. 61. 2.7. Life cycle scheme after Rousseau et al. (2007) extended by the fourth 63 morphotype found (AA=grey box).. 3.1. Schematic diagram showing the construction of the AFLP fragments (from 77 Mueller, 2005).. 3.2. AFLP electropherograms (resolution from 50-350 base pairs) of eight 79 different primer sets for P. antarctica A1-3 (Prydz Bay, Fig. 3.6).. 3.3. AFLP electropherograms (resolution from 0-500 base pairs) of the primer set 81 MseI+CT/EcoRI+AT for the P. antarctica isolates (Table 3.2).. 3.4. AFLP electropherograms (resolution from 0-500 base pairs) of the primer set 83 MseI+CT/EcoRI+AA for the P. antarctica isolates (Table 3.2).. 3.5. Most parsimonious phylogram of +/- presence of fragment lengths of the P. 84 antarctica isolates obtained from the primer set MseI+CT/EcoRI+AT. 3.6. Geographic origin of the four P. antarctica strains (Table 3.2). To indicate the 85 position of the Antarctic Circumpolar Current (ACC) encircling the Antarctic a schematic presentation was chosen (Lange et al. 2002, Olbers et al. 1962). [1: SK21, 2: SK22, 3: D5, 4: A1-3]. 4.1. The diagram illustrates possible workflows for microsatellite marker design in 103 the STADEN package using the STAMP extension modules.. 4.2. GAP4 dialog to configure microsatellite detection with PHOBOS. 4.3. Dialog to start the design of flanking primers for user defined tags present in a 107 GAP4 database. 4.4. Combined display of trace, sequence and tags. From top to bottom: table of 109 results from a flanking primer design process; contig editor window with tagged tandem repeat (green) together with a forward primer candidate (on the left); trace display of the corresponding sequence segment. 4.5. Top: Multiplex primer design dialog. Bottom: Table showing similarities 111 between primers and other contigs to detect possible cross-hybridizations. 4.6. Overview of SQLite entries and associated flanking primers. Top: Dialog to 113 load data from SQLite, listing available analysis results. Bottom: Table of tandem repeats and their flanking primers loaded from an SQLite database v. 105.

(18) 6.1. Sampling sites of P. antarctica in the Southern Ocean (Prydz Bay, diamond; 145 ACC, triangle; Amundsen Sea/Ross Sea, star; Scotia Sea/Weddell Sea, circle; Transit to Prydz Bay, square). Polar front systems according to Orsi et al. (1995) and Stewart (2007). STF=Subtropical Front, ACC=Antarctic Circumpolar Current, PF=Polar Front, SAF=Subantarctic Front. Arrowheads indicate the direction of the current.. 6.2. Allele length frequencies of eight microsatellite loci genotyped for the 147 populations of P. antarctica from Prydz Bay (Pop1 dark blue); ACC (Pop2 red); Amundsen Sea/Ross Sea (Pop3 green); Scotia Sea/Weddell Sea (Pop4 purple) and Transit to Prydz Bay (Pop5 light blue).. 6.3. Structure program calculation on MS data set (f) a) without prior, b) with 149 prior. 1=Pop1 (Prydz Bay), 2=Pop2 (ACC), 3=Pop3 (Amundsen Sea/Ross Sea), 4=Pop4 (Weddell Sea/Scotia Sea) and 5=Pop5 (Coastal Region). 6.4. UPGMA dendrogram of the five populations based on pairwise population 151 matrix of Nei unbiased genetic distance (Nei’s 1972) for a) MS data set (a) and b) MS data set (f).. 6.5. Current system in the Southern Ocean (redrawn from Rintoul et al. 2001). 153 White arrows indicate the Antarctic Coastal Current, acting as counter-current of the ACC.. 7.1. Dendrogram of Phaeocystis antarctica isolates from the ACC (=dark grey 177 box), Weddell Sea (=marmoured box), Scotia Sea (=white box) and from Prydz Bay (=light grey box), visualized by brackets, obtained by AFLP technique. The dendrograms were constructed by using UPGMA (PAUP), the ACC sample (SK22) was used as an outgroup. Isolates used in the physiological experiments are labeled with boxes (PrydzBay_1; PrydzBay_2; ScotiaSea_1 and ScotiaSea_2). 7.2. a) Map of sample region of the Phaeocystis antarctica isolates used in 179 physiological experiments (boxed=genetically close isolates; also see Table 1), b) Ocean currents around Antarctica ACC= Antarctic Circumpolar Current, [a) The grounding and coastlines are taken from MODIS Mosaic of Antarctica (MOA) (Haran et al. 2006), b) Map modified after Rintoul, Hughes and Olbers, 2001. ESRI world map, Bathymetry GEBCO_08 Grid sampled on 10km x 10km raster.]. 7.3. Microscopic overview of exponential growth phase in all three salinities used 181 for the five different Phaeocystis antarctica strains during the experiment. Except of PrydzBay_1 (18psu, day 8), RossSea_1 (18psu, day 36), ScotiaSea_1 (18psu, day 16) and PrydzBay_1 (70psu, day 36), all photos are from day 12 of the experiment. Photos: F. Hinz and S. Gäbler-Schwarz. 7.4. Box plot of Chl a measurements of the five Phaeocystis antarctica isolates in 183 the different salinity regimes generated with R.. 7.5. Box plot of PAM measurements of the five Phaeocystis antarctica isolates in 185 the different salinity regimes generated with R. vi.

(19) 7.6. Box plot of DMSP measurements of the five Phaeocystis antarctica isolates 187 in the different salinity regimes generated with R.. 7.7. a) Map of Phaeocystis antarctica sample origin , b) Mean 1979 – 2007 sea ice 189 coverage January, c) Mean 1979 – 2007 sea ice coverage July [a) The grounding and coastlines are taken from MODIS Mosaic of Antarctica (MOA) (Haran et al. 2006), b-c) Sea ice trends taken from Stroeve and Meier (1999, updated 2008).]. 8.1. Microscopic overview of growth of the two Prydz Bay isolates in 18 psu. a-d) 209 isolate PrydzBay_1 on days 4, 8, 16 and 28, e-h) isolate PrydzBay_2 on days 4, 8, 16 and 28. PrydzBay_1 is genetically close to ScotiaSea_1. Photos: F. Hinz and S. Gäbler-Schwarz. 8.2. Microscopic overview of growth/death of the two Scotia Sea isolates in 18 211 psu. a-c) isolate ScotiaSea_1 on days 4, 8, and 16, c-e) isolate ScotiaSea_2 on days 4, 8, and 16. PrydzBay_1 is genetically close to ScotiaSea_1. Photos: F. Hinz and S. Gäbler-Schwarz. 8.3. Microscopic overview of growth of the two Prydz Bay isolates in 33 psu. a-d) 213 isolate PrydzBay_1 on days 4, 8, 16 and 28, e-h) isolate PrydzBay_2 on days 4, 8, 16 and 28. PrydzBay_1 is genetically close to ScotiaSea_1. Photos: F. Hinz and S. Gäbler-Schwarz. 8.4. Microscopic overview of growth of the two Scotia Sea isolates in 33 psu. a-d) 215 isolate ScotiaSea_1 on days 4, 8, 16 and 28, e-h) isolate ScotiaSea_2 on days 4, 8, 16 and 28. PrydzBay_1 is genetically close to ScotiaSea_1. Photos: F. Hinz and S. Gäbler-Schwarz. 8.5. Microscopic overview of growth of the two Prydz Bay isolates in 70 psu. a-d) 217 isolate PrydzBay_1 on days 4, 8, 16 and 28, e-h) isolate PrydzBay_2 on days 4, 8, 16 and 28. PrydzBay_1 is genetically close to ScotiaSea_1. Photos: F. Hinz and S. Gäbler-Schwarz. 8.6. Microscopic overview of growth/death of the two Scotia Sea isolates in 70 219 psu. a-c) isolate ScotiaSea_1 on days 4, 8, and 16, c-e) isolate ScotiaSea_2 on days 4, 8, and 16. PrydzBay_1 is genetically close to ScotiaSea_1. Photos: F. Hinz and S. Gäbler-Schwarz. vii.

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(22) Summary The objective of this thesis was to estimate the genetic diversity of Phaeocystis antarctica and to correlate it with its physiological diversity. To conduct this study, the largest P. antarctica culture collection worldwide was assembled. Our final collection contains 210 isolates and is, to our knowledge, a unique collection of P. antarctica strains and of great scientific interest. To analyse the population structure of P. antarctica and to study the genetic diversity inside populations from different locations and the gene flow between them, two fingerprinting techniques were used. Amplified Fragment Length Polymorphisms (AFLPs) were used as a pre screening tool in the beginning of this study, but later rejected because it needed axenic DNA material for amplification. Thus, DNA from 34 axenic cultures as well as DNA from earlier projects obtained from Weddell Sea, Prydz Bay and the Antarctic Circumpolar Current (ACC) was investigated. Results showed that some isolates from Weddell Sea/Scotia Sea were closely related to Prydz Bay isolates showing that the two oceanographic regimes were not isolated from each other genetically. To test if P. antarctica shows distinct population structures in Antarctica and to estimate the magnitude of gene flow between the populations, an analysis of genetic diversity using microsatellites was conducted. Eight species specific microsatellite markers for P. antarctica were developed by screening a microsatellite enriched library established using chromosomal DNA from P. antarctica. Nearly 100% of the sequenced clones contained microsatellites. The STAMP package, an improved software tool, was used to design primer pairs to regions flanking repeat units. This new software allowed 1) to improve repeat detection using the state of the art repeat detection software, 2) to separate repeat detection for assembly and for marker design and 3) to permit interactive primer design allowing manual improvements if results generated automatically are judged unsatisfying. This pipeline provides the possibility to analyze data quickly, which is an improvement over existing software. With the primers developed, a high number of isolates (110) were genotyped for five geographical Antarctic regions: Prydz Bay, ACC, Amundsen Sea/Ross Sea, Weddell Sea/Scotia Sea and Coastal region (Transit from Weddell Sea to Prydz Bay area). Microsatellites were highly polymorphic in all five populations. The number of alleles per microsatellite locus for all isolates screened, ranged from Na=5 (LOC7) to Na=25 (LOC2). Observed heterozygosity (HO) ranged from 0.250 (LOC1, Pop4) to 1.000 (LOC2, Pop3; LOC5, Pop2&5; LOC8, Pop4). The length range of alleles was extremely broad for locus LOC7 and locus LOC8 (70 bp to 404 bp) indicating that other mutations than simple repeat unit extensions have contributed to the extension. P. antarctica populations were not strictly isolated in the oceanic regimes they x.

(23) inhabit and the ACC provides a dispersal mechanism among populations to all other oceanographic regimes of the Southern Ocean. But these regimes still maintain “their own genetically distinct” populations. To test whether genetically closely related strains originating from different geographic regions in the Southern Ocean would react similarly to changes in environmental conditions or whether the environment would set physiological constraints (phenotype) on the genotypes, the salinity tolerance of five different isolates from three ecologically different locations was tested. The specific responses reflected the geographical origin of the tested isolates. Genetically close isolates from different regions did not respond similarly to conditions tested, whereas isolates from the same region, although genetically distant, reacted similarly. Thus, certain ecotypic responses predominate over genotypic differences in each geographic region sampled. Each oceanographic regime contained a highly diverse population that ensured adaptive respond mechanisms to specific environmental parameters of a given ecosystem.. In addition to the insights gained from these studies, a new cell stage of P. antarctica was discovered, which has been interpreted to be a zygote thus completing the unresolved sexual stage of the haplo-diploid life cycle of this species., The zygote functions either as a refuge from grazers, or as an overwintering stage. Further studies are required to determine the significance of this new cell stage in the ecology of P. antarctica.. This thesis provides a detailed insight into genetic diversity among and between P. antarctica populations from different regions of the Southern Ocean and the gene flow between them. Genetic, physiological and ecological results are discussed in a broader view giving valuable insights into the ecology of this keystone species in the Southern Ocean. This work is opening a new field of research on P. antarctica, which can be termed 'ecophysiological genetics'. By combining genetic data with results from ecological investigations and physiological experiments, a broader understanding of ecosystem functioning will also direct future studies.. xi.

(24) Zusammenfassung Zielsetzung dieser Arbeit war die Bestimmung der genetischen Diversität von Phaeocystis antarctica aus dem Südpolarmeer und außerdem diese genetische Variabilität mit ihren physiologischen Eigenschaften zu korrelieren. Zur Durchführung dieser Studie wurde die bisher weltweit umfangreichste Kulturensammlung von Phaeocystis antarctica erstellt. Sie enthält heute 210 Isolate und stellt eine einzigartige Sammlung von Stämmen dieser Mikroalge dar. Diese Sammlung ist deshalb von großem wissenschaftlichem Interesse. Mithilfe der Bestimmung der genetischen Diversität und des Genflusses unter-, sowie innerhalb von P. antarctica Populationen verschiedenen Ursprungs im Südpolarmeer, kann die Populationsstruktur dieses ökologisch wichtigen Phytoplanktonorganismus analysiert werden. Um populationsgenetische Untersuchungen durchzuführen, wurden molekulare Marker zur Herstellung von sogenannten genetischen Fingerabdrücken etabliert und entwickelt. Eine dieser molekularbiologischen Methoden ist die Amplified Fragment Length Polymorphisms, kurz ALFPs. AFLP wurde zum Beginn der Arbeit als Werkzeug zur Vorauswahl genutzt, später aber verworfen, da nur axenisches DNA-Material zur Durchführung genutzt werden konnte. 34 Kulturen, sowie Probenmaterial aus vorhergegangenen Projekten, welche aus dem Weddellmeer, dem Scotiameer, der Prydz Bay und aus dem Antarktischen Zirkumpolarstrom (ACC) isoliert worden waren, wurde mittels AFLPs untersucht. Die Isolate aus dem Weddelmeer und dem Scotiameer zeigten nicht nur genetische Ähnlichkeiten zu ihrem Ursprungsort, sondern waren ebenfalls genetisch ähnlich zu Isolaten aus der Prydz Bay. Dies zeigt, dass die P. antarctica Populationen in diesen Gebieten nicht genetisch voneinander isoliert zu sein scheinen. Mit der Entwicklung von acht artspezifischen Mikrosatelliten wurden weiterere genetische Marker zur Analyse der Populationsstruktur von P. antarctica in der Antarktis eingeführt, welche unabhängig vom Reinheitsgrad des Probenmaterials spezifisch eingesetzt werden können. Eine mit Mikrosatelliten angereicherte Klonbank (microsatellite enriched library) wurde für anhand von chromosomaler DNA von P. antarctica angelegt. Zur schnelleren Analyse der daraus sequenzierten Klone konnte das verbesserte Software Tool STAMP benutzt werden, mit dem Mikrosatelliten in den Sequenzen schnell detektiert und automatisch Primer zu den flankierenden Sequenzbereichen entwickelt werden konnten. Diese neue Programmerweiterung erlaubt eine schnelle und zügige Verarbeitung großer Datenmengen. Eine große Anzahl der vorhandenen Kulturen aus fünf geographischen Regionen in der xii.

(25) Antarktis wurde mit den acht neu entwickelten Mikrosatelliten untersucht. Hierbei zeigte sich, dass alle der acht Marker hoch polymorph sind. Die Anzahl der Allele pro Locus variierte für alle untersuchten Isolate von Na=5 (LOC7) zu Na=25 (LOC2). Die beobachtete Heterozygosität (HO) variierte ebenfalls von 0.250 (LOC1, Pop4) zu 1.000 (LOC2, Pop3; LOC5, Pop2&5; LOC8, Pop4). Die Fragmentlängen der Allele waren besonders divers bei zwei der untersuchten Mikrosatelliten, LOC7 und LOC8, was darauf zurückzuführen sein könnte, dass andere Mutationen neben den zu erwartenden einfachen Repeats diese Längenunterschiede zu verantworten hatten. Die Untersuchungen zeigten, dass die P. antarctica Populationen nicht strikt in ihren Ursprungsorten isoliert vorkamen und dass die ACC eine Verbreitung in andere Gebiete fördern könnte. Dennoch beherbergen die jeweiligen Gebiete ihre eigenen genetisch distinkten Populationen. In einem physiologischen Experiment wurde die Salzgehaltstoleranz von fünf verschiedenen Phaeocystis antarctica Isolaten, die aus drei ökologisch unterschiedlichen Gebieten in der Antarktis stammten, untersucht. Es sollte getestet werden, ob Stämme, die genetisch nah sind, gleich reagieren, oder, ob die physiologischen Bedingungen des jeweiligen Lebensraums, aus dem sie isoliert wurden, die Reaktion auf unterschiedliche Salzgehalte bestimmt. Die Ergebnisse zeigen, dass hier die geographische Herkunft ausschlaggebend für die jeweiligen Wachstumsreaktionen war. Isolate, die sich genetisch nah standen, aber aus verschiedenen Gebieten kamen, reagierten unterschiedlich. Dagegen reagierten Isolate, die genetisch weit voneinander entfernt waren, sehr ähnlich auf die unterschiedlichen Salzgehalte im Experiment. Daher scheinen ökologische Faktoren die Physiologie von P. antarctica stärker zu prägen als ihren genetischen Unterschied. Jedes der ozeanographischen Gebiete im Südozean beherbergt eine hoch diverse P. antarctica Population, welche mit ihren gegebenen Möglichkeiten sich an spezifische Umweltbedingungen anpassen kann. Außerdem konnte während dieser Arbeit ein neues Zellstadium für P. antarctica entdeckt werden. Es wird angenommen, dass es sich hierbei um eine Zygote handeln könnte, die das fehlende sexuelle Stadium des bisher ungeklärten haplo-diploiden Lebenszyklus darstellen könnte. Die Bedeutung einer solchen Zygote kann einerseits ein Schutz vor Wegfraß sein, da sie vorwiegend an großen Diatomeenschalen haftend im Pelagial gefunden wurde, oder, andererseits als ein Überwinterungsstadium dienen. Weitere Untersuchungen sind notwendig, um die Bedeutung dieses neu entdeckten Zellstadiums für die Ökologie von P. antarctica im Südozean zu verstehen.. xiii.

(26) Die vorliegende Arbeit liefert einen detailierten Einblick über die genetische Diversität und vorkommenden Genfluss unter- und innerhalb von Populationen der ökologisch wichtigen Mikroalge P. antarctica im Südpolarmeer. Genetische, physiologische und ökologische Ergebnisse werden diskutiert, um die Lebensweise dieser antarktischen Schlüsselart im Pelagial des Südpolarmeeres besser zu verstehen. Diese Arbeit eröffnet ein neues Forschungsfeld, das ökophysiologische Genetik bezeichnet werden könnte. Diese Arbeit zeigt weiter, dass tiefere Einblicke und Direktiven für weitere Forschungsvorhaben durch die Kombination von Genetik, ökologischen Untersuchungen gekoppelt mit physiologischen Experimenten ermöglicht werden könnten.. xiv.

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(29) . . General introduction.

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(31) Chapter 1 Southern Ocean There are many unique ecosystems around the world, but none is as diverse and still undiscovered as the world’s ocean. It covers 70% of the earth surface and is divided into five major oceans. One of them, the Southern Ocean (SO), is known to be one of the most stable but also most harsh environments on earth playing a disproportionately important role in climate control in both the past and the present (Sarmiento et al. 1998, Broecker & Henderson 1998, Moore et al. 2000, Boyd 2002). The Southern Ocean exhibits a unique oceanographic regime as evidenced from its current pattern (Fig. 1.1a). Because of the circumpolar character of its main current, the Antarctic Circumpolar Current (ACC), the Southern Ocean is climatically isolated and characterised by low surface temperatures and a strong seasonality in light regime and sea ice coverage. Albeit climatically isolated, the Southern Ocean plays a central role in the global ocean conveyor belt since it connects the Atlantic, Pacific and Indian Oceans and therefore constitutes a major driver of heat and mass transport across the ocean basins. The ACC is driven partly by the vigorous mid-latitude westerly winds and affected by adjacent landmasses and submarine topography. It is the main current, transporting a water volume of 130 Sv (1 Sverdrup = 1 Sv = 1x106 m3 s-1) along a 24 000 km path, varying in depth and width (Rintoul et al. 2001, Boning et al. 2008, Thompson 2008). Close to the Antarctic continent, the Antarctic Coastal Current, a counter-current to the ACC, flows westward parallel to the Antarctic coastline (Gyory et al 2003). Between these current systems, hydrographical circulations form well defined gyres, the two largest being the Weddell Gyre and the Ross Gyre (Fig. 1.1a). Most of the macronutrients upwelled at the Antarctic Divergence are returned unused to the ocean`s interior with Antarctic Intermediate Water (AAIW) at the northern rim of today’s Southern Ocean with the exception of silicic acid which is sequestered as biogenic silica in Circumpolar Deep Water (CDW) and the sediments surrounding Antarctica (Levitus et al. 1993, Treguer et al. 1995 Boyd 2002). Despite this abundant availability of nutrients, chlorophyll levels are low throughout most of the ACC. This apparent paradox can be explained by the lack of the essential micronutrient iron that limits phytoplankton growth rates in the land remote Southern Ocean. The Southern Ocean is thus termed a High Nutrient Low Chlorophyll (HNLC) region (Hense et al. 2003). Despite its iron limited character the Southern Ocean is the largest source of dimethyl sulphide (DMS), a potential climate gas affecting indirectly, via clouds, the global albedo (Kettle et al. 1999).. 3.

(32) Chapter 1 Ocean boundaries in the Southern Ocean, caused by the current patterns, generate specific Antarctic oceanographic regimes, such as the ACC system and the gyres. The effects of the water mass movements are greater on smaller organisms than on larger ones. Small organisms, such as phytoplankton, can have a distribution largely shaped by water circulation and thus a circulation related gene flow (Patarnello et al. 1996). The role of the oceanic currents in the dispersal and distribution of Antarctic marine organisms has been investigated for a long time. However, gene flow around the Antarctic continent and between gyres is documented only in a few cases (Marr 1962; Amos 1984; Deacon 1984, Patarnello et al. 1996, Zane et al. 1998, Rogers et al. 1998, Bargelloni et al. 2000, Copley & Young 2006, Copley et al. 2007). However, especially the Southern Ocean, because of its unique current system and its ecological importance, offers the opportunity to gain valuable insight into the gene flow of phytoplankton. A good example of this process will be presented in this thesis with the prymnesiophyte Phaeocystis antarctica.. 4.

(33) b. Ocean. Sea surface temperature (SST) is given (Smith et al. 2005).. coast line show the Antarctic Coastal Current, Map modified after Rintoul et al. (2001) b) Scheme on Currents, zones & fronts in the Southern. Fig. 1.1 a) Overview showing the main ocean currents in the Southern Ocean: ACC (Antarctic Circumpolar Current), thin white arrows close to the. a.

(34) Chapter 1 Sea ice, temperature and light Antarctic sea ice is an important factor in the global climate system and exhibits a great seasonal variation (King & Turner 1997, DeLiberty et al. 2004). The sea ice coverage in the Southern Ocean can reach up to approximately 60° S during austral winter and drastically retreats southward in summer. Thus in winter, up to 20 million square kilometres are covered by sea ice compared to only about 4 million square kilometres in summer (http://nsidc.org/seaice/characteristics/difference.html, Fig. 1.2). In the Southern Ocean, sea surface temperatures (SST, Figure 1.1b) range from 5°C in the northern ACC to –1.86°C further south. In sea ice, lowest temperatures are found in brine channels and pockets ranging from –2 to below –20°C.. Fig. 1.2 Southern Hemisphere 28-year average ice concentration maps for February and September, the months of average minimum and maximum sea ice extents, respectively (after Cavalieri & Parkinson 2008) Sea surface salinities are relatively stable varying from 33.5 to 34.9 psu (Smith et al. 2005) in the open Southern Ocean compared to very high salinity fluctuations in sea ice covered regions with up to 200 psu within the sea ice as a function of temperature and sea ice freezing. The highest salinities can be found in the brine system of the Antarctic sea ice (Thomas & Dieckmann 2003). Only organisms adapted to such a wide range in salinity and temperature can live in such an environment. The light regime is also rather extreme with no or little light in winter and continuous light during the summer months, especially in the far south. Thus, photoperiods are highly variable, showing wide ranges in photon flux densities (Tang et al. 2009). 6.

(35) Chapter 1 Southern Ocean regimes The Southern Ocean harbours a great diversity of life. Ocean boundaries may limit exchange of subpopulations and therefore support the development of subspecies in the different biological provinces or oceanographic regimes of the Southern Ocean. These oceanographic regimes also offer the possibility of establishing genetically distinct populations within a species. Among the primary producers, diatoms are generally regarded as the main contributors for biomass build-up in the Southern Ocean. However, investigations carried out during the last 25 years have revealed, that nanoflagellates as well as eukaryotic picoplankton contribute significantly to biomass and primary production. Within the group of nanoflagellates the prymnesiophyte P. antarctica dominates the phytoplankton standing stock in certain regions of the Southern Ocean and thus contributes significantly to primary produced biomass in the Antarctic pelagic system (e.g. Arrigo et al. 1999, Nöthig et al. 2009).. The genus Phaeocystis The genus Phaeocystis plays a crucial role in the ecology and biogeochemistry of almost all marine ecosystems. Species of the genus share the ability to produce nearly monospecific blooms of large colonies that have a flexible but tough skin, in several coastal and oceanic waters (Schoemann et al. 2005). It is a nanoflagellate that contributes approximately 10% of annual global marine primary production. Several Phaeocystis species have been identified world-wide, but only three of the described species have been documented to form colonies and dominate blooms (P. pouchetii, P. globosa and P. antarctica) (Medlin et al. 1994). Two species are dominant in Polar Regions: P. antarctica Karsten in the Southern Ocean and P. pouchetii Scherffel in the Arctic Ocean. P. antarctica can form huge blooms in seasonal ice zones and coastal Antarctic waters (El-Sayed et al. 1993). Blooms of P. antarctica have been shown to dominate both in deep (Arrigo et al. 1999) and shallow mixed layers in the Southern Ocean (Bodungen et al. 1986). This illustrates the ability of this species to adapt to varying light regimes. Only relatively few species play such fundamental roles in the trophic structure and biogeochemical cycles of the Southern Ocean (Smetacek et al. 2004). Its peculiar physiology can profoundly influence tropho-dynamics, community composition and biogeochemical carbon and sulphur cycles in the Southern Ocean (Davidson & Marchant 1992). P. antarctica produces high amounts of dimethylsulfoniopropionate (DMSP), which can act as cryoprotectant in algae (Kirst et al. 1991, Karsten et al. 1996, Stefels 2000), serve as an antioxidant system (Sunda et al. 2002) or possibly maintain intracellular osmotic pressure (Dickson et al. 1982, Vairavamurthy et al. 1985, Dickson & Kirst 1986, 1987a, 7.

(36) Chapter 1 1987b), but also deter herbivores (Wolfe et al. 1997). Whether Phaeocystis is grazed down by herbivores zooplankton has been a point of discussion for decades and is as yet unresolved. The single celled flagellates may serve as food for smaller organisms such as ciliates within the microbial loop. Colonies can provide nutrition for larger zooplankton. But especially the hatching success of the grazer community that feed on Phaeocystis is discussed controversially (see i.e. Turner et al 2002). Despite P. antarctica´s pivotal role in pelagic ecosystems, little is known about its life cycle and sexual reproduction. Four morphotypes have been proposed for P. antarctica: colonial cells, two types of flagellates and one attached aggregate, which develops after the fusion of two scaled haploid flagellates forming a zygote (Fig 1.3, Rousseau et al. 2007, Chapter 2 Gäbler-Schwarz et al. subm.). The existence of a haploid-diploid lifecycle is well-supported for P. globosa and should exist within the other colonial Phaeocystis species (Rousseau et al. 2007, Medlin and Zingone 2007), because haploid-diploid life cycles are common in most other haptophytes in the class Coccolithophyceae (Edvardsen et al. 2000).. 8.

(37) Chapter 1. Fig. 1.3 Different P. antarctica morphotypes (developmental stages) a) flagellates, b) new cell stage, c) overview of various colony stages, d) young colony, e) middle-sized colony and f) large colony. (Scale bar for a, b, d = 20 μm; c = 50 μm, e, f = 100 μm). In the past, the morphology of the genus Phaeocystis has led to much taxonomic confusion assigning a species name to the similar colony stages (Sournia 1988). Traditional methods for taxonomy, such as microscopy for phytoplankton, need high expertise and proper identification of phytoplankton smaller than 20 μm is often impossible. Furthermore the lack 9.

(38) Chapter 1 of morphological characters makes the delimitation of species difficult for most microalgae (Müller 2005). Therefore molecular techniques have become a valuable additional tool during the past decades to study the genus Phaeocystis by providing the possibility to identify phytoplankton even down to species level, regardless of their sizes and developmental stages.. Molecular methods Molecular biology offers a broad toolbox of techniques that address different questions in ecology and evolution (de Bruin 2003), such as taxonomic affiliation, genetic diversity, gene flow or dispersal (Medlin 2003). Thus it is critical to choose the appropriate molecular technique for the research questions to be addressed. In the past, the global distribution of the genus Phaeocystis was unravelled taking advantage of three genetic markers, the nuclearencoded 18S rDNA genes and two non-coding regions – the internal transcribed spacer 1 (ITS1) and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO). However, only 18S and ITS1 were informative (Lange et al. 2002a, 2000b). Those phylogenetic analyses have given valuable information about the systematic relationships of taxa/species within the genus Phaeocystis. Furthermore they supported the hypothesis, that ancestral populations of Phaeocystis in Antarctica gave rise to present day polar P. antarctica and P. pouchetii populations. These two species within the genus are more closely related to each other than either is to warm temperate or tropical populations of present day P. globosa as supported in all the analyses from the 18S and ITS1 regions. Molecular clock calculations from 18S rDNA and the fossil record from the haptophyte coccolithophorid species indicate that the divergence of the warm water from the cold water Phaeocystis species coincided with the opening of the Drake passage and thus with the formation of the ACC approximately 30 MY ago (Figure 4, Medlin et al. 1994, Lange et al. 2002a, b, Medlin & Zingone 2007).. 10.

(39) Pavlova CCMP 1394 Pavlova gyrans Chrysochromulina throndsenii Chrysochromulina scutellum 100/100 Chrysochromulina strobilis 100/100 90/93 Chrysochromulina campanulifera -/58 76/Chrysochromulina leadbeaterii Imantonia rotunda 98/94 Chrysochromulina chiton Prymnesium parvum 83/87 70/84 Chrysochromulina brevifilum 98/100 Chrysochromulina ericina 93/91 Coccolithus pelagicus 80/97 Reticulosphaera japonensis Pleurochrysis carterae 70/81 -/45 Isochrysis galbana 100/100 Emiliania huxleyi 97/99 Phaeocystis sp.PLY 559 Clade 1 100/100 Phaeocystis jahnii Phaeocystis cordata 100/100 75/65 Phaeocystis antarctica Phaeocystis antarcticaACC Phaeocystis antarctica 98/100 Phaeocystis antarctica Phaeocystis antarctica 97/100 Phaeocystis pouchetii P360 Phaeocystis pouchetii SK34 99/100 Clade 2 Phaeocystis globosa North Sea Phaeocystis globosa South Africa Phaeocystis globosa Surinam 99/100 Phaeocystis globosa Gulf of Mexico Phaeocystis globosa Thailand Phaeocystis globosa Eastern Atlantic Phaeocystis globosa Galapagos. 95/100. Pavlova cf. salina Pavlova CCMP1416. b). redrawn from Lange et al. (2002) and Medlin & Zingone (2007).. strain of Phaeocystis are connected by the arrows. P. antarctica exhibited a single sequence per strain and these are collapsed into a triangle. Both. from a single strain of P. globosa and P. pouchetii. Each different sequence comes from a different bacterial vector clone. All haplotypes from one. bar corresponds to two base changes per 100 nucleotides. b) Maximum-likelihood phylogeny of the ITS regions showing the multiple sequences. The class Pavlovophyceae was used as an outgroup. Bootstrap values are placed on the nodes that are identical from ML/NJ/MP analyses. The scale. Fig. 1.4 a) Maximum-likelihood phylogeny (fastDNAml) of 17 Phaeocystis species/strains and other prymnesiophytes inferred from 18S rDNA.. a). 2%. 100/100. 55/85. non-colony forming cells colony forming cells.

(40) Chapter 1 The molecular clock calculations were also supported by ITS1 data of the two cold water Phaeocystis species P. antarctica and P. pouchetii. They were more closely related to each other as to the warm water species P. globosa. All P. antarctica strains sampled showed only a single type of ITS sequence in each strain. In contrast P. pouchetii and P. globosa showed multiple ITS variants. The similarity among ITS sequences from Antarctic isolates indicates that P. antarctica could be a single species rather than a species complex as considered for P. pouchetii and P. globosa. Despite its ecological importance in the Southern Ocean there is still very little information on the genetic structure of P. antarctica populations. The genetic markers used reached their limit of phylogenetic resolution. In order to gain deeper insights into P. antarctica’s life cycle and population structure, highly polymorphic nuclear genetic markers, such as amplified fragment length polymorphisms (AFLP) and microsatellites, are needed. These methods have the potential to provide intraspecific information and the resolving power to distinguish rates of migration and can estimate the relatedness of individuals within a population.. Genetic diversity is any variation in the nucleotides, genes, chromosomes or whole genomes of organisms. It can be measured at many different levels, is a fundamental component of biodiversity and encompasses all of the genetically determined differences that occur between individuals of a species. Genes create genotypes and genotypes in turn compose populations that collectively belong to species. Finally, ecosystem diversity derives from species diversity (Bagley et al. 2002, Reusch & Hughes 2006). Thus, in order to assess genetic diversity within populations the most often used fingerprinting techniques are AFLPs and microsatellites (Chavarriaga-Aguirre et al. 1999, Mariette et al. 2001, De Bruin et al. 2003, Lamote et al. 2005).. Amplified Fragment Length Polymorphism (AFLP) AFLPs rely on the restriction enzyme digestion of whole genomic DNA followed by the PCR amplification of these fragments using special oligonucleotide adapters to the DNA fragments to generate specific fingerprint patterns (Fig. 1.5, Box 1 AFLP in detail). This method offers a lot of advantages, e.g. there is no need for sequence data to design primers, we find random distribution of the fragments throughout the genome, and it is stable and reproducible (Vos et al. 1995, Wang et al. 2007). AFLP is based on the digestion of DNA with restriction enzymes. The fragments obtained are ligated to specific adaptors which act as target sites for primer annealing in subsequent polymerase chain reaction (PCR) steps. Therefore, regardless 12.

(41) Chapter 1 whether axenic cultures or field material are used, AFLP generates a fingerprint pattern consisting of all the different organisms present in a respective sample. In a field sample or contaminated culture all fragments would get amplified, resulting in a false fingerprint. Because of this, AFLPs are not reliable for field samples or contaminated cultures, because the resulting fragments cannot be assigned with certainty to the organism in study or to the contamination (Müller et al. 2005). In regard to this high sensitivity of AFLPs, only axenic microalgal cultures should used. But there are exceptions; Müller et al. (2005) performed AFLP on a wildtype strain and mutants of Dunaliella salina that were contaminated with bacteria and/or fungi. They showed that if the same contamination is present in all samples, then it is actually possible to use AFLP. But field samples, especially from the marine environment, would still be problematic. In comparison to microsatellite markers (described below) AFLPs can reveal higher diversity in recently diverged populations, because they show polymorphic genetic loci over the entire genome (Alpermann et al. 2009). Box 1: Explanation AFLP work flow (a) AFLP template preparation: 1. Isolation of whole genomic DNA. 2. We need two restriction enzymes and 3. Adaptors. (b) Digestion overnight with the two restriction enzymes. This combination consists of a rare cutter (EcoRI – six base pair recognition sequence) and a frequent cutter (MseI – four base pair recognition sequence). Subsequent to the restriction, restriction site-specific adapters (EcoRI-adaptor, MseI-adaptor) are ligated to the ends of the digested fragments. (c) One to six additional nucleotides can be added to the primer set consisting of the adapter and restriction site sequence as target sites for primer annealing (Vos et al. 1995) to amplify a subset of DNA fragments. With two additional PCR steps, the preselective and the selective amplification, only a specific subset of fragments is amplified, so that the number of the resulting fragments provides sufficient resolution among the strains. (d) To be able to visualize the amplification products (electropherogram), one primer (EcoRI) is fluorescently-labelled for the second amplification step.. 13.

(42) Chapter 1. Fig. 1.5 Workflow scheme showing how to generate AFLP markers (Vos et al. 1995, Müller & Wolfenbarger 1999, Müller 2005). Description Box 1.. 14.

(43) Chapter 1 Microsatellites Microsatellites have been applied to population genetic studies in a wide range of organisms (Weber & May 1989, Hughes & Queller 1993, Lagercrantz 1993, Ostrander et al. 1993, Schlotterer & Pemberton 1994, Jarne & Lagoda, 1996, Roy et al. 1994, Scribner et al. 1994, Wattier et al. 1997, Goldstein & Schlotterer 1999, Procaccini et al. 2001). Microsatellites are codominantly (= alleles are equally strong) inherited markers consisting of repeating units (simple sequence repeats). Due to the often great number of alleles per locus, microsatellite show very high levels of heterozygosity. Heterozygosity, the proportion of individuals with two alleles at a locus (heterozygous) (for a diploid specimen), is a common measure of genetic diversity and might be beneficial in a changing environment. A high degree of heterozygosity means high genetic diversity. In contrast, if heterozygosity is low it indicates low genetic diversity. Microsatellite analysis involves the assessment of the observed (HO) and the expected (HE) level of heterozygosity. If HO > HE there might have been an isolatebreaking effect (the mixing of two previously isolated populations). If HO < HE then e.g. inbreeding could have been involved or the sampled population consists in fact of two or more cryptic populations (Hamilton 2009). Because microsatellites are not universal, they must be developed for each species of interest. For this, microsatellite enriched libraries have to be constructed. Therefore DNA is digested with a restriction enzyme and subsequent to the restriction, a restriction site-specific adapter is ligated to the ends of the digested fragments. After PCR with a primer set, consisting of the adapter and restriction site sequence as target sites for primer annealing, the PCR product gets purified. Two biotinylated microsatellite oligonucleotides Bio-GA and Bio-GT (Thermo Electron GmbH, Germany) are immobilized on magnetic beads (Dynabeads, Dynal Biotech Invitrogen) and are used to bind and capture CT and CA repeats in restriction fragments to which oligonucleotide adapters have been ligated. A second round of enrichment achieves higher amounts by re-capturing postamplification Dynabead products and repeating all subsequent steps in the protocol (according to Evans et al. 2004). Enrichment PCR fragments are then cloned into Escherichia coli. This established microsatellite enriched clone library from the species DNA of interest is now used for generating microsatellite markers. Clones from that library are picked, sequenced and primer pairs spanning the microsatellites are designed (Fig. 1.6). For this purpose several freeware tools can be used, such as STAMP or PRIMER3 (Kraemer et al. 2009, Rozen & Skaletsky 2000). These primer sets are then tested on the species of interest (Edwards et al. 1996, Evans et al 2004).. 15.

(44) Chapter 1. Sequenced clone Flanking region. 5’-. ATCGGCCAGTGC. 3’-. TAGCCGGTCACG -5’. Core sequence(repeat). New primer designed. Flanking region. GTCTTGGAACTG. -3’. 3’- CAGAACCTTGAC. -5’. New primer designed. Fig. 1.6. Scheme on how to design a microsatellite marker. Clones from that library are picked, sequenced, and primer pairs (red) spanning the microsatellites are designed.. 16.

(45) Chapter 1 Aim of the thesis The distribution of P. antarctica varies throughout different regions of the Southern Ocean. This study was designed to resolve the population structure of P. antarctica and estimate genetic diversity among populations from different locations. Previous phylogenetic (18S, ITS1) analyses have given valuable information to the systematic relationships of taxa/species within the genus Phaeocystis. They exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the strains. Among nine P. antarctica strains, only one type of ITS1 was found per strain, and therefore P. antarctica was genetically differentiated as a distinct species. Populations of P. antarctica within continental boundary water masses around Antarctica appear to be well-mixed because they are transported by currents around the Antarctic continent, which may prevent an isolation of populations (Medlin & Zingone 2007). One objective was to establish a large culture collection consisting of a large number of clonal P. antarctica isolates from each of the major gyres in the Southern Ocean in order to have statistically reliable estimates of genetic diversity. To estimate the genetic diversity and the gene flow between the different samples taken for this study another objective was to establish and develop AFLP and microsatellite markers.. The main research questions are: •. How high is genetic diversity within P. antarctica in the Antarctic Region?. •. Are bloom-forming populations composed mainly of one clone or do we find genetically different individuals?. •. Are the distinct P. antarctica populations around the Antarctic continent isolated from one another or does the ACC provide a vehicle for dispersal among populations in all other oceanic regimes of the Southern Ocean?. •. If so, what is the magnitude of gene flow around the Antarctic continent?. •. Do genetically closely related strains originating from different geographic regions in the Southern Ocean react similarly to changes in environmental conditions or is the environment more important to set physiological constraints (phenotype) than the genotype?. Answers to these questions will provide valuable insight to a possible link between environmental changes and biodiversity of organisms in the aquatic ecosystem.. 17.

(46) Chapter 1 Outline of the Thesis In order to address the research questions discussed in this thesis it was necessary to isolate and culture a large number of clones of P. antarctica from each of the major oceanographic regimes in the Southern Ocean, in order to have statistically reliable estimates of genetic diversity (Fig. 1.7). New isolates of P. antarctica were obtained during several cruises of RV Polarstern with the help of many cooperation partners from other research institutes. Samples originating from various regions in Antarctica were obtained and unialgal clonal cultures were established by single colony isolation. About 586 cultures (x3=1758) were isolated, but because of a very low survival rate (20%), this part had its drawbacks, was time-consuming and introduced an unavoidable bias into the study. To our knowledge, we have assembled, the largest P. antarctica culture collection worldwide which is of great scientific interest.. Fig. 1.7 Overview of the Southern Ocean showing the sampling locations (light blue) where P. antarctica isolates were obtained for this thesis. The ACC (grey area) and its current flow pattern (red arrows) are shown highly schematically.. This dissertation is based on six individual manuscripts (chapters 2 to 7), two of which are published, two submitted and two prepared for submission. The following sections give a short overview of the aims and outlines of the individual manuscripts. The contributions of authors are noted.. 18.

(47) Chapter 1 Chapter 2 “A new cell stage in the haploid-diploid life cycle of the colony-forming haptophyte Phaeocystis antarctica and its ecological implications” During the biological programme on-board RV Polarstern on cruise leg ANT XXII-4 in the Antarctic region (April 05 – May 05), it was possible to isolate several clones of P. antarctica. Field samples of P. antarctica taken on this cruise showed a previously unrecognised cell stage of this microalgae. After discussion with other scientists and evidence from cultures, genetic and flow cytometric identification, it was concluded that this was a new cell stage in addition to the three already described by Rousseau et al. (2007): the colonial cells and the two types of flagellates. This new cell stage is proposed to be a zygote, thus completing the sexual life cycle in P. antarctica. This cell stage may also occur in the life cycle of other Phaeocystis species because van Breemen (1905) observed colonies of P. globosa being produced by cells attached to Chaetoceros willei. The ecological function of the zygote either as a refuge from grazers, or as an overwintering stage, was unspecified at that time. Further studies are required to determine the significance of this new cell stage to the ecology of P. antarctica. Authors’ contributions Gäbler-Schwarz S, Davidson A, Assmy P, Chen J, Henjes J, Lunau M, Nöthig EM, Medlin LK (2009). A new cell stage in the haploid-diploid life cycle of the colony-forming haptophyte Phaeocystis antarctica and its ecological implications. J. Phycol. submitted. Steffi Gäbler-Schwarz combined her own data with those provided by the co-authors, interpreted and wrote the manuscript in discussion with the co-authors. J. Chen helped in producing additional genetic data. The flow cytometric experiments were planned and performed together with M. Lunau.. Chapter 3 “Methods used to reveal genetic diversity in the colony-forming prymnesiophytes Phaeocystis antarctica, P. globosa and P. pouchetii – preliminary results (short communication)” After attending the SCOR Phaeocystis working group meeting in 2005, I was asked to write a short communication about the progress to establish and develop new molecular markers to assess the genetic diversity within the colony forming Phaeocystis species. This involved the application of two fingerprinting techniques. First, AFLPs were established as a prescreening tool to assess clone diversity prior to physiological investigations. Second, the. 19.

(48) Chapter 1 more powerful microsatellite markers were established to assess population structure and biogeography more accurately. Preliminary results showed differences in the AFLP patterns between a subset of P. antarctica isolates from different geographical regions, and that a wide variety of microsatellite motifs could be obtained from the microsatellite enriched clone libraries established for all three Phaeocystis species.. Authors’ contributions Gaebler S, Hayes PK, Medlin LK (2007). Methods used to reveal genetic diversity in the colony-forming prymnesiophytes Phaeocystis antarctica, P. globosa and P. pouchetii— preliminary results. Biogeochemistry 83:19-27. The experiments were planned together with Linda K. Medlin and performed in collaboration with Paul K. Hayes. I conducted all laboratory experiments, interpreted the data and wrote the manuscript in discussion with the co-authors.. Chapter 4 “STAMP: Extensions to the STADEN sequence analysis package for high throughput interactive microsatellite marker design” The manual analysis of sequences of microsatellite enriched libraries and marker design is time consuming and tedious. To speed up this process, a co-operation was established with the AWI bioinformatics group (Prof. Stephan Frickenhaus) that was already working on testing and developing software for automated microsatellite marker design. My role in this cooperation was to provide data and act as an end-user. After testing several already published pipelines e.g.: •. Phred, Phrap (http://www.phrap.org/phredphrapconsed.html),. •. MSATFINDER (Thurston & Field 2005),. •. STADEN package (Staden et al. 1998, http://staden.sourceforge.net/),. •. TROLL pipeline (Wellington et al. 2006),. with no acceptable results, we developed a consortium to create a pipeline based on the Staden package for easier and better microsatellite marker design. Chapter 4 presents a set of new tools implemented as extensions to the Staden package, which provides the backbone functionality for flexible sequence analysis workflows. The possibility to assemble overlapping reads into unique contigs (provided by the base functionality of the Staden package) is important to avoid developing redundant markers, a feature missing from most other similar tools.. 20.

(49) Chapter 1 Authors’ contributions Kraemer L, Beszteri B, Gäbler-Schwarz S, Held C, Leese F, Mayer C, Pohlmann K, Frickenhaus S (2009). STAMP: Extensions to the STADEN sequence analysis package for high throughput interactive microsatellite marker design. BMC Bioinformatics, 10:41, 11pp, doi:10.1186/1471-2105-10-41 All authors contributed to writing the manuscript. I performed the laboratory tests on P. antarctica individually and provided user feedback during development of the extensions.. Chapter 5 “PRIMER NOTE: Microsatellite markers for the polar prymnesiophyte species Phaeocystis antarctica KARSTEN” This note gives insight into the established microsatellite marker, their heterozygosity levels and amplification rate within the following geographic locations: Amundsen Sea and McMurdo Sound, Scotia Sea and Weddell Sea, Antarctic Circumpolar Current, and Prydz Bay. Authors’ contributions Gäbler-Schwarz S, Evans KM, Hayes PK and Medlin LK (to be submitted). PRIMER NOTE: Microsatellite markers for the polar prymnesiophyte species Phaeocystis antarctica KARSTEN. I conducted all laboratory experiments, interpreted and wrote the manuscript in discussion with the co-authors.. Chapter 6 “Genetic structure and diversity of Phaeocystis antarctica assessed by fastevolving microsatellite markers” In this study, genetic diversity and distribution of genetic polymorphisms in the circumpolar prymnesiophyte Phaeocystis antarctica were studied using eight fast-evolving nuclear microsatellite loci. Analyses were conducted for 110 P. antarctica isolates representative of five major oceanic regions in the Southern Ocean. These results reveal unexpected strong differentiation among several populations, whereas some geographically very distant populations are more closely related. The data are mainly in agreement with the Antarctic Circumpolar Current as well as the Antarctic costal current acting as major transport systems.. 21.

(50) Chapter 1 Authors’ contributions Gäbler-Schwarz S, Leese F, Evans KM, Vermeulen F and Medlin LK (Prepared for submission). Genetic structure and diversity of Phaeocystis antarctica assessed by fastevolving microsatellite markers. The experiments were planned together with Linda K. Medlin. I conducted all laboratory experiments and wrote the manuscript in discussion with the co-authors. F. Leese and I performed the computational analyses.. Chapter 7 “Responses of Different Antarctic Genotypes of Phaeocystis antarctica to three salinities: Evidence for Ecosystem Resilience” Relying on genetic data I started to consider physiological parameters. The four regions sampled for genetic analysis were not genetically isolated. This finding led us to assess (1) whether genetically closely related strains originating from different geographic regions in the Southern ocean react similarly to changes in environmental conditions or (2) whether the environment is more important in setting physiological constraints (phenotype) than the genotype. In order to test both hypotheses, we conducted experiments on growth, photosynthetic efficiency (Fv/Fm) and DMSP content from five P. antarctica strains from three different Antarctic regions: Prydz Bay, Ross Sea (both ice covered most of the year) and Scotia Sea (open water, little influence from sea ice). Authors’ contributions Gäbler-Schwarz S, Beszteri B, Gindulis JS, Hinz F, Nöthig EM, Wesche C, Kirst GO and Medlin LK (2008). “Responses of Different Antarctic Genotypes of Phaeocystis antarctica to three salinities: Evidence for Ecosystem Resilience. Marine Ecology Progress Series. submitted. The experiments were planned together with Janina S. Gindulis, Eva M. Nöthig, Gunther O. Kirst and Linda K. Medlin and performed by me. I conducted all laboratory experiments, interpreted and wrote the manuscript in discussion with the co-authors. Friedel Hinz helped with microscopic photo documentation during the laboratory experiments. Bank Beszteri provided help in bioinformatics analysis and in producing the R-figures. Christine Wesche provided help with glaciological data interpretation and redrawed the maps for Fig. 2 & 7 in this chapter.. 22.

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