Contribution to the Study of the Present and Past Diversity of Antarctic Cyanobacteria

Contribution to the study of the present and past diversity of Antarctic cyanobacteria

Rafael Fernández Carazo

July 2011

Dissertation submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Sciences.

Promotor: Dr. Annick Wilmotte

University of Liège. Faculty of Sciences. Department of Life Sciences.

Centre for Protein Engineering.

Printed: Centre for Protein Engineering.

Fernandez-Carazo, R. (2011) Contribution to the study of the present and past diversity of Antarctic cyanobacteria. PhD Thesis. University of Liège.

Publicly defended in Liège, July 1, 2011.

This PhD work was financially supported by the Belgian Science Policy Office (BelsPO), the Foundation Alfonso Martin Escudero (Spain) and the University of Liège.

Examination committee

Prof. Jacques Piette (President)

V irology and Immunology Unit. GIGA-R. University of Liege. Liege. Belgium.

Dr. Annick Wilmotte (Promotor)

CIP. University of Liege. Liege. Belgium.

Prof. Vincent Demoulin

Algology, mycology and experimental systematics. Department of Botany. University of Liege. Liege. Belgium.

Prof. Bernard Joris

Bacterial physiology and genetics. CIP. University of Liege. Liege. Belgium.

Prof. Jacques Coyette

Bacterial physiology and genetics. CIP. University of Liege. Liege. Belgium.

Dr. Elie Verleyen

Protistology and aquatic ecology. University of Ghent. Ghent. Belgium.

Dr. Anne Jungblut

Department of Botany. The Natural History Museum. London. United Kingdom.

Curriculum vitae

Personal

Rafael Fernández Carazo.

Born May 22 nd 1976.

Bachelor of Pharmacy . University of Granada (1994-1999).

Advanced Masters Degree ( DEA ) in “Biochemistry, Molecular Biology and

Microbiology”. University of Granada (2002-2003).

List of publications

Accepted

Fernández-Carazo, R., Hodgson D.A., Convey, P., Wilmotte, A. (2011). Low cyanobacterial diversity in biotopes of the Transantarctic Mountains and Shackleton Range (80-82ºS), Antarctica. FEMS Microbiology Ecology .

Fernández-Carazo, R., Namsaraev, Z., Mano, M.J., Ertz, D., Wilmotte, A. Cyanobacterial diversity for an anthropogenic impact assessment in the Sør Rondane area (Antarctica). Antarctic science .

Namsaraev, Z., Mano, M.J., Fernández-Carazo, R., Wilmotte, A. (2011). Biogeography of terrestrial cyanobacteria from Antarctic ice-free areas. Annals of Glaciology . 51: 171-177

Hodgson D., Convey, P., Verleyen, E., Vyverman, W., McInnes, S., Sands, C., Fernández- Carazo, R., Wilmotte, A., Peeters, K., Willems, A. (2010). Observations on the limnology and biology of the Dufek Massif, Transantarctic Mountains 82° South.. Polar Science. doi:10.1016/j.polar.2010.04.003.

Fernández-Carazo, R., Wilmotte, A. Ancient cyanobacterial DNA from sediment cores at Beak Island (Antarctic Peninsula): Challenges for a molecular approach. IPY Proceedings’ book . In press.

309

Curriculum vitae

In preparation

Fernández-Carazo, R., Gibson, J.A.E., Wilmotte, A. A compilation of molecular studies (16S rRNA) of Antarctic lacustrine cyanobacteria, including new data from the Mac.Robertson Land and Kemp Land.

Fernández-Carazo, R., Verleyen, E., Hodgson, D.A., Roberts, J.R., Vyverman, W., Wilmotte, A. Holocene changes in cyanobacterial communities in Maritime Antarctic lake sediments.

Popularization articles

Des cyanobactéries découvertes près du Pôle Sud . Popularization article in the electronic journal of the University of Liege “ Reflexions” (written by the Journalists) (http://reflexions.ulg.ac.be/cms/c_27953/des-cyanobacteries-decouvertes-pres-du-pole- sud).

Papers not related to this thesis

Pereira, P.J., Vega, M.C., Gonzalez-Rey, E., Fernández-Carazo, R., Macedo-Ribeiro, S., Gomis-Ruth, F.X., Gonzalez, A., Coll, M. (2002). Trypanosoma cruzi macrophage infectivity potentiator has a rotamase core and a highly exposed alpha-helix. EMBO Reports . 3(1): 88-94.

Argandoña, M., Fernández-Carazo, R., Llamas, I., Martinez-Checa, F., Caba, J.M., Quesada, E., del Moral, A. (2005) The moderately halophilic bacterium Halomonas maura is a free-living diazotroph. FEMS letters . 244(1):69-74.

Selected communications to conferences

Oral presentations

A Belgian collection of polar cyanobacterial: novel diversity and pharmaceutical screening of isolates. Fernández-Carazo, R., Lassence, C., Taton, A., Vincent, W., Storms, V., Marinelli, F., Wilmotte, A. 24 November 2006. Bruxelles (Belgium) Belgian Society for Microbiology (BSM) “Novel Compounds and strategies to combat pathogenic microorganisms”

Diversity and distribution of cyanobacterial in Antarctica. Fernández-Carazo, R., Zakhia, F., Taton, A., Verleyen, E., Vyverman, W., Hodgson, D.A., Wilmotte, A. 25-29 June 2007. Merida (Mexico). 17th Symposium of the International Association for Cyanophyte Research.

310

Curriculum vitae

Cyanobacterial diversity and distribution in the very extreme environments of the Pensacola Mountains (82°S, Antarctica). Fernández-Carazo, R., Hodgson, D., Wilmotte, A. 15 December 2006. Gembleux (Belgium). Ecole doctorale « Biodiversité, Ecologie et Evolution » PhD students’ day.

Posters

Exploring the Holocene through fossil cyanobacterial sequences from Antarctic lake sediments. Fernández-Carazo, R., Waleron, K., Hodgson, D.A.,Verleyen, E., Vyverman, W., Wilmotte, A. 26-31 July 2009. Sapporo (Japon). 10th SCAR Biology Symposium.

Baseline data on cyanobacterial diversity near the new Princess Elizabeth Antarctic research station. Fernández-Carazo, R., Namsaraev Z., Ertz D., Mano MJ., Wilmotte A. 26-31 July 2009. Sapporo (Japon). 10th SCAR Biology Symposium.

Biogeographical distribution and diversity of cyanobacteria in Antarctica, a polyphasic approach. Wilmotte, A., De Carvalho Maalouf, P., Zakhia, F., Taton, A. , Fernández- Carazo, R., Mano, MJ. , Namsaraev, Z., De Wever, A., Verleyen, E. 14 August 2009. Montreal (Canada). 13 th International Symposium on Phototrophic Prokaryotes.

Cyanobacterial diversity in the Transantarctic Mountains (Antarctica). Fernández-Carazo R., Hodgson D., Wilmotte A. 31 August-4 September 2008. České Bud ějovice (Czech Republic). 7th European Workshop on the Molecular Biology of Cyanobacteria.

Fossil cyanobacterial sequences in Antarctic lake sediments. Fernández-Carazo R., Waleron K., Hodgson D., Verleyen E., Vyverman W, Wilmotte A. 31 August-4 September 2008. České Bud ějovice (Czech Republic). 7th European Workshop on the Molecular Biology of Cyanobacteria.

Cyanobacterial diversity at Utsteinen: The impact of the “zero emission” research station in Antarctica. Fernández-Carazo, R; Ertz, D., Wilmotte, A. 11 October 2007. Liège (Belgium). 11 th Bioforum.

Forlidas Pond and Lundström lake: cyanobacterial diversity in the Antartctic at 82ºS. Fernández-Carazo, R., Hodgson, D., Wilmotte, A. 9-13 June 2007. Roscoff (France). Environmental genomics: from individual genomes to genomes of complex communities.

Antarctic cyanobacterial communities: baseline data on their diversity and distribution. Fernández-Carazo, R., Zakhia, F., Taton, A., Wilmotte, A. 21-22 Mai 2007. Brussels (Belgium). Belgian Biodiversity Platform conference: Biodiversity and Climate Change.

311

Curriculum vitae

Cyanobacterial diversity in “extremely extreme” environments of the Pensacola Mountains (82°S, Antarctica). Fernández-Carazo, R., Hodgson, D., Wilmotte, A. 28 August 2006. Pau (France). ISPP. 12 th International Symposium on Phototrophic Prokaryotes.

Do you like this place to live? Cyanobacterial diversity in extreme environments of the Transantarctic Mountains, Antarctica (82°S). Fernández-Carazo, R., Hodgson, D., Wilmotte, A. 17 May 2006. Liège (Belgium). 10 th Bioforum. Prize “Original research”.

Cyanobacterial diversity in extreme environments of the Pensacola Mountains (Transantarctic Mountains). Fernández-Carazo, R., Hodgson, D., Wilmotte, A) 23-25 Mars 2006. Brussels (Belgium). Be-Poles Workshop.

Courses, memberships and prizes.

Participation in the research projects HOLANT (BELSPO SD/CA/01A), AMBIO (BELSPO SD/BA/01A), and collaboration in the research projet BCCM (BELSPO C3/00/14) and the expertise pole « ANTAR-IMPACT» (www.antar- impact.ulg.ac.be)

Determination Course of Freshwater and Terrestrial Cyanobacteria. University? České Bud ějovice (Czech Republic). 24-28 July 2006.

Bioinformatics course “ BIO-computing with BEN ”. Belgian EMBnet Node. March 2006.Bruxelles.

Prize “Original research” at the 10 th Bioforum , Liège (Belgium). Granted by the biotechnology company “Eurogentec”.

Member of Belgian Society for Microbiology ( BSM ) (2006-2010).

312

Table of content

ACRONYMS AND ABBREVIATIONS………………………………………………………9

RESUMÉ………………………………………………………………………..…….…13

PART 1: GENERAL INTRODUCTION………………………………………………….…15

1. History of the Antarctic continent ...... 17 1.1. The Antarctic expeditions...... 17 1.2. The Antarctic Treaty and the International Polar years...... 18 1.2.1. Antarctic Treaty...... 18 1.2.2 International Polar Years...... 20 2. The Antarctic continent...... 21 2.1 Extreme continent...... 21 2.2. Biogeographical zones...... 23 2.3. Ice free areas and Ice Sheets...... 24 2.3.1. Ice-free areas...... 24 2.3.2. The Antarctic Ice Sheet ...... 24 2.3.3. The Continental Shelf ...... 26 2.3.4. The Ice Shelf ...... 26 2.3.5. Sea Ice ...... 26 2.4. Considerations about the Antarctic isolation...... 27 2.4.1. The climate of the Antarctic...... 27 2.4.2. The Polar vortex...... 28 2.4.3.1. Thermohaline circulation...... 28 2.4.3.2. The Antarctic Circumpolar Current (ACC)...... 29 2.4.4. Evolution of the Antarctic Ice sheets...... 30 3. Antarctic habitats...... 35 3.1. Antarctic lakes...... 35 3.2. Cryoconites...... 37 3.3. Rocks...... 38 3.4. Soils...... 39 3.5. Snow...... 41 4. Endemism and species adaptation...... 42 4.1. Everything is everywhere versus endemism...... 42

4.2. Adaptation strategies...... 46 5. Micropaleolimnology...... 47 5.1. Cores...... 48 5.1.1. Lake sediment cores...... 48 5.1.2. Sea sediment cores...... 49 5.1.3. Ice cores...... 49 5.2. Cyanobacterial microfossils...... 50 5.3. Pollen and mosses...... 53 5.4. Diatoms...... 54 5.5. Algae and bacteria...... 54 5.6. Pigments...... 55 5.7. Fossil DNA...... 57 5.7.1. Degradation process of DNA...... 57 5.7.2. Preservation of aDNA...... 59 6. Cyanobacteria...... 62 6.1. Generalities...... 62 6.2. Cyanobacterial structures...... 62 6.3. Classification...... 65 6.5. Cyanobacterial survival abilities...... 69 6.5.1. Desiccation...... 69 6.5.2. UV resistance...... 70 6.5.3. Freezing tolerance...... 72 6.6. Cyanobacterial diversity in the Antarctic continent…………………………….74

PART 2: RESEARCH OBJECTIVES…………………………………………………………………………………….….79

PART 3: RESULTS…………………………………………………………………………………………………………….……85

CHAPTER 1: Low cyanobacterial diversity in biotopes of the Transantarctic Mountains and Shackleton Range (80-82ºS), Antarctica…………………………………………….……87

CHAPTER 2: The limnology and biology of the Dufek Massif, Transantarctic Mountains 82º South.……………………………………………………………………………………………………….….127

CHAPTER 3: Cyanobacterial diversity for an anthropogenic impact assessment in the Sør Rondane Mountains area (Antarctica)….…….……………………………………………….…147

CHAPTER 4: A compilation of molecular studies (16S rRNA) of Antarctic lacustrine cyanobacteria, including new data from the Mac.Robertson Land and Kemp Land...... 185

CHAPTER 5: Biogeography of terrestrial cyanobacteria from Antarctic ice-free areas….225

CHAPTER 6: Holocene changes in cyanobacterial communities in Maritime Antarctic Lake sediments. ……………………………………………………………………………………………………235

CHAPTER 7: Ancient cyanobacterial DNA from sediment cores at Beak Island (Antarctic Peninsula): Challenges for a molecular approach. …………………………………………271

PART 4: GENERAL DISCUSSION……………………………………………………..……………………………………275

PART 5: FUTURE RESEARCH……………………………………………………..…………………………………………285

PART 6: REFERENCES…..…………………………………..………………………………………………………………….291

ACKNOWLEDGMENTS……………………………………………………..…………………………………………………..307

AUTHOR’S curriculum vitae……………………………………………………..…………………………………………309

Résumé

En Antarctique, les cyanobactéries jouent un rôle primordial dans le fonctionnement de l’écosystème. Elles sont responsables d’une grande partie de la production primaire aussi bien dans les lacs que dans les habitats terrestres. De fait, les cyanobactéries sont souvent les organismes photosynthétiques les plus abondants. Elles forment des biofilms laminés dans le fond et sur les bords des lacs et des mares. On les trouve également format des tapis dans les zones terrestres où l’on peut trouver assez d’eau liquide disponible.

Les deux objectifs principaux de cette thèse sont :

i) Augmenter les connaissances sur la diversité des cyanobactéries antarctiques des habitats lacustres et terrestres ainsi que leur profil de distribution globale et intra-antarctique.

ii) Étudier les communautés cyanobactériennes fossiles, en reliant la structure de ces communautés à d’autres marqueurs pour comprendre les facteurs sous- jacents influençant leur distribution dans le passé.

En ce qui concerne le premier objectif, nous avons analysé 23 échantillons environnementaux provenant des Montagnes Transantarctiques, de la chaîne Shackleton, des montagnes Sør Rondane et des régions de Mc.Robertson Land et de Kemp Land (les collines Stilwell et la chaîne Chapman).

En ce qui concerne le deuxième objectif, nous avons procédé à l’étude de deux carottes sédimentaires provenant de deux lacs de l`Ile Beak (péninsule antarctique). Ces lacs se situaient sous le niveau de la mer pendant une période de l’Holocène. Après la déglaciation de la croûte continentale, l’île remonta et surpassa le niveau de la mer. De cette manière, des lacs d’isolation se formèrent. Ces lacs contiennent un registre paléolimnologique exceptionnel, qui peut permettre de reconstruire les changements dans les communautés des cyanobactéries et d’autres microorganismes.

Pour répondre à ces objectifs, nous avons utilisé une approche polyphasique dans la plupart des cas. Cette approche inclut des observations microscopiques des échantillons environnementaux et des souches isolées, l’analyse des pigments par HPLC et l’analyse des ADN extraits de ces échantillons à l’aide de techniques de biologie moléculaire. Pour ces dernières, sont inclus des bibliothèques de clones, des DGGEs et des profils ARDRA basés sur des gènes codant pour l’ARNr 16S. Les séquences de l’ARNr 16S sont comparées avec d’autres séquences des bases de données (BLAST, RDPII). Elles sont analysées à l’aide d’outils bioinformatiques comme Bioedit, Geneious, ARB et DOTUR ainsi que des programmes détectant des chimères. Ces séquences sont ensuite organisées en unités taxonomiques opérationnelles (OTUs), définies comme des

13 groupes de séquences des gènes codants pour l’ARNr 16S qui partagent plus de 97.5% de similitude.

Des méthodes d’analyse ont été développées dans le cadre de cette thèse:

-une nouvelle amorce (16S369F) a été testée pour améliorer la spécificité de l’amplification par PCR de l’ADN cyanobactérien.

-deux méthodes d’extraction d’ADN on été développées et comparées pour essayer d’extraire un maximum d’ADN cyanobactérien des échantillons environnementaux, spécialement des morphotypes à gaines très épaisses.

-deux méthodes d’amplification isothermale on été dessinées et comparées pour améliorer l’amplification de l’ADN fossile présent en faibles concentrations dans des carottes sédimentaires.

Les résultats globaux de cette thèse suggèrent un nombre relativement bas des OTUs potentiellement endémiques dans l’Antarctique (8 OTUs sur un total de 32). Par contre, l’étude des séquences des ITS montre un niveau d’endémicité plus élevé que celui obtenu par les gènes codant pour l’ARN 16S.

Ce sont dans les Montagnes Sør Rondane que nous avons trouvé le nombre le plus élevé d’OTUs potentiellement endémiques. Ces montagnes ont pu jouer un rôle de refuge durant les périodes de glaciations, hypothèse émise dans cette thèse. La plupart des OTUs décrits dans les chapitres de cette thèse ont montré une distribution restreinte à des zones particulières (les montagnes Sør Rondane, les montagnes Transtantarctiques et Shackleton ou les régions de Mc.Robertson et Kemp Lands). L’analyse des séquences présentes dans les bases de données montre que la diversité des cyanobactéries terrestres a été particulièrement bien étudiée dans seulement trois régions Antarctiques (Southern Victoria Land, les Montagnes Sør Rondane et l’ile Alexander) alors que deux régions étaient très bien représentées pour les cyanobactéries lacustres (les régions déglacées à l’Est de l’Amery Ice Shelf et les vallées déglacées de South Victoria Land). Le reste de l’Antarctique reste encore peu étudié et plus de données sont nécessaires pour avoir une vision globale de la distribution des cyanobactéries dans ce continent.

Dans cette thèse, nous établissons une base de référence qui concerne la zone où la base polaire « Princesse Elizabeth » a été construite. Cela permettra l’étude des impacts futurs dus aux activités anthropogéniques dans la base et ses alentours.

Finalement, nous avons étudié des carottes sédimentaires provenant des lacs antarctiques. L’analyse des pigments montrait une concentration des caroténoïdes élevée pour la période climatique optimale de l’Holocène. L’étude de l’ADN fossile a révélé un changement dans la composition des OTUs pendant les 2-3 dernières décennies, peut- être en relation avec une augmentation de la température liée au changement climatique. General Introduction

PART 1: GENERAL INTRODUCTION .

General Introduction

1. History of the Antarctic continent .

1.1. The Antarctic expeditions.

The period between the ends of the 19 th century and the first decades of the 20 th century were years of intensive scientific and geographical exploration, including Antarctica. This period was denominated the “Heroic Age of Antarctic Exploration”. As a result of this intense activity, much of the continent's coastline was discovered and charted, and significant interior areas were explored. These expeditions generated large quantities of scientific data across a wide range of scientific disciplines.

James Cook (1728-1779) became the first explorer to traverse the Antarctic polar circle (66º 33’) the 17 th of January 1773. He also accomplished the circumnavigation of the Antarctic continent. Later, James Clark Ross (1800-1862), which localized the Magnetic Nord Pole (1831), discovered the Transantarctic Mountains and observed the Mont Erebus in eruption (1841). In 1820, Fabian von Bellingshausen, captain of the Russian royal navy, discovered the Alexander Island (Antarctic Peninsula). Several other explorers tried to land in the continent in those years, e.g; Charles Wilkes, John Boscie or James Weddell. Finally, a Norwegian expedition leaded by Henryk Bull in 1893- 1895, first reached the Antarctic continent in Cap Adare.

Belgium possesses a long Antarctic expedition history. The “Heroic Age of Antarctic Exploration” was inaugurated in 1897 by the Belgian Geographical Society’s expedition. Adrien de Gerlache de Gomery (1866-1934), Lieutenant of the Royal Belgium Marine, put forth the port of Anvers in 1897 aboard of the Belgica (Fig.1) with destination to the Antarctic Peninsula. The ship was trapped by the ice in the Bellingshausen Sea for one complete year, moving to the west following the shelf movements. Adrien de Gerlache collected the first annual cycle of Antarctic observations. He also reached the 71°30'S latitude and discovered the Gerlache Strait (Antarctic Peninsula). Later, Roald Amundsen (1872-1928) and Robert Falcon Scott organized parallel expeditions to reach the Geographic South Pole. Roald Amundsen first arrived on the 14 th December 1911. The Scott’s expedition was 3 days later. All the members of the Scott’s crew deceased during the way back to the coast (Simion, 2007).

More recently (1946-1948), the United States of America organized two expeditions to study the feasibility of the building permanent bases in Antarctica, thus improving the previous knowledge about hydrographic, geographic, geological, meteorological and electromagnetic conditions in the area.

General Introduction

Figure 1. The Belgica, prisoner of the ice on the 19 th November 1898. (Picture from the book “Voyage of the Belgica - Fifteen Months in the Antarctic by Adrien de Gerlache”).

In 1957, Gaston de Gerlache (Belgium) started an expedition for the construction of the first Belgian Antarctic base (King Baudouin). These years were very productive for the Belgians, with 3 expeditions between 1958 and 1961. In 2004, the Belgian government started the construction a new research station to replace the King Baudouin base, buried under several meters of snow, and abandoned 40 years before. With the coordination of the International Polar Foundation, several expeditions were organized from 2004 to 2009 to finally inaugurate (15 February 2009) the Princess Elisabeth research station, the first polar base ever designed to run entirely on renewable energy (BelSPO).

1.2. The Antarctic Treaty and the International Polar years.

1.2.1. Antarctic Treaty.

The Antarctic Treaty was signed in Washington on 1 December 1959 by twelve countries involved in the International Geophysical Year (1957-58). No date was indicated in the Treaty’s text specifying the end of the agreement. The total number of Parties to the Treaty is now 48. The treaty defines the delimitation of Antarctica as the area which extends below the 60 th parallel of the Southern hemisphere and therefore including continental and marine areas.

Thanks to this treaty, Antarctica shall be used for peaceful purposes only (Article I), and the freedom of scientific investigation and cooperation is encouraged (Article II). Scientific observations and results shall be exchanged and freely available (Article III) and all installations open to inspection by third countries.

General Introduction

Numerous claims of territory were expressed by several countries over the Antarctic continent before the entry into force of the Washington Treaty. The continent was divided in concentric sections as seen in Fig. 2. Among the signatories of the Treaty, seven countries (Argentina, Australia, Chile, France, New Zealand, Norway and the United Kingdom) expressed territorial claims which sometimes overlap. These claims were done on the grounds of their discovery and occupation of certain areas, as well as the principle of proximity. However, the claims are frozen by the Article IV, while the present Treaty is still in effect.

Figure 2. Main claims over Antarctica. Figure from the Antarctic Geological Drilling website.

The Canberra convention (signed in1980, entered into force on 7 April 1982) designed the importance of safeguarding the environment and protecting the integrity of the ecosystem of the Antarctic surrounding seas. The treaty concerned the protection of the Antarctic marine flora and fauna, including birds (Zegers, 1983).

The Madrid Protocol (signed in 1991, entered into force in 1998) was considered the further step of the Washington treaty (http://www.aad.gov.au/default.asp?casid=826). It was adopted to harmonize and legally bind form to the different provisions related to the protection of the Antarctic environment. The Protocol designates Antarctica as a 'natural reserve, devoted to peace and science', establishes environmental principles for the conduct of all activities, requires the development of contingency plans to respond to

General Introduction environmental emergencies and potentially blocked the exploitation of mineral, oil and gas resources.

The Convention for the Conservation of Antarctic Seals (signed in 1972, entered into force in 1978) was created to promote and achieve the objectives of protection, scientific study and rational use of the Antarctic seals and to maintain a satisfactory balance within the ecological system. It defines protection areas as “sanctuaries”, defines the maximal number of individuals to capture of each of the species concerned in the convention and designs protected species which could not be captured (British Antarctic Survey website).

The Convention on the conservation of Antarctic living marine resources (CCAMLR) (signed in 1980, entered into force in 1982) integrates the Antarctic Treaty System (http://www.ccamlr.org). The Convention requires that the conservation of Antarctic marine living resources and decisions about their rational use must be based on an ecosystem approach. Unlike other fisheries agreements that focused only on the status of the commercial target species, the CCAMLR requires that all species in the ecosystem are taken into consideration to conserve ecological relationships. This was a very far-sighted and unprecedented approach at the time the Convention was negotiated.

1.2.2 International Polar Years.

The first IPY was organized in 1882-1883 with the participation of 12 nations, including Belgium. The first IPY was undertaken under the assumption that geophysical phenomena could not be surveyed by one nation alone. An undertaking of this magnitude would require a coordinated international effort. This first IPY settled a precedent for international science cooperation. In this frame, 15 expeditions were organized, 2 to Antarctica and 13 to the Arctic.

The second IPY (1932-1933) was proposed and promoted by the International Meteorological Organization as an effort to investigate the global implications of the newly discovered “Jet Stream”. Forty nations participated and it produced significant advances in meteorology, magnetism, atmospheric science and the “mapping” of ionospheric phenomena that advanced radioscience and technology. Forty permanent observation stations were established in the Arctic. In Antarctica, the U.S.A constructed an inland winter-long meteorological station on the Ross Ice Shelf. This was the first research station inland from the Antarctic coast.

The third IPY (1957) was really the First International geophysical year (IGY). The International Council of Scientific Unions, a parent body, broadened the proposals from polar studies to geophysical research. More than 70 existing national scientific

General Introduction organizations formed IGY committees and participated in the cooperative effort. The first Antarctic treaty was born in this frame.

Finally, the last IPY (2007-2008) involved more than 60 participant countries holding 228 scientific projects. The main themes proposed aimed i) to determine the present environmental status of the polar regions by quantifying their spatial and temporal variability; ii) to quantify, and understand, past and present environmental and human change in the polar regions in order to improve predictions; iii) to advance the understanding of polar-global interactions by studying teleconnections on all scales; iv) to investigate the unknowns at the frontiers of science in the Polar Regions; v) to use the unique location of the polar regions to develop and enhance observatories studying the Earth's inner core, the Earth's magnetic field, geospace, the Sun and beyond; vi) to investigate the cultural, historical, and social processes that shape the resilience and sustainability of circumpolar human societies and identify their unique contributions to global cultural diversity and citizenship.

2. The Antarctic continent.

2.1 Extreme continent.

Antarctica is a continent isolated by the Southern Ocean, hidden by an enormous cover of snow accumulated over the years. This is the highest, driest, windiest and coldest continent on Earth. With a maximum total area of 14x10 6 km 2, including the surrounding ice, Antarctica comprises 10% of continental Earth. In some places, the deepest ice is more than one million years old. Older ice has been squeezed out by compression from above and it is nowadays very thin and practically undatable (Hodgson et al., 2009). The Antarctic continent is an inhospitable place for life. In addition to the low temperatures, dry air and high speed winds, life must cope with the lack of solar radiation from March 22 to September 22, when the sun sets down and the polar night begins (British Antarctic Survey website).

The mean elevation is ca. 2200 m with several mountain peaks rising over 4000 m. The highest elevation is the Mount Vinson in the Ellsworth Mountains (4892 m). Due to this particular elevation above sea level, downslope winds (katabatic winds) flow from the high elevations of the plateau trough the coast (Fig. 3). When the air enters in contact with the ice it is cooled and increases its density, flowing downhill due to the gravity and the pressure gradient along the valley bottom. Katabatic winds reach high speeds (ca. 320 km/h) and play an important role in the geography and biogeography of the Antarctic continent. For instance, it blows snow away in some coastal areas, playing a role in the maintenance of ice-free areas such as the McMurdo Dry Valleys. Winds also can mix the lake’s water columns. The formation of blue ice areas is also influenced by the action of winds. Blue ice areas are formed when the snow is blown away, the surface ice evaporates and it is replaced by the glacier flow from upstream.

General Introduction

Unlike the Arctic, Antarctica is a continent surrounded by the Ocean. This implies that the continental interior does not benefit from the moderating influence of the water. The high elevation, the reflection of the sunlight by the white snow and the ice cover of the ice shelves together with the low concentration of vapour molecules of water in the atmosphere, enhance low temperatures in the Antarctic plateau ranging from -70ºC to -40ºC in winter and from -35ºC to -15ºC in summer. The lowest temperature record (-89.6ºC) was registered at the Vostok Station (East Antarctica) on the Polar Plateau. Nevertheless, in some punctual locations temperatures exceed the freezing point, ie. in some areas on the Antarctic Peninsula or the sub Antarctic islands. Interestingly, exposed rocks can experience higher temperatures than the surrounding air or white snow, because rocks absorb the heat more efficiently (see 3.4). For instance, in the Sør Rondane mountains, the air temperature is -1.6ºC (1.7 m above the rock surface) whilst the temperature on the surface of rock is 15.5ºC (data measured on 24 th December at 12:00 pm) (Zorigto Namsaraev, personal communication).

Antarctica is one of the driest places all over the world, despite the fact that it harbours more than 70% of the world’s fresh water. The average precipitation is only (100-150) mm - (350-500) mm of water equivalent (in continental Antarctica and the Maritime Antarctic Peninsula respectively) (Weller et al, 1987).

Figure 3. Katabatic winds blowing from the inland plateau to the Ocean. Figure from Hannes Grobe (Creative Commons CC-BY-SA-2.5).

General Introduction

2.2. Biogeographical zones.

Antarctica has been commonly divided into three biogeographical zones (Holdgate et al., 1977; Kennedy, 1995) (Fig.4): i- Sub-Antarctic Islands, into the Indian and Pacific Oceans. ii- Maritime Antarctica, comprising the West coast of the Antarctic Peninsula and the surrounding islands. iii- Continental Antarctica, comprising the rest of the continent as well as the near coast Islands.

Figure 4. Division of Antarctica in 3 biogeographical zones: subantarctic, maritime Antarctic and continental Antarctica (Holdgate et al., 1977). (Figure from Pete Convey; BAS).

General Introduction

2.3. Ice free areas and Ice Sheets.

2.3.1. Ice-free areas. Exposed rock and soils represent ca. 40000 km 2 which is only the 0.3% of the total area of the Antarctic continent. Although the Antarctic continent is almost completely covered by snow and ice, there are some regions called 'oasis', consisting of valleys and ridges and some borders of the polar plateau which are ice- free. These regions can be considered as cold deserts, due to the combination of low temperatures and low precipitations (Tedrow and Ugolini, 1966).

Permafrost, the earth material with temperatures below 0ºC for at least two consecutive years, is an important feature in the Antarctic’s environment. Permafrost temperatures in Antarctica range from -12ºC to -24°C in continental Antarctica with warmer temperatures in maritime Antarctica. Although less extensive than in the Arctic, permafrost covers extensive areas of the continent, ie. inland ice-free regions, some parts of the Antarctic Peninsula and it is also present in the maritime Antarctic islands. Thickness ranges from 240-900 mm in continental Antarctica, to 15-180 mm in maritime Antarctica (Summerhayes et al., 2009).

2.3.2. The Antarctic Ice Sheet is the mass of ice and snow which covers 99.7% of the Antarctic continent (Fig. 5). It is the largest freshwater world’s reservoir, with ca. 30 million km 3 of ice, representing ca. 70% of the Earth’s freshwater (Fox and Cooper, 1995). The ice sheet is nourished at its surface by deposition of snow and frost, which does no melt but accumulates year-on-year. The Antarctic ice is a dynamic mantle which flows from the inland to the edge of the continent at an average speed of 500 m per year, transporting ca. 2000 billion tonnes of ice per year to the Southern Ocean (Summerhayes et al., 2009). The ice sheet is an important source of nutrients in the Southern Ocean, as the ice and snow of the ice sheet contain high concentration of nutrients such as nitrate and ammonium. Biggs (1978) estimated that 1-2% of the total dissolved inorganic nitrogen within the pelagic waters south of the polar front comes from the Antarctic ice sheet.

Three distinct geographical zones are distinguished on the continent: East Antarctica, West Antarctica and the Antarctic Peninsula (Fig. 6). Antarctica is divided in two unequal parts, separated by the Transantarctic Mountains. This range started to uplift some 50-55 Ma ago (Cande et al., 2000). With a total length of about 3500 km and 100- 300 km wide, the Transantarctic Mountains are one of the longest mountain ranges on Earth.

General Introduction

Figure 5. Antarctic map with the main ice-free areas (gray color).

Figure 6. Regional map of Antarctica (from Mayewski et al., 2009).The maps represent the three Antarctic geographical areas: East Antarctica, West Antarctic and the Antarctic Peninsula. Some of the major Regions (Wilkes Land, Enderby Land, Dronning Maud Land and Marie Byrd Land) are also indicated

General Introduction

The greater zone is East Antarctica (EA) with an approximated area of 10.35x10 6 km 2 and the smaller is the West Antarctica (WE) with ca. 1.97x10 6 km 2. The Antarctic Peninsula (0.52x10 6 km 2) is often considered as an individual zone because of its climatic differences. The Antarctic Peninsula is the only region above 63ºS, with abundant ice-free areas, milder temperatures and climatic conditions due to its geographical position (Hodgson et al., 2009).

The East Antarctic landmass under the snow is mostly above the sea level. The East Antarctic ice sheet has an approximated age of 12-15 Ma (references in Convey et al., 2008) and forms a more or less uniform plateau.

The West Antarctica (WA) landbed is mostly under the sea level, sometimes several hundred to one thousand meters deep. Because the mean elevation is 850 m.a.s.l., its behaviour significantly differs from the EA. The WA’s ice sheet is less cold, sometimes with temperatures reaching the melting point. The WA’s ice sheet formed between 6 and 17 Ma ago (references in Convey et al., 2008).

The Antarctic Peninsula (AP) ice sheet is quite different from the other two zones. Ice depth is lesser and it moves much faster trough the ocean in form of Alpine-type glaciers. Ice melts in summer more abundantly than in EA and WA, and comprises a significant component of the ice lost in this area.

2.3.3. The Continental Shelf is the extended perimeter of the continent and its associated coastal plain. Due to the isostatic rebound, an uplift of the landbed during warmer periods when ice melts, the continental shelf has been sometimes part of the continent during the glacial periods, and located under water during interglacial periods as nowadays. The Antarctic Continental Shelf is deeper than in other continents, due to the weight of the massive ice covers that push down the continent. Because of this depth, the majority of the continental shelf is in the aphotic zone. Floating ice shelves cover about one third of the shelf, while the rest is covered by sea-ice for about half the year.

2.3.4. The Ice Shelf is the thick ice layer floating on the ocean surface formed when a glacier or ice sheet flows down to the coastline and then to the ocean’s surface. The Antarctic ice shelf constitutes 11% of the Antarctic area. The boundary between the floating ice shelf and the grounded ice it is called the grounding line . There are over 44 ice shelves in Antarctica, from which 19 measure 10000 Km 2 or more. The two largest Antarctic ice shelves are the Ronne-Filchner ice shelf and the Ross ice shelf (Vincent, 1988). Ice shelves have an important participation in the maintenance of the Antarctic currents, as they produce cold and dense water that flows into them (see section 2.4.3).

2.3.5. Sea Ice . The ice from the Sea ice has a different origin than the ice from the ice shelves, as sea ice is formed by freezing of sea water. Because of its salinity, temperatures enabling sea ice formation are under 0ºC. Antarctica doubles its size

General Introduction during the austral winter, when sea ice is formed and surrounds the continent, covering an area at least of the same size than the continent itself. Sea ice is relatively thin with 1-2 m in average (Summerhayes et al., 2009).

2.4. Considerations about the Antarctic isolation.

Antarctica has been isolated from the rest of the continent at least ca. 40 Ma ago. Several factors are involved in the Antarctic’s isolation and are important to explain the existence of endemic biota which has potentially inhabited the Antarctic continent since the pre-Gondwana era (Convey et al., 2009). Among these factors we are going to cite the local climate, the oceanic and atmospheric currents, the last involved in the high UV radiation levels which affects the Antarctic’s surface, and the processes involved in the formation of the ice sheets and their deglaciation during the interglacial periods.

2.4.1. The climate of the Antarctic.

Snowfalls over the Antarctic continent are very sparse and the distribution of precipitation is heavily biased by the geographical location. Thus, several meters of snow fall near the coast every year but only several centimetres further inland (British Antarctic Survey website). Due to the low temperatures, precipitations remain in the field because the water evaporates and sublimates in low quantities, building up the ice sheet. Antarctica experiences long periods of night followed by periods of almost continuous daylight. This is because of its southern position, at very low latitudes. When the southern end of the Earth is pointing towards the sun in summer the sun never sets down but appears to circle around the sky. During the Antarctic summer, which occurs during the northen hemisphere’s winter, the sun irradiates the continent more than the tropics. However, the high albedo properties of the ice shelves reflect much of this radiation back to the space. Therefore, only small quantities of energy are absorbed by the continent and the temperatures do not considerably increase. In addition, the atmosphere contains low concentration of water vapour, which contributes to the escape of the radiation, as water vapour reflects radiation back to the Earth (Summerhayes et al., 2009). This process does not occur in the ice free areas or in the ocean (and in smaller proportion, in the lakes) because their darker colour allows higher solar radiation absorption and therefore increased temperatures.

The coldest Antarctic region is East Antarctica (EA). The lack of clouds and water vapour in the atmosphere together with the higher elevation and the isolation of the EA from the warmer maritime air masses found over the Southern Ocean, influence the lower EA temperatures. The highest Antarctic temperatures are recorded in the western side of the AP, influenced by a northwesterly wind. In some places, temperature monthly means are positive for 2-4 moths per year (Hodgson et al., 2009).

General Introduction

The Antarctic coastal region is less extreme than the plateau. However there are some exceptions where inland zones benefits of mild climate, such as the Schirmacher Oasis, in the Dronning Maud Land (EA), which can be climatically classified as a coastal zone (Richter and Bormann, 1995).

2.4.2. The Polar vortex.

The Antarctic polar vortex is a persistent large-scale cyclonic circulation pattern in the middle and upper troposphere and the stratosphere. This phenomenon occurs during the polar winter when stratospheric air moves in a circular motion, with an area of relatively still air in its centre. The vortex acts like an atmospheric barrier which impedes the warmer coastal air entering the interior of the continent. The vortex is encircled by a strong eastward flowing jet, the “Polar night jet”. Because of the lack of solar radiation, temperature of the air is extremely low over the Antarctic. The temperature in the vortex is approximately -80ºC, which improve the formation of polar stratospheric clouds

(PSCs), composed of HNO 3 or pure ice (Tabazadeh et al., 1994). The Polar Vortex plays an important role in the formation of the ozone (O 3) hole. The nitric acid from PSCs reacts with Chlorofluorocarbons (CFCs) to form chlorine, which catalyzes the photochemical destruction of ozone. Chlorine is accumulated by the Polar Vortex in the stratosphere over the Antarctic continent during the Antarctic winter and then the ozone destruction reaches a maximum peak when the sunlight comes back in spring. The ozone depletion over Antarctica implies high ultra-violet radiation levels which constitute one of the inconvenient to which the Antarctic life forms must face.

2.4.3 Main Antarctic Currents.

2.4.3.1. Thermohaline circulation.

All the oceans are interconnected by the thermohaline circulation or global conveyor belt (Fig. 7). The thermohaline circulation is one of the two major mechanisms of water’s ocean circulation, which influences the dynamics of deeper water columns. The second major mechanism is the wind force that usually moves the upper meters of the water column. The warm water flows poleward near the surface and is converted into cold and saline water that sinks and flows equatorward. When sea water arrives to the poles, it freezes. In this process, cold and highly saline brines are expelled. As this water is characterized by a higher density, it sinks. Warm surface water is transported towards the northern area of the North Atlantic, where it is cooled and sinks to form the North Atlantic Deep Water that then moves southwards. In the Southern Ocean, water is cooled and this deep-water mass moves to the South Indian and Pacific Oceans where they start to move up to the surface (Gordon et al., 1986).

The thermohaline circulation plays a crucial role in the world’s climate because it moves huge quantities of energy. Biotic systems are also highly influenced by this current as nutrients and oxygen are removed from the deeper waters and then driven to

General Introduction the surface. This thermohaline circulation is nowadays menaced by the global warming because it favors the addition of high volumes of freshwater to the system and thus could collapse the conveyor (Rahmstorf, 2000).

Figure 7. Schematic picture of the global ocean circulation. Warmer superficial currents are in red and cold and deeper currents are in blue. Figure from the Intergovernmental Panel on Climate Change (IPCC) 2001 report.

2.4.3.2. The Antarctic Circumpolar Current (ACC).

The ACC is the major oceanographic feature of the Southern Ocean. It freely flows unrestricted around the continent, isolating the cold high latitude land and sea areas from temperate mid- and low-latitude warm surface waters.

The ACC is one of the most powerful oceanic currents (Fig. 8). It is composed of a series of eastward-flowing jets as the Subantarctic Front (SAF), the southern ACC front (SACCF) and the Polar Front (PF). The Antarctic Polar Front (PF), or Antarctic Convergence, is characterized by high-speed currents and strong horizontal gradients in density, temperature and salinity. It is formed by Antarctic waters that sink below subantarctic waters (Moore et al., 1999). The PF is visible at the surface due to a strong horizontal temperature (up to 6ºC) and salinity gradients, and it sinks at least 1000 m deep. The PF acts as a barrier, impeding the exchange of water between northern and southern waters (Clarke et al., 2005; Xiaojun and Martinson, 2004).

It has been suggested that the ACC was formed during the late Eocene through Early Oligocene (30-34 Ma ago) when the Tasmanian Seaway (between Antarctica and Australia) and the Drake Passage (between South America and Antarctica) opened. The

General Introduction formation of the ACC has been related to the cooling mechanisms which isolated Antarctica and favored the ice sheet formation. As Antarctica became isolated from warmer waters and glaciers, the ice sheets gradually covered the formerly forested continent (Lyle, 2007).

Figure 8. Main oceanic currents in the Southern Hemisphere, including the Antarctic Circumpolar Current (ACC) (from Rintoul et al., 2001).

2.4.4. Evolution of the Antarctic Ice sheets.

Antarctica was the centerpiece of the Gondwana supercontinent ca. 200 Ma ago before the Gondwana broke up into several fragments ca. 180 Ma ago. The different continental fragments spread during 100 to 65 Ma ago, until Antarctica reached its actual position at the South Pole. Even at this southernest position, Antarctica was forested and inhabited by dinosaurs in the Cretaceous and mammals during the Tertiary. Once the Drake Passage and the Tasmanian Seaway opened, the circumpolar currents started to flow and temperatures decreased drastically over the last 40 million years. A temperate adapted flora was replaced by cold adapted species. Their fossils allow to interpret the cooling dynamics of the continent. Climatic events moving from green to icehouse conditions started 42 Ma ago which is concordant with the Drake Passage opening. Later, 33-34 Ma ago, the ice-sheets started to cover the entire continent, a drastic change which coincides with the Tasmanian Seaway opening (Fig. 9) (Hodgson et al., 2009).

To explain the Antarctic cooling, some authors supported the role of the greenhouse gases, which atmospheric concentrations dropped at that period but neglected the importance of the ACC formation (references in Hodgson et al., 2009, Tripati et al.,

General Introduction

2005). Nevertheless, paleoclimatic reconstructions mostly support that the oceanographic and climatic processes leading to the current glacial conditions started at the same period of time. Volcanic activity was intense during the Early cretaceous (130 Ma BP) until the Early Cenozoic (45 Ma BP). These volcanoes expulsed high quantities of CO 2 (3000 and 1000 ppm in the Cretaceous and the Cenozoic, respectively). The levels of the CO 2 (greenhouse gas) influenced the rise of temperatures which were 6-7ºC warmer than today. CO 2 levels further decreased leading to cool conditions, with the exception of a CO 2 peak observed in the last century (EPICA 2004). The average temperature during the interglacial cycles was 15ºC and CO 2 and CH 4 concentrations averaged 300 ppm and 800 ppbv respectively. During the glacial episodes, temperatures and CO 2 and CH 4 levels decreased (10ºC, 180 ppm and 350 ppbv, respectively and the sea level dropped by 120 m. Currently, CO 2 levels are around 380 ppm, the highest value in the last 850 Ka (EPICA 2004) and surely of the last 25 million years. CH 4 levels are also elevated (1770 ppbv) nowadays, exceeding the natural range of the past 650 ka. If this trend continues, an increase of the sea levels is expected, as it already happened during the Pliocene where sea level was 15-25 m higher than today (review in Hodgson et al., 2009).

Marine sediments from Cape Roberts (Ross sea) contained evidence of 55 glacial- interglacial cycles covering the last 33-17 Ma. The shifts from glacial to interglacial periods are somehow predictable as they are related to the Milankovitch cycles (Milankovicht, 1941). However, other changes are not predictable by these approximations and some events are still not well understood to provide the basis for future predictions. Several factors other than CO 2, the ACC formation or the Earth’s orbit, also influenced the formation of the ice sheets such as global atmospheric circulation changes, albedo dynamics and solar variability (Hodgson et al., 2009). In the last 33 million years, the fluctuations were explained by cycles of 41 Ka. However, recent studies on Antarctic ice cores indicated 8 global glacial cycles for the last 800 ka. This means that recent oscillations are more spaced in time, which is related to lower atmospheric CO 2 concentrations. However, despite the strong correlation between CO 2 variations and Antarctic temperature, the mechanisms involved are still not well elucidated (Hodgson et al., 2009)

The Last Glacial Maximum (LGM), around 22–17 ka, is the most recent interval when global ice sheets reached their maximum levels (Convey et al., 2009). The transition from the LGM to the present interglacial period has been exceptionally abrupt, at least concerning the last 800 Ka. It first started with a rapid warming in the North hemisphere 15 Ka ago, followed by a return to cooling conditions (Younger Dryas) and a final rapid warming to reach the Holocene (11.7 Ka). The Holocene is the actual interglacial period. The Antarctic Early Holocene climate optimum has been estimated between 11.5 and 9 ka BP, from several ice cores from coastal and continental sites (references in Verleyen et al., 2010).

General Introduction

Signals from several ice core records indicate that the Holocene was of relative climate and environmental stability, although it has been marked by many, often rapid, global temperature and precipitation excursions (Hodgson et al., 2009). The relationships between the ice sheets, the Ocean, the sea ice and the atmosphere resulted in complex climatic changes events which were sufficient variable to cause major changes in the Antarctic ecosystems during the Holocene. For instance, the Antarctic Peninsula has recently experienced one of the most rapid warmings on the Earth. By contrast, lower temperatures have been recorded in other Antarctic areas at the same time (HOLANT project, www.holant.ugent.be). Several ice shelves from the Antarctic Peninsula gradually deglaciated between 13.5-4 Ka BP (e.g. Hodgson et al., 2001) (Fig. 10) because of temperature rise, resulting in rapid loss of ice masses. Although the Antarctic Peninsula ice sheet contributed in a minor way to the post-LGM sea-level rise (1-2 m), it has been the most sensible of the Antarctic ice sheets to climate change. The East Antarctic ice sheet showed a multi stage gradual deglaciation pattern. Thus, the Stornes area (Larsemann Hills) and some parts of the Lützow Holm Bay region only deglaciated in Mid to Late Holocene (Verleyen et al., 2010a) whilst several areas were already ice- free during the LGM such as the Bunger Hills, the Larsemann Hills, the Vestfold Hills and the Amery Oasis and probably some parts of the Lützow-Holm Bay area (Hall, 2009; Verlyen et al, 2010 and references therein). The retreat of the grounding line to the present coastal position of the Wilkes Land started 9 Ka ago and was complete by 2 Ka BP. Prydz Bay retreated around 11.5 Ka. The western Ross Sea region ice sheet started its retreat shortly after the LGM to reach its actual position about 7 Ka ago (Fig. 12). Regarding East Antarctica, there are several evidences pointing to wet conditions in the Larsemann Hills at 11.5-9.5 Ka BP with the development of microbial mats ca. 11 Ka BP in Lake Reid (Larsemann Hills) (Hodgson et al., 2005a), diatoms and rotifers colonizing lakes in the ice-free areas of the Vestfold Hills since 13 Ka BP, or increased nutrient inputs in a lake from the Mac.Robertson Land 13 ka BP, resulting in the establishment of faunal community (Verleyen et al., 2010a). Regarding the West Antarctic ice sheet, the available data do not allow to accurately determine the exact ages of ice retreat. However, for the Marie Land, the ice sheet retreat reached its actual position some 10 Ka ago (Hodgson et al., 2009 and references therein).

Figure 9. Summary of the main geological and glaciological events in Antarctica, 555 Ma to present (non-linear timescale). (Figure from Hodgson et al., 2009).

Figure 10. Main events occurred during the East Antarctic’s deglaciation (from Verleyen et al., 2010a).

General Introduction

3. Antarctic habitats.

The cryosphere, the collection of all ice environments, includes sea ice, lake ice, streams beneath glaciers, the junction between ice crystals in the Antarctic ice sheets, permafrost soils, supercooled water in ice clouds and ice shelves (Vincent et al., 2004 and references therein). Extreme cold habitats are typical in the High Arctic, Alpine sites and Antarctica. The ecology of these environments is mostly microbial. Among the microbial communities, cyanobacteria play an important role in the ecosystem functioning. In Antarctica, cyanobacteria are one of the dominant phototrophic organisms due to their adaptation to low temperatures, thaw-freeze cycles, variable light intensities, UV radiation, low nutrient and osmotic regimes (Vincent, 2000a). Recently, Jungblut et al. (2009a) suggested a cold biogeographical distribution of some cyanobacterial taxa, as 68% of the ribotypes analyzed in that study were only related to cyanobacteria inhabiting cold habitats.

Cyanobacterial global biomass has been estimated to ca. 232x10 12 - 300x10 12 g C (Garcia-Pichel, 2003). 75% of all global cyanobacterial biomass is present in the oceans whilst the remaining 25% is found in terrestrial habitats. Comparatively, the total biomass of land plants corresponds to 560x10 15 g C (Schlesinger, 1997).

Despite the harsh life conditions in Antarctica, several environments are able to host living communities. We propose here a brief enumeration of the main biotopes within the Antarctic continent.

3.1. Antarctic lakes.

Antarctic lakes are typically oligotrophic although some eutrophized lakes can be found near colonies of marine mammal, birds, or scientific stations (Butler, 1999). In general, Antarctic lakes are protected from direct human contamination, except for lakes near scientific stations which can be used as water supply or be influenced by human activities. However, indirect human activities also affect Antarctic lakes. These environmental changes may alter the delicate lake’s ecosystems due to their quick response to local perturbations (Quayle et al ., 2002; Smol and Douglas 2007; Smal et al . 2005). However, Antarctic lakes are one of the most pristine ecosystems on earth. Lake systems in Antarctica provide a natural laboratory for studying microorganism’s evolution, which can teach us important facts about the influence of environmental and historical factors in the biodiversity and the dynamics of microbial communities.

Antarctic lakes are found within the ice sheet (epiglacial ) or glacier depressions (supraglacial ), against nunataks or on ice free areas (Pienitz et al., 2008). Many lakes are covered by a thick ice cap, underlying liquid water despite the low (minus 0ºC) external temperature. Ice lids protect the bottom of the lake from the cold Antarctic winds and release heat during the freezing process by the latent heat of fusion.

General Introduction

Therefore, buffered lake waters can absorb solar radiation during summer reaching up to 24ºC in the bottom as it occurs in the Lake Vanda (South Victoria Land) (Takii et al., 1986). Small lakes or ponds sometimes freeze completely up to their base in winter and melts total or partially in summer, gathering the conditions to develop microbial communities. Perennial ice covers results in hypersaturation of atmospheric gases in the water columns, altered sedimentation and reduced wind-driven circulation (Wharton et al., 1999 and references therein). However, the presence of the ice cover does not impede the development of microbial communities, as the solar radiation traverse the translucent ice and allow the photosynthesis of primary producers.

Some lakes possess freshwater stream inputs (open lakes) whilst others do not receive any external inputs ( closed lakes). In the latter, the balance of water is dominated by sublimation and evaporation. Open lakes are generally freshwater whilst closed lakes trends to be meromictic and/or saline. Meromitic lakes are chemically stratified, with an incomplete circulation between the water column (Hakala, 2004). These lakes are quite abundant in Antarctica. Gibson et al. (1999) described 34 meromictic lakes in the Vestfold Hills area, providing anoxic habitats for photosynthetic sulphur bacteria, other anoxic bacteria and methanogenic Archaea. Some saline lakes are seawater, generally located at the coastline, isolated after the isostatic rebound (Gibson et al., 2006). Other sources of salinity are windblown salts, dissolved inorganic compounds from ground and rocks, evaporation of water and consequently concentration of salts, or extruded salts during the freezing process. Saline waters possess higher freezing points than freshwater lakes. These lakes are typical from Antarctic oases as Larsemann, Vestfold Hills, Bunger Hills, Antarctic Peninsula and maritime Antarctica (Gibson et al., 2006).

Low altitude lakes were formed after the isostatic rebound following the postglacial retreat of ice. Commonly, the sea water trapped was flushed out in open lakes or has been diluted by water from melting ice in Closed lakes. High altitude lakes, located at higher elevations than the Holocene’ maximum sea level, were formed by glacial retreat and are typical from the maritime Antarctic Islands (Hodgson et al., 2004a).

Lakes formed in marine embayment or fjords dammed by advancing glaciers and isolated from the sea, are denominated epishelf lakes. They are almost restricted to Antarctica, with only one or two similar lakes described in the Arctic (Veillette et al., 2008; Mueller et al., 2003). In epishelf lakes, sea water has been gradually replaced by freshwater from glaciers and snow. In some cases epishelf lakes are connected to the sea under and became stratified with freshwater underlying a layer of saline water. This is due to an “ice conveyor belt” system. In some places, the ice shelf is in contact with the seabed. In the surface, freshwater lakes are formed by partial melting of the ice shelf. When water evaporates, because of sublimation or evaporation, new ice is formed by seawater freezing on the bottom and ice gradually moves upwards, carrying the materials that are trapped into it such as microorganisms, animals, and other seafloor debris (Fig. 11). This dark colour debris increases the melting as they possess a higher ratio of sunlight absorption (Hawes and Howard-Williams, 2003).

General Introduction

Lakes are dominated by benthic cyanobacteria and diatoms. Planktonic cyanobacteria have also been found to be abundant in some Antarctic lakes. Other phytoplankton and bacterioplankton are present; with important seasonally changes in their composition and richness (Powell et al., 2005; Vincent, 1988 and 2000b).

Figure 11. Features involved in the formation and evolution of the ice shelf ponds. (Figure from Hawes and Howard-Williams, 2003).

3.2. Cryoconites.

Particles and debris can be windblown and travel long distances before falling down to the snow. The darker color of these particles increases the caption of solar radiation which leads to heating and melting of the surrounding ice, producing a cylindrical basin, a cryoconite, filled with liquid water. During the winter, ice lids can close the crycononite (“sealed crycononites”) which can persist for several years (Fig. 12). Fountain et al. (2004) reported closed cryoconites with lids up to 30 cm that persist for 10 years or more. Viable cells from cryoconites can be transported by the same mechanisms as particles and debris, and therefore colonize new habitats. Several authors have found cyanobacteria, photosynthetic algae, heterotrophic bacteria, rotifers, tardigrades and other microorganisms within cryoconites (Wharton et al., 1985, Mueller et al., 2001, Porazinska et al ., 2004).

Cryoconite holes can cover 0.1-10% of the surface area of glaciers and they can act as refuge of microorganisms. Cryoconites are autotrophic ecosystems with an important role in primary production during summer, comparable to soils in warmer and nutrient-

General Introduction rich regions (Anesio et al., 2009). For instance, Hodson et al. (2008) highlighted their + 3- role in the biogeochemical cycling of key nutrients such as NH 4 and PO 4 in glacial - ecosystems. Cryoconites are also an important input of NO 3 due to the capacity of heterocystous cyanobacteria to fix nitrogen directly from the atmosphere.

Figure 12. Composition of a cryoconite hole showing (a) closed, (b) open and (c) submerged morphologies (from Hodson et al., 2008).

3.3. Rocks.

The lithobionts are microorganisms inhabiting in, on or under hard rock substrates (Golubic et al., 1981). Covered by a mineral layer, the microorganisms are protected from the wind and the harsh ultraviolet radiation whilst freeze-thaw cycles are also mitigated (Cockell and Stokes, 2004). In general, the colonization of the underside and/or rock fissures by photosynthetic microorganisms requires a sufficiently translucent material, such as quartz, to allow the penetration of photosynthetically active radiation (PAR).

Epilithic microorganisms inhabit the surface of the rock. Temperatures are several degrees higher than the air temperature due to the different energy absorption ratio which depends of the mineral properties. If they colonize the interior of the rocks, they are considered as endoliths . There are 3 types of endoliths (Fig. 13): i) chasmoendoliths , which colonize fissures and cracks in the rock; ii) cryptoendoliths , which colonize structural cavities within porous rocks, including spaces produced by euendoliths; and iii) euendoliths , which penetrate actively into the interior of the rocks

General Introduction forming tunnels. Meltwater is the main source of water for these communities. Dew or rime has been recently suggested as a water source in addition to the rare snowfalls, in chasmoendolithic cyanobacterial communities living inside granite rocks in the Taylor valley (McMurdo Dry Valleys) (Büdel et al., 2008). Finally, there are some microorganisms that colonize the surfaces under rocks ( hypoliths ).

Figure 13. Schematic representation of various types of endoliths (from Golubic et al., 1981)

3.4. Soils.

Antarctic soils show different stages of development but they are often characterized by extreme cold and aridity. The richest soils are usually located in the coastal shoreline. Coastal soils usually possess higher levels of water content, but always subjected to rapid freeze-thaw cycles and large shifts in moisture seasonal availability. The high concentrations of soluble salts are often a restricting factor for microbial growth.

Three kinds of soils can be distinguished in Antarctica (Vincent, 1988): i) high latitude soils (ie. the Transantarctic mountains). Here, the low temperatures rise over the freezing point only a few times per year and the permafrost persists all the year. Soils are ahumic and are covered by lag gravel, fine gravel, sand and stained-oxidized material with up to 4% clay and salts. Chemical weathering is minimal in these soils. ii) Soils at the margin of the continent . The temperatures are less severe, with warmer summers reaching temperatures up to 5ºC. The moisture content and chemical weathering are slightly higher than in the previous kind of soils, although salts and clay are present in high concentrations. Patches of microbial mats usually underlies a primary humus layer with sand. iii) Continental coastline and offshore islands . These soils are characterized by relatively high moisture content and organic deposits from seabirds and mammals. The chemical weathering is higher as well as the organic nitrogen and carbon contents, normally due to birds nesting or plant growth. Mosses,

General Introduction lichens and algae can grow in quite extensive patches, which are sometimes slowly degraded and form thick (1.25 m) peat deposits.

The organic content of the soils spans between the extremely carbon-poor soils of the Transantarctic Mountains to the rich soils colonized by penguins or birds (Vincent, 1988). The Dry Valley region contains low carbon contents while the coastal areas and islands, as Signy Island, contain higher organic content due to the decomposition of lichens and bryophytes. On the contrary, nitrogen is unusually highly concentrated, due to marine and atmospheric inputs. Significantly high N 2 proportions are found in ornithogenic soils (Vincent, 1988).

Moisture content is extremely low in Antarctic soils. The Dry Valleys of Victoria Land contain moisture values comparable to temperate latitude deserts. Snowfalls are very rare in the Dry Valleys but play an important role activating short periods of microbial activity. Antarctic soils, as other temperate deserts, are usually highly saline. Evaporation or sublimation exceeds precipitation. Soils are often divided in polygons due to repetitive freeze-thaw cycles (Fig. 14). Temperature shifts are considerable, crossing the 0ºC threshold several times per day. Dark soils catch sunlight at a higher ratio than white snow. Therefore, temperatures over rock can reach higher temperatures than the air. For instance, the soil temperature at Mount Melbourne (2700 m a.s.l.) can reach 40ºC despite the -20ºC of the air temperature (Broady et al., 1987).

Despite these unfavorable conditions, Antarctic soils harbor a variety of microorganisms such as microalgae, fungi, yeast, actinomycetes and protozoans (less abundant), springtails, mites and bacteria, including cyanobacteria which are usually the dominant phototrophs (Vincent, 1988).

Soil polygons are common part of Antarctic soils (Fig. 14). Polygons are opaque but they can also host microbial life. Colonization of polygons located in Alexander Island ice-free areas (Antarctic Peninsula) was of 5% under the polygons, but 100% on their edges. This is due to the higher light intensities on the edges whilst lower PAR levels are measured in the interior of the polygons because some light can penetrate and reach parts of the interior during freeze-thaw cycles. The light intensity threshold to perform photosynthesis has been estimated in 0.06% of the ambient light intensity (considering the value of 2000 µmoles m -2· s -1 at midday) (Cockell and Stockes, 2006). Meltwater can be wind-dried by the winds at the surface, but remains in the cavities around the polygons (Cockell and Stokes, 2004). The crust thickness regulates the amount of light and water that can be used by the microbial communities. In fact, dew deposition is strongly affected by the surface crust due to its strong influence on substratum heat flux.

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Figure 14. (a) Polygonal pattern soils in Beacon Valley (Department of Geography, University of Sheffield). (b) Frost-sorted polygon with central fines in Cierva Point (Antarctic Peninsula) (picture from Mataloni et al., 2000).

3.5. Snow. Antarctic snow is generally deep cold and dry, providing little opportunities to develop microbial structures which most likely occurs in maritime zones and near of ice-free areas, where melting is less rare and provides liquid water. In high-altitude or high- latitude areas, colored patches (red, orange, and green) appear sporadically on snowfields during the snow-melting period (Fujii et al., 2010). Algae growing in or on the surface of snow and ice are well known from other latitudes but rare in the Antarctic region (Vincent, 1988). “Red snow” is formed by snow algae (eukaryotic phototrophs adapted to cold habitats) which produce the red colored astaxanthin pigments (Fig. 15). They have been detected all over the world, including Antarctica (Langhovde, Lützow- Holm Bay, East Antarctica). Fujii et al. (2010) detected cyanobacterial sequences by clone library from this red snow, related to Oscillatoriales sequences from other cold habitats and other Antarctic locations. However, no occurrence of red snow were reported from these locations. Snow algae are adapted to harsh environments including low temperature, nutrient depletion, and high UV irradiation. As snow is nutrient depleted, they play an important role as primary producers and sustain a community often based on ice worms, collembola, and bacteria. However, little is known about the factors controlling the occurrence and distribution of red snow in Antarctica or in other continents.

General Introduction

Figure 15. Red snow pictures. a) Red algae on snowfield at Newcomb Bay, East Antarctica (picture from the Australian Antarctic Division). b) Microphotograph of a red snow sample from Lützow-Holm Bay, East Antarctica (picture from Fujii et al., 2010).

4. Endemism and species adaptation.

4.1. Everything is everywhere versus endemism.

In 1934, Professor Lourens Gerhard Marinus Baas Becking stated his famous sentence, “Everything is everywhere but, the environment selects” which has been largely used in studies of prokaryotic and protist biodiversity and their biogeographical patterns. This concept is clearly opposed to other biodiversity theories that pointed the importance of geographical obstacles and dispersal limitation, claiming a higher role of stochasticity in the dispersal and installation of microbial communities (de Wit and Bouvier, 2006 and references therein).

Although Antarctica has been long considered as an isolated system surrounded by the Southern Ocean, invasion of exogenous species are being observed nowadays. For instance, marine species such as the Hyas areneus spider crab, original from the North Atlantic, and some larvae of subpolar invertebrates are now colonizing some benthic areas of the Antarctic Peninsula (Clarke et al., 2005). This can be due to a certain permeability of the barriers that isolate the Antarctic continent.

Vincent (2000) indicated that microbiota could be dispersed in Antarctica by the action of the ocean currents. Several transport mechanisms have been proposed to explain the passage across the Antarctic circumpolar current (ACC). Formation of eddies, carrying picoplankton, larvae or benthic or epipelagic organisms has been pointed out, although they do not seem to be strong enough to transport benthic forms as the spider crab larvae (Clarke et al., 2005). Other mechanisms that facilitate the transport of certain biota are the pumice, wood and kelp rafts, and more recently, human activities. Organisms can travel for long distances and reach the Antarctic continent attached to ships, ballast water, or human mediated, attached to the scientists or tourists who visit

General Introduction the continent. Most of the ships destined to the navigation in the southern ocean are not painted with antifoulant because it is quickly scraped off by the ice. Therefore, macro and microorganisms easily attach to the vessels’ surfaces, becoming an excellent medium of transport to cross the ACC. In any case, not all the potential colonists that cross the ACC are able to establish and develop in the Southern Ocean or the Antarctic continent, specially due to the decrease of temperatures when going south of the Polar Front (Clarke et al., 2005).

Other studies considered the possibility of transportation of bacteria by wind, oceanic currents or macroorganisms as birds or humans. Thus, Schlichting and Speziale (1979) detected several microorganisms including algae, Protozoa and fungi attached to Antarctic flying birds. Moss spores, pollen, fungal spores, algal and lichen propagules, viruses, tardigrade cysts, nematodes, arthropods, cyanobacteria and other bacteria could be transported by air propagules to the Antarctic continent (Pearce et al., 2009 and references therein).

Recent ice sheet modeling of the Last Glacial Maximun (LGM) estimated previous ice maxima indicating that all or almost all the currently ice free areas have been overridden during the previous glaciations. This implies that all pre-glacial (Mesozoic) Antarctic terrestrial life should have disappeared and therefore that all the actual biota should have been installed after glacial retreat. However, the actual glaciological models used for simulations of the extension of the Antarctic ice sheets have not enough resolution to recreate the situation of outlet glaciers or ice streams smaller as 20-40 km. Thus, the extension of Antarctic refugia in the LGM could be greater than previously accepted (Convey et al., 2009). In addition, biological evidences support the endemicity and the existence of glacial refugia in low-altitude locations. Antarctic terrestrial biota from the pre-Gondwana era has been observed in some regions (Pugh and Convey, 2008). This suggests that some areas escaped from the glaciations and remained isolated in long time-scales (Fig. 16). At the time when Antarctica was part of the Gondwana supercontinent, the climate was temperate or sub-tropical. Fossil proxies indicate that the change from the sub-tropical to the actual Antarctic climate, did not automatically lead to the extermination of all the fauna and flora. For instance, tundra communities survived the formation of the permanent ice sheets, persisting until the Miocene, mid- Pliocene or even later (Ashworth and Cantrill, 2004, and references therein) and the existence of nunataks which have been exposed from the pre-LGM e.g. at the Ellsworth Mountains (Fogwill et al., 2007) could provide refugia for some microorganism which could spread to other ice-free areas when ice and glacier retreated.

Several species from different groups have been recently studied for their potential endemicity to the Antarctic continent. For example, chironomid midges endemic to tectonically distinct parts of the AP and the Scotia Arc persisted ca. 50 million years (Allegrucci et al., 2006). Some nematode species are also endemic to Antarctica, in spite of their great capability for long distance dispersal and gene flow (Maslen and Convey, 2006).

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The ratio of endemism of the major photosynthetic microorganisms in Antarctica is still being discussed. Diatoms have undergone erroneous identifications until the late 1980’s because the standard keys for identification of diatoms were developed for European identifications (e.g. Krammer and Lange-Bertalot, 1991) and therefore, many species were considered of cosmopolitan distribution. In addition, the plasticity of the diatom phenotypes caused by the environment led to species misidentification. For example, the marine planktonic diatoms Thalassiosira gravida Cleve and T. rotula Meunier were originally considered to be two distinct species (Hasle, 1976). Culture experiments showed however that valve morphology changed from T. rotula to T. gravida when the growth temperature was decreased (Syvertsen, 1977). However, in other studies several diatoms sharing similar morphologies were considered as the same species and therefore the diversity was reduced. Taking into account these factors and using more fine grained taxonomy , Sabbe et al. (2003) recognized a greater percentage of endemism in diatoms than previously known. Furthermore, the diatoms also appear to be distributed in biogeographical provinces which correspond to the distribution of invertebrate faunas and higher plants within the Antarctic continent. This suggests that the long-term and large-scale mechanisms applying to the biogeography of macroorganisms and diatoms may be similar (Vyverman et al., 2010).

Regarding green algae, De Wever et al. (2009) identified 14 chlorophycean and trebouxiophycean lineages which apparently showed an Antarctic endemic distribution. Time-calibrated phylogenies indicated isolation times of 84-2.7 Ma for some chlorophycean isolates and 708-330 Ma for a trebouxiophycean isolate. Following the proposed De Wever’s chronogram, they mostly diverged at the time of the opening of the Drake passage (84-30 Ma) but even at pre-Gondwana time-scales (100-65 Ma).

Regarding the cyanobacteria, most of the studies to date have worked on samples from coastal regions of East Antarctica (Taton et al ., 2006a&b), the McMurdo Dry Valleys (Taton et al ., 2003) and the McMurdo Ice Shelf (Bratina Island) (Jungblut et al. 2005, 2006, 2009a, 2009b). The Taton’ studies suggested that potential Antarctic endemic OTUs (57%) were more abundant than previously estimated by morphological methods (35%). However, recent update of the database with new cyanobacterial sequences has drastically changed this proportion, which dropped to 28.5% in 2011. Recently, Jungblut et al. (2009a) observed high similarity levels between alpine, Arctic and Antarctic genotypes, including some sequences previously considered as endemic to Antarctica, as Leptolyngbya antarctica sequences which were 99.8% similar to Arctic sequences. In general, ca. 68% of the genotypes analyzed in the Jungblut et al.’ study were (only) related to sequences from perennially cold terrestrial ecosystems, indicating a possible global distribution of cyanobacteria adapted to low temperatures throughout the cold terrestrial biosphere.

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Figure 16. Map showing the identification of pre-LGM refuges (Picture from Convey et al., 2008).

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4.2. Adaptation strategies.

Most Antarctic marine species are stenothermal, with maximal temperatures between 5 and 10ºC. In general, increase in the organismal complexity reduces thermal tolerance. Invertebrates seem quite reluctant to acclimate to elevated temperatures. The bivalve mollusc Laternula elliptica shifts to an anaerobic metabolism at around 6°C and has an upper lethal temperature of 9°C. The scallop Adamussium colbecki loses the ability to swim at 1-2°C and dies at 5-6°C. Fifty percent of the population of the limpet Nacella concinna loses the ability to turn themselves when turned over at ca. 2°C and has an upper lethal temperature of 9.5°C (Fig. 17) (review in Hodgson et al., 2009).

Temperature, viscosity, density, oxygen saturation of water and the formation of ice crystals, are the main factors influencing the evolution of Antarctic fishes. Near of the continent, the water temperature is always under the freezing point (-1.87ºC for sea water). At that temperature, the concentration of oxygen in the seawater is 1.6 times higher than at 20ºC. This water is also very viscous: viscosity at 0ºC is twice than at 25ºC. When liquids are in supercooled state, small ice crystals act as seeds, which can start an instantaneous freezing chain reaction. In this environment, Antarctic fishes developed a series of antifreeze molecules such as glycoproteins and peptides (Devries, 1982). Among the physiological adaptations of these fishes, we can observe an absence of swim bladder and very cartilaginous bones. Their kidneys are modified to avoid the elimination of glycoproteins and their cardiovascular system possesses a low number of red blood cells. The Channichthydes, from the Notothenoids family, show a special adaptation related to the oxygen concentration in the Antarctic waters. They do not produce haemoglobin nor myoglobin because the oxygen diffuse directly from gills to the blood and then to the different tissues. Their blood is transparent, the heart is bigger than in other fish species, possess an especially spongy myocardium and mitochondria are more numerous (Hamoir, 1988).

Mechanisms involved in the adaptation of cyanobacteria to cope with the extreme Antarctic habitat conditions are discussed in section 6.5.3.

General Introduction

Figure 17. Antarctic species with special adaptations. a) Chionodraco hamatus b) Adamussium colbecki c) Laternula elliptica d) Nacella concinna (pictures from the SCAR-MarBIN web site. Authors are cited in each picture).

5. Micropaleolimnology.

Antarctica is a fantastic archive of past climate changes and paleolimnological records. Instrumental data from the Antarctic continent are quite recent, only covering the last four decades. However, the thick ice sheet archived very old ice, as the 800 Ka ice core studied by Wolff et al. (2010) from the Epica Dome C ice core. Thus, the Antarctic ice sheet is a unique depository of paleoclimatic and paleomicrobiological information that reflects previous ecological events at planetary scale. Dust particles, spores, pollen and different microorganisms brought by the wind, were gradually buried under ice depositions and frozen, thus being preserved for hundreds of thousands years (Abyzov et al., 2004). Ratios of different isotopes such as H2 or O2 in water molecules or CO 2 and CH 4 gas molecules in the ice, enable the reconstruction of past temperatures (Hodgson et al., 2009). Salt concentrations can be related to sea ice levels or the extension of the ice sheets in the past. A wide range of molecules such as pollutants and other proxies, are accumulated in the ice and then compacted by new layers of snow and ice. The Ice cores can then be used, for instance, to infer the influence of anthropogenic impacts.

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Most fossil microorganisms are preserved in cherts, phosphorites, evaporate deposits (halites) (Fish et al., 2002) or ice (Segawa et al., 2010). However, freshwater ecosystems have been less studied for cyanobacteria by paleolimnologists because vegetative cells are not well preserved in lake sediments and their small size and lack of morphological diacritical characters make identification difficult (Krings et al., 2007). It is not the case of other biological proxies such as diatoms, which silica valves (frustules) are detected intact after long timescales (Räsänen et al., 2006). Another interesting proxy is the ancient or fossil DNA (aDNA) preserved on sediment core layers. aDNA can provide information about past communities and their link with the environmental factors at that time (Coolen et al., 2008) (see section 5.7).

5.1. Cores.

Lake and marine sediment cores together with ice cores can provide detailed records of climate change and its influence on the biosphere, hydrosphere and cryosphere (Fig. 18). For instance, Rohling et al. (2004) combined these 3 core types to determine that Antarctic and northen ice sheets equally contributed to the glacial sea level changes during the period between 75000 and 20000 years ago.

5.1.1. Lake sediment cores.

Sediments are accumulated during centuries at the bottom of Antarctic lakes, including microfossils, pigments or aDNA. Most of Antarctic lakes which possess stratigraphic records are from Holocene origin. However not all lakes contains stratigraphic records, as small or shallow lakes can completely evaporate and then lose their sediment records blown by the strong Antarctic winds. In general, Antarctic lakes are useful for paleolimnological studies as their biota strongly responds to the environmental changes. In Maritime Antarctica, the recent temperature rise has resulted in enhanced lacustrine primary productivity (Quayle et al ., 2002), whereas in Arctic lakes, the recent warming has led to a negative precipitation-evaporation balance (Smol and Douglas, 2007) and marked changes in the planktonic community structure (Smal et al ., 2005). Antarctic lakes can also contain records from the isostatic lift and the relative sea level change, making lakes a useful proxy together with ice cores and marine cores to investigate how changes in the ice sheets influenced the sea levels. Wagner et al. (2010) analyzed the level changes and past evaporation cycles in Lake Bonney (Taylor Valley, Antarctica) by the study of the variation in halite crystal sizes and the composition of sedimentary rocks. The low and stable proportion of non-water soluble clastic material (i.e. mirenals and organic matter) was related to the absence of major desiccation or dissolution events, although the gravel at the core and the top parts of the core were related to more saline conditions and the small salt crystal sizes in the middle part of the core indicated more freshwater conditions.

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5.1.2. Sea sediment cores.

The ocean floor contains important sediment records which are usually sampled from beneath the surface of the deep sea. Sediments contain macro and micro fossils (Yasuhara and Cronin, 2008), metals, humic substances (Calace et al., 2005) and other proxies. For instance, sands can indicate the presence of ocean currents, ancient shorelines, submarine earthquakes, etc. Bijl et al. (2009) reconstructed the temperatures of the southwest Pacific by the data contained in a sea core from the East Tasman Plateau. Temperatures rose above present-day tropical values during the Early Eocene and gradually decreased to ca. 21ºC by the early Late Eocene.

Sea sediment cores allow the reconstruction of the extent ice cover. In a recent study, Shevenell et al. (2011) highlighted the importance of regional summer duration as a driver of Antarctic seasonal sea-ice fluctuations from a sea surface temperature proxy from a western Antarctic Peninsula sea sediment core.

5.1.3. Ice cores.

Snow is buried by new snowfall at the surface of the ice sheets and then compressed and eventually transformed into solid ice, a process which captures chemical records of past climates and environments. Organisms trapped in the ice might remain viable in a dormant state, or maintain metabolic activity as shown by thymidine and leucine incorporation experiments on E. coli , which showed metabolic activity at -15ºC but not at -70ºC (Carpenter et al., 2000). Ice cores from glacial ice are an intriguing habitat to study life, because it represents an extreme environment with low nutrient concentrations and low temperatures. This habitat could provide insights into microbial survival and serve as exoplanets analogues (Miteva and Brenchley, 2005). DNA and amino acids from dead organisms, buried in ice, can be also recovered from the basal sections of deep ice cores and allow reconstructions of past flora and fauna (Willerslev et al., 2007). The last glacial termination chronology has been reconstructed using a Greenland ice core. Ice cores from both Antarctica and Greenland suggested temperature between 2-5ºC over the past 400 Ka and that sea levels were ca- 4-6 m higher than today (Hodgson et al., 2009; Rasmussen et al., 2006).

General Introduction

Figure 18. a) Research vessel JOIDES, adapted for sea sediment coring (Picture : William Crawford, IODP, Texas A&M University); b) Lake sediment core from Beak Island; c and d) Ice cores from the Epica project (Science cover).

5.2. Cyanobacterial microfossils.

Cyanobacteria are one of the oldest organisms in Earth history, and therefore traces of their fossils are abundant all over the World. However, due to the small size of cyanobacteria, their microfossils are subjected to textural transformations of sediments, such as recrystallization, mineral replacement and metamorphism. Due to these transformations, cyanobacteria microfossils are usually deformed (Golubic and Seong- Joo, 1999).

Cyanobacterial fossil records can be recognized on the basis of their fossilized cell structures such as vegetative cells, akinetes or heterocysts; chemical fossils such as the molecules produced during cyanobacterial activity (e.g. exopolysaccharides, pigments, bacteriohopanepolyols); the traces of their living structures (e.g. stromatolites, tunnels into the rocks, annual growth layers) and their DNA, when preserved.

For instance, the presence of 2-methylhopanes characteristic of cyanobacteria, in molecular fossils preserved in 2700 Ma shales in Australia, provided evidence that oxygenic photosynthesis evolved before the atmosphere became oxidizing (Brocks et al., 1999). Analysis of the glycolipids which envelops the heterocysts in N2-fixing

General Introduction cyanobacteria, from Arctic Sea sediments, indicates that the symbiosis with the Azolla fern was already effective during the Eocene (56-40 Ma) (Bauersachs et al., 2010).

Stromatolites are macrofossils structures related to cyanobacteria. These laminated organosedimentary structures are sedimentary deposits that are the direct results of interactions between microorganisms and their surrounding environment, but microorganisms are rarely preserved (Walter et al., 1992). Stromatolites dominated the Earth for over 3 billion years (Foster et al., 2009) but modern stromatolites are relegated to a few locations as the Island of Highborne Cay (Bahamas) and the Shark Bay (Fig. 19).

The earliest microfossils of cyanobacteria were claimed to be recorded in Australia (3500 Ma old) by Schopf et al. (2006). However, Brasier et al. (2002) contradicted the Schopf’s findings, suggesting that the geology and the shape of the microfossils were misinterpreted in the Schopf publication. Brasier suggested that the Schopf’s microfossils where not preserved on marine sediments but in hydrothermal vents and that therefore the development of cyanobacteria could not be possible. Schopf et al.’s finally suggested that the microfossil structures were indistinguishable from mineral growths, and therefore the presence of cyanobacteria in those samples was doubtful.

More recent findings include the works of Krings et al. (2007) and Jiang et al. (2008). Krings et al. described a new aquatic filamentous fossil cyanobacterium showing structured colonial growth from the Early Devonian Rhynie chert (400 Ma) (England) (Table 1). Jiang et al. recorded filamentous cyanobacterial fossils morphologically similar to Rivularia from the Permian-Triassic (ca. 250 Ma ago) in the Laolongdong region (Southwest China). Several authors have suggested an anoxic event in the Laolongdong section because of pyrite formation. Considering that this cyanobacterium can survive in dysoxic conditions, the bloom of this organism and the absence of others corroborates that the environment was oxygen-deficient at that time.

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a c

e f

Figure 19. a) Modern stromatolites in Shark Bay, Australia. Picture from the University of Wisconsin- Madison; b) cross sections of 1.8 billion year old fossil stromatolites at Great Slave Lake, Canada (picture from David Ward, Montana State University); c&d) oscillatorian cyanobacterium-like archean microfossils (ca. 2500-2700 Ma) (pictures from Klein et al., 1987; Buick, 2001); e&f) oscillatorian cyanobacterium-like microfossils from the 2723 Ma old Tumbiana Formation of Western Australia (Schopf and Walter, 1983).

Certain cyanobacteria are able to produce resistance forms called akinetes. Akinetes are resting spores that can be reactivated under optimal conditions. In lacustrine environments, it has been postulated that they act as reservoirs of life, reinoculating the lake when the nutrients and appropriate conditions come back. Several papers studied the germination limits of the akinetes found in sediment cores. Thus, Wood et al. (2008) extracted a sediment core from Lake Okaro (New Zealand). By ARISA and clone library, they found two filamentous cyanobacterial sequences related to Pseudanabaena and Anabaena , and observed akinete germination from 120 years old sediments. Nevertheless, there are no studies about cyanobacterial microfossils in Antarctica.

The studies of the cyanobacterial past communities based on fossil DNA are very sparse and in general cyanobacteria were collaterally found in metagenomic studies from different samples such as evaporites (Panieri et al., 2010). However, there are no references in the literature based on the study of cyanobacterial fossil DNA from sediment cores from the Antarctic continent apart from Coolen et al. (2008), where only two Synechococcus related spp. sequences were detected.

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Eon Location Estimated age (10 9 years BP) Phanerozoic Rhynie Chert, England. 0.3-0.4

Neoproterozoic Doushantuo formation, China. 0.5-0.6 Bitter Springs formation, Australia. 0.8

Mesoproterozoic Wumishan formation, China. 1.2-1.3 Billyakh group, Russia 1.3-1.6 Paleoproterozoic Balbirini dolomite, Australia. 1.7 Belcher supergroup, Canada. 1.9-2.1 Late Archean Transvaal supergroup, South Africa. 2.5 Early Archean Warrawoona group, Australia. 3.4-3.5

Table 1. Location of the major sites containing fossil cyanobacteria (after Golubic and Seong-Joo, 1999).

5.3. Pollen and mosses.

Angiosperms are not present in continental Antarctica and only 2 ( Deschampia antarctica and Colobanthus quitensis ) live in maritime Antarctica (Fig. 20). However, pollen from plants from other continents has been found in Antarctic sediment cores, which has been used to detect the different dynamics of air masses (Björck et al., 1993).

The presence of mosses, which are able to produce spores, is a good indicator of stable conditions because limiting factors for moss growth include increased salinity or influxes of silty material and unstable bottom lakes (Verkulich et al., 2002).

Figure 20. a) Deschampia antarctica and b) Colobanthus quitensis , the two only Antarctic Spermatophyta (pictures from the SCAR-MarBIN website).

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5.4. Diatoms.

Diatoms are one of the major components of the benthic microbial communities in Antarctica (Sabbe et al., 2003). They have been successfully used as proxies to reconstruct changes in lake salinity, ice cover, sea level, UV radiation and other phenomena indicative of climatic change. This can be done due to the great sensitivity of diatoms to changes in water chemistry, salinity, nutrients depth and other physical parameters (Spaulding and McKnight, 1999). Several regions of Antarctica have been studied for the ecology and distribution of diatoms. However, the diatom flora of some regions is still poorly known. Fossil diatoms are a good indicator of exotic species introduction by human-mediated activities. They provide a strong indication that geographic dispersal by human vectors was limited in the past. For instance, Asterionella formosa appears in the sediment records of numerous lakes in New Zealand after 1880 but no records were detected before this date, indicating their absence before the European colonization (Vanormelingen, 2008).

Lacustrine diatoms are usually combined with transfer functions to quantitatively reconstruct past changes in the moisture balance and nutrient levels (Verleyen et al., 2010a). Regarding polar microfossil diatoms, Michelutti et al. (2002) tracked recent eutrophication in high Arctic lake (Lake Meretta, Canada) using fossil diatoms assemblages. Microfossils and pigments from diatoms were used to reconstruct the primary production in Antarctic lakes. Verleyen et al. (2004) showed that benthic diatom production was greatest in the lacustrine core section of the Pup Lagoon and the Heart Lake (Larsemann Hills, East Antarctica) than in the marine section. In the first section, diatoms were associated with the benthic microbial mats which dominated the flora at that time, whilst diatoms were associated with the water column and sea ice during the marine periods.

5.5. Algae and bacteria.

Macrofossils of algae have been studied in several studies. For instance, Hu et al. (2010) observed fossils from the eastern Yunnan (China), similar to Margaretia from the Middle Cambrian of Utah which was assigned to the green algae. This indicates that the water environment was in the photic zone at that time. Li et al. (2009) reconstructed the history of a lake in Zhejiang (East China) using green algae microfossils among other proxies and established that the lake was a shallow, mesotrophic freshwater waterbody on the Early Pliocene. However, only a few studies have been focused on Antarctic locations. For instance, Coolen et al. (2004) analyzed the haptophyta from a sediment core from Ace Lake. Using newly developed primers, they found haptophyta-related sequences in layers containing high and low alkenone concentrations (traditional lipid biomarkers from haptophytes). In total, 6 different haptophyte sequences were retrieved. The haptophtyes were mainly detected in the deeper layers of the sediment core but not

General Introduction in the surface layers. This is in concordance with the high saline conditions in Ace Lake, which impedes the growth of these algae and suggests that more freshwater conditions existed in the past.

Bacterial fossil 16S rRNA genes (sulfur bacteria) from the Holocene have been recorded in Mahoney Lake (Canada) (Coolen and Overmann, 1998) or from Mediterranean sapropels (217 Ka) (Coolen and Overmann, 2007). Nelson et al. (2007) observed marked different bacterial communities composition in the Early, Middle and Late Holocene in a northern temperate lake. Regarding Antarctica, Segawa et al. (2010) analyzed two ice cores from the Mizuho Base (Enderby Land) and a glacial age sample from the Yamato Mountains (Dronning Maud Land). They observed that a large part of the bacteria were possibly ancient airborne bacteria, transported in the past by the air or carried by animals from different environments.

5.6. Pigments.

Fossil pigments are widely used in lacustrine sediment cores (see Leavitt and Hodgson 2001 for an overview). In some cases, biota does not leave microfossils because of degradation processes. However, pigment molecules as carotenoids and chlorophylls are preserved in the sediment layers when low temperatures and limited grazing activities occur. For instances, the carotenoid pigment zeaxanthin was detected in 340 Ka old marine sediments (Watts and Maxwell, 1977) whilst other carotenoid derivates (ie. perhydrocarotene) were detected in 50 Ma old lake sediments (Murphy et al., 1967).

The structure and dynamics of past communities can be tracked by the analysis of fossil pigments (Leavitt et al., 2003). Fossil pigments from sediment cores have been studied in Antarctic lakes to assess the contribution of cyanobacteria to primary production (Hodgson et al., 2005a), to reconstruct past changes in UV radiation penetration (Hodgson et al., 2005b; Verleyen et al., 2005), and to detect transitions from marine to lacustrine conditions and construct relative sea level curves. Hodgson et al. (2004b) analysed the pigment composition related to several factors as lake depth, conductivity, turbidity, dissolved oxygen, sulphate and geographical location. Lake depth was identified as the principal factor, presumably as a result of its influence on light regimes. That study showed that chlorophylls dominated the deeper lakes whilst chlorophylls and carotenoids dominated intermediate depth lakes and scytonemin was the most abundant pigment in shallow lakes, due to its function as UV-screen. Therefore, samples with high levels of scytonemin can be related to periods of increasing UV radiation (see section 6.5.2).

Some pigments are common to several taxa, as the beta-carotene which can be found in algae and some phototropic bacteria. Chlorophyll-a is the most common photosynthetic pigment in both eukaryotic and prokaryotic organisms. Chlorophyll-b is also found in many organisms and microorganisms, i.e. algae, plants and some cyanobacteria.

General Introduction

Bacteriochlorophylls (BChl a, b, c, d, e, and g) are indicative of prokaryotic microorganisms. Carotenoids, divided in xanthophylls and carotenes primarily act as photoprotective pigments but can also function in light harvesting, are found in plants, lagae, fungi and some bacteria (Leavitt and Hodgson 2001; Gomez Maqueo Chew and Bryant, 2007)

Some pigments are taxa-specific, such as the carotenoids alloxanthin (specific of cryptophyta) or peridin, specific of Dinophyta. Some carotenoids are unique to cyanobacteria, such as the ketocarotenoids, the echinenone and the 4-ketomyxol (Takaichi and Mochimaru, 2007). Interestingly, scytonemin (see 6.5.2) is specific of cyanobacteria (Proteau et al., 1993) (Fig. 21). Regarding the cyanobacteria, only chlorophyll a is present, with the exception of the genus Acaryochloris which also produces chlorophyll d (Kühl et al., 2005), and the genera Prochloron , Prochlorococcus and Prochlorothrix which also produce chlorophyll b (Larkum and Kühl, 2005). Cyanobacteria are also characterized by the presence of biliprotein pigments called phycobilines, accessory pigments which enhance photosynthetic efficiency. Among the phycobilines, the phycocyanine confers a typical blue color which, together with the green color from chlorophyll, provides the characteristic blue-green color of cyanobacteria. However, some cyanobacteria also possess phycoerythrin, a red phycobiline which confers a red-brownish coloration to the cells (Madigan et al., 1998).

Figure 21. Scytonemin and scytonemin derived molecules (from Sinha and Häder, 2008).

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5.7. Fossil DNA.

Most ancient or fossil DNA (aDNA) studies have focused on tissues or bones, but only a few focused on sediments (D’Andrea et al., 2006). Willerslev et al. (2007) revealed DNA and aminoacids from organisms buried under 2 km of ice in Southern Greenland, providing the first direct evidence supporting a forested Greenland. Permafrost cores from Siberia (400-10 Ka BP) contained at least 19 different plant taxa, including the oldest authenticated aDNA sequences known, and megafaunal sequences including mammoth, bison, and horse (Willerslev et al., 2003). Haloarcheal 16S rDNA amplicons have been detected in halite samples, evaporite deposits of NaCl. For instance, Mormile et al. (2003) detected DNA from the Archaea Halobacterium salinarum , from 97 Ka old halite brine inclusions. Halites represent the desiccated remains of saline lakes or ancient seas which became extinct by long water evaporation. Several microorganisms and especially haloarchea can be trapped in droplets of brine within the solid crystals (Fish et al., 2002). External contamination in halite crystal must be treated carefully, as halites can be fractured and reorganized over time and therefore novel microorganisms can enter the structures and can be erroneously identified as ancient inhabitants.

Unfortunately, DNA molecules are relatively unstable in geological systems, limiting the effectiveness of aDNA as biomarker. However, several attempts retrieved rather ancient DNA, such as the 400 Ka old DNA preserved in Siberian permafrost successfully amplified by Willerslev et al. (2003). Other studies detected much older aDNA (see chapter 5.7.2).

5.7.1. Degradation process of DNA.

DNA is an unstable molecule in comparison with other cellular components. Living cells possess DNA repairing mechanisms operated by complex enzymatic pathways. On the contrary, post mortem decay starts immediately after cell death, favored by the release of cellular enzymes in the environment. Cell rupture also releases nutrients to the medium, which are substratum for other microorganisms which subsequently continue the degradation of DNA from dead cells. As soon as the DNA molecules are not subjected to reparation mechanisms, they can suffer spontaneous degradation processes such as hydrolysis, alkylation and oxidation (Fig. 22). Crosslinking is a typical reaction occurring in DNA from dead cells. Crosslinking includes intermolecular crosslinks between different DNAs or between DNA and proteins but also interstrand crosslinks (i.e. between two DNA strands). The crosslinking can stabilize DNA molecule but impedes their correct amplification by PCR methods (Hebsgaard et al., 2005). These DNA damages are source of incomplete DNA recovery, incorrect amplification and erroneous identification. For instance, hydrolysis of DNA strands implies cleavage of DNA in short fragments. This can lead to contamination by DNA

General Introduction from living cells also present in the samples or coming from external sources or produce short PCR products (Fig. 22a). Crosslinks hinder the PCR amplifications and therefore increase the possibility of amplification of contaminants (Fig. 22b). Oxidative processes cause blocking lesions which imply that the polymerase enzyme “jumps”, producing chimeric sequences (Fig. 22c). Finally, hydrolysis produce miscoding lesions, as some bases can be modified, causing base misincorporations and therefore errors in sequencing (Fig. 22c).

General Introduction

Figure 22. DNA modification processes in fossil samples and problems related to the amplification of aDNA: (a) hydrolysis, (b) alkylation, (c) oxidation/hydrolysis. (Figure from Willerslev and Cooper, 2005).

The hydrolytic damages are responsible for breaks of the sugar backbone (Fig. 23.1.), base losses (Fig. 23.2) and deamination of bases (Fig. 23.3). The oxidative damages lead to alteration of the DNA integrity. Thus, oxygen attacks the double bond of carbons C5 and C6 of pyrimidines (cytosine and thymine) (Fig. 23.4) or the C4, C5, and C8 carbons of purines (Fig. 23.5, 23.6, 23.7) (Hebsgaard et al., 2005). Oxidative damage can also perturb the sugar backbone (23.8).

Figure 23. Main damages suffered by DNA after extinction of DNA repairing complexes. Arrows indicate the position where hydrolytic and oxidative damages occur (from Hebsgaard et al., 2005).

5.7.2. Preservation of aDNA.

Interestingly, decay of DNA is not only a time function but the preservation conditions play also a crucial role (Hebsgaard et al., 2005). Low temperature is the most important factor for aDNA stability (Willerslev 2005 and references therein) whilst rapid desiccation and high salt concentrations may also prolongate DNA survival (Lindahl, 1993).

General Introduction

Most of the fossil samples analyzed in laboratory do not contain amplifiable DNA because favorable conditions for aDNA preservation do not occur systematically. In order to predict the presence of aDNA in these samples, several methods based on aminoacids analysis have been proposed (total amount, composition and racemization, pyrolysis coupled with gas chromatography and mass spectrometry). For instance, the relative amounts of pyrolisis products of proline-containing dipeptides are a useful index of the amount of peptide hydrolysis and DNA preservation (Poinar and Stankiewicz, 1999).

Recent studies predicted the life-time of aDNA. Kinetic calculations estimated that 100- 500 bp fragments might survive 10 Ka in temperate regions or 100 Ka in cold regions (Poinar et al., 1996; Smith et al., 2001). The survival limit for aDNA has been estimated to be 1 Ma (Hofreiter et al., 2001). aDNA older than 1 Ma is called ‘antediluvian’ (Lindahl, 1993). Although several studies claimed retrieval and amplification of antediluvian DNA, reproducibility was often incomplete. Nevertheless, several studies claimed much older ages for DNA recovery. Panieri et al. (2010) described ancient cyanobacteria from mediterranean gypsum crystals dating back to 5910-5816 Ma. However, although their results were not validated in an independent laboratory, their methodology seems correct and the results unambiguous. Other examples of claimed antediluvian DNA are from Fish et al. (2002), which detected 16S ribosomal RNA gene fragments in 425-11 Ma halite crystals. Greenblatt et al. (2004) described the isolation of the non-spore-forming cocci Micrococcus luteus from a 120 Ma old block of amber. The most striking case is that of Vreeland et al. (2007) which describes the isolation of six strains of halophilic Archaea from the Cretaceous (121– 112 Ma) that were still alive and isolated in culture.

For some authors, a more rigorous validation should be applied to antediluvian DNA. For example, Hebsgaard et al. (2005) proposed a 15 points table of authentication criteria for antediluvian DNA, including intra and inter laboratory verification, extensive decontamination of samples, or the use of evolutionary rate tests. In Table 2, the authors summarized the results from several studies and the authenticity points that failed some of their criteria. Similarly, Hofreiter et al. (2001) enumerated 7 criteria to validate aDNA sequences, including biochemical assay for macromolecular preservation, inverse correlation between amplicon length and amplification efficiency, quantification of numbers of template molecules and exclusion of nuclear insertions of mitochondrial DNA.

Specimen Source Age Gene Size Culture Decontamination Indenpendent Clean Cloning Molecular Relative Reference (Ma) (bp) viability b replication c room test d test d a Amber Weevil 120- 18S 315, - + - - - Fail Fail Cano et al., 135 226 1993 Amber Legume 35-40 rbcL 348 - - - - - Pass Fail Poinar et al., 1993 Clarckia Magnolia 17-20 rbcL 820 - - - - - Pass Fail Golenberg et al., 1990 Halite Bacillus 250 16S 1560 + + - + - Fail Fail Vreeland et al., 2000 Halite Haloarchaeal/ 11- 16S 693- - + - + + Pass/Fail Fail Fish et al., Proteobacteria 415 1009 2002

Table 2. Summary of antediluvian DNA results. a Viable cells were obtained b Specimen was decontaminated before the extraction/culturing c Replication in an independent laboratory d Sequences passing or failing molecular-distance rates test and relative rates test. (From Hebsgaard et al., 2005)

General Introduction

6. Cyanobacteria.

6.1. Generalities.

Cyanobacteria are a large and heterogeneous group of oxygenic prototrophs from the Bacteria domain. Cyanobacteria differ from all the other prokaryotes in their fatty acid composition. They often produce unsaturated fatty acids with two or more double bonds whilst other bacteria produce almost exclusively mono-unsaturated fatty acids.

Cyanobacteria are one of the oldest organisms in Earth history, possibly appearing ca . 3500 Ma ago (Schopf et al., 2006) or maybe 2300 Ma ago (Blank, 2002). This implies a margin of ca. 1500-300 Ma (considering the Schopf or the Blank’s hypotheses) from the prebiotic soup to the RNA world and then to cyanobacteria. Lazcano and Miller (1994) proposed that assuming that gene duplication rate of ancient prokaryotes was comparable to present days and considering the gene recombination processes, gene sharing, fusion events, duplication and high error rates in duplication of DNA as non- self-correcting systems were yet present, the evolutionary time for the construction of cyanobacterial-like genomes should require ca. 7 Ma.

Cyanobacteria induced the change from the anoxic atmosphere of the Precambrian to the actual oxygenated atmosphere (Tomitani et al., 2006). The oxygenic photosynthesis at the beginning of this change originated monophyletically in the Bacteria, within the cyanobacterial cluster (Golubic and Seong-Joo, 1999) and was therefore transferred to other phylogenetic groups by endosymbiosis (Golubic and Seong-Joo, 1999).

Cyanobacterial symbiotic associations are an important component of the ecology of many cyanobacterial lineages. This implies associations with plants, fungi, animals, and eukaryotic algae. For instance, Nostoc sp. shows a wide number of symbiotic associations with lichenized and non-lichenized fungi, bryophytes, cycads, flowering plants and ferns (O’Brien et al., 2005 and references therein). Although symbioses of cyanobacteria with plants are restricted today, it occurs in most of the lineages of land plants (Krings et al., 2008). Some cyanobacteria are able to fix nitrogen from the atmosphere, which is shared with the host plants whilst cyanobacteria profit of a stable source of nutrients from the plants. There is direct evidence of cyanobacteria growing inside plants as early as 400 Ma ago from filamentous cyanobacteria colonizing mycorrhiza axes on Aglaophyton major (Krings et al., 2008).

6.2. Cyanobacterial structures.

Cyanobacteria possess several typical structures which can be used as diacritical characters for the identification of the species

General Introduction

Cytoplasmatic inclusions and reserve products : Several cytoplasmatic structures are visible by electron microscopy as glycogen granules (source of carbon and energy), lipid globules (lipid stores for use in the synthesis of the membrane), polyphosphate bodies (reserve of phosphate) or cyanophycin . Cyanophicin is a polypeptide composed of 2 aminoacids (arginine and aspartic acids) produced by a non-ribosomal mechanism and it is involved in nitrogen turn-over. It is a source of nitrogen and possibly carbon storage, located in the periphery of cells. Carboxysomes are reserves of the enzyme ribulose-1,5-biphosphate carboxylase which catalyses photosynthetic CO 2 fixation into ribulose-1,5-biphosphate.

Gas vacuoles : Cyanobacteria produce gas vesicles, surrounded by a protein membrane. These vesicles regulate the buoyancy in planktonic or marine cyanobacteria controlling their position in the water column, to optimize photosynthesis.

Heterocysts : Some cyanobacteria are able to fix the atmospheric nitrogen, reducing it to ammonia (NH 3) in a reaction catalyzed by the enzyme nitrogenase. Nitrogenase is quickly inactivated by oxygen. Heterocysts are oxygen-free cells, as they do not realise the photosynthesis, an elegant mechanism to avoid the presence of oxygen and perform nitrogen fixation. They occur in two orders, the Nostocales and the Stigonematales . Heterocysts evolved from vegetative cells. Their cells develop thick walls with a refractive polar nodule at one or both ends. Heterocysts are sometimes induced under nitrogen absence in the environment and suppressed when nitrogen concentration increases. Anabaena mutants lacking PBP2 cannot produce heterocysts and so cannot fix nitrogen. PBP2 is a class A Penicillin Binding Protein, a group of enzymes involved in the synthesis and cross-linking of the cell wall’s peptidoglycan. In addition, a mutation in the hcwA gene inactivates the heterocyst’s formation pathway. The hcw A protein is a putative N-acetylmuramoyl-L-amidase which removes short peptide side chains from glycan polymers of the peptidoglycan. Thus, it has been suggested that shape changes precede nitrogen fixation in cyanobacteria, in order to adapt the cell wall to produce the heterocysts (Young, 2006).

Akinetes : Several species from the Nostocales and Stigonematales orders can produce akinetes or resting spores under adverse environmental conditions such as low temperature, desiccation, high levels of salt or iron depletion (Adams and Duggan, 1999). As heterocysts, akinetes develop from vegetative cells which suffer profound changes in their biochemistry and physiology. Akinetes are often several times bigger than vegetative cells and accumulate large reserves of cyanophycin and glycogen (Whitton, 1992; Stanier and Cohen-Bazire, 1977). Akinete formation and germination may play an important role in the survival of cyanobacterial species in water bodies and they have been reported to survive for several hundreds of years (Wood et al., 2008). They can also play a role in withstanding cold winters (Sukenik et al., 2007) (Fig. 24).

General Introduction

Fig. 24. Schematic representation of the life cycle of a lacustrine filamentous cyanobacterium producing akinetes during winter (picture from Kaplan-Levy et al., 2010).

Hormogonia : Short and motile chains of cells. Hormogonia differ from the parent trichomes in their cell size and shape, gas vacuolation and often absence of heterocysts. They are produced when the cyanobacterium is exposed to environmental stress or hormogonium-inducing factors secreted by some symbiotic plants.

Necridial cells : Some cells from filamentous cyanobacteria are “sacrificed” to break up the trichomes and form new hormogonia.

Hair cells : Hair cells are cells which metamorphose into long colorless, unicellular hairs. These cells act as centers of phosphatase activity in some species of the Rivulariaceae (Nostocales). Hair cells also are present in three phyla of eukaryotic algae (green, brown and red algae) (Whitton, 1992).

Baeocytes : Baeocytes are formed by successive radial and tangential divisions of the protoplast resulting in the formation of many small daughter cells which immediately begin to grow. Unlike binary fission, which results in two daughter cells approximately half the size of the parent, multiple fission is a rapid process of division in three planes producing many small cells. Baeocytes are produced by some species of the Pleurocapsales . They were previously described as endospores (Kunkel, 1984).

General Introduction

6.3. Classification.

Cyanobacteria are currently divided into five subgroups according to the Bergey’s Manual of Systematic Bacteriology following the Stanier method (Waterbury, 2006).

I. Unicellular or non-filamentous aggregates of cells held together by outer walls or a gel-like matrix (colonies).

A-Binary fission in one, two or three planes, symmetric or asymmetric; or by budding.

Subgroup 1 (Order Chroococcales): Unicellular cyanobacteria occurring as single cocci and rods or cell aggregates. Division occurs in one, two, or three planes at right angles or in irregular planes. Cells range from 0.5 to 30 µm.

B. Reproduction by internal multiple fissions with production of daughter cells smaller than ½ the parent; or by multiple and binary fission.

Subgroup 2 (Order Pleurocapsales): Unicellular forms that divide exclusively by multiple fission or that produce cell aggregates by vegetative binary fission. Cell aggregates range in complexity from groups of a few cells to complex pseudofilamentous cell assemblages. Some species from this order can release baeocytes.

II . Filamentous; trichome of cells branched or unbranched, uniseriate or multiseriate.

A. Binary fission in one plane only giving rise to uniseriate, unbranched trichomes, although false branching may occur.

1. Trichomes composed of cells which do not differentiate into heterocysts or akinetes.

Subgroup 3 (Order Oscillatoriales): Includes all the undifferentiated filamentous cyanobacteria. Reproduction occurs by trichome fragmentation or by the production of undifferentiated hormogonia. Cell diameters vary from 0.5 to 100 µm.

2. One or a few cells of each trichome differentiate into heterocysts, at least when concentration of external combined nitrogen is low, some also produce akinetes.

Subgroup 4 (Order Nostocales): Filamentous cyanobacteria which are able of cell differentiation, ie. heterocysts, akinetes, hormogonia and tapered trichomes.

B. Binary fission periodically or commonly in more than one plane, giving rise to multiseriate trichomes or trichomes with true branches or both.

General Introduction

Subgroup 5 (Stigonematales): Filamentous cyanobacteria capable of generate the same differentiate cells than the Nostocales order. In addition, Stigonematales are able to divide by binary fission in multiple planes. The filaments may display true branching.

6.4. Cyanobacterial nomenclature and taxonomy.

Cyanobacteria were primarily considered as algae and are still called ‘green-blue algae’. As a heritage, the nomenclature of cyanobacteria was traditionally governed by the International Code of Botanical Nomenclature (ICBN). As cyanobacteria are typical bacteria, Stanier et al. (1978) proposed that their nomenclature should follow the International Code of Nomenclature of Bacteria (now the International Code of Nomenclature of Prokaryotes, ICNP). The procedure to publish the description of nomenclatural type of a cyanobacterial species differs between the ICBN and the ICNP. For instance, the ICNP includes the registration in the International Journal of Systematic and Evolutionary Microbiology (IJSEM) and the obligatory deposit of two subcultures from a living pure strain in two public culture collections. The ICBN, on the contrary, demand for permanently preserved material (e.g. herbarium specimen) but it may not be living cultures. The Judicial Commission of the International Committee on Systematics of Bacteria (ICSB) recommended that the cyanobacterial names described under the ICBN should be accepted by ICNP but no rules were established to really reconcile both Codes. Indeed, most of those names are not legitimated by the ICNP because of different basic requirements. Because both codes are still ongoing, Oren and Tindall (2005) proposed that the ideal solution should use a “joint nomenclature and the use of identical methods for the description of species and designation of types”. Due to this problematic and to the difficult process to validate new names of cyanobacteria, only 43 articles on cyanobacteria have been published in the International Bulletin of Bacteriological Nomenclature and Taxonomy/ International Journal of Systematic Bacteriology/ IJSEM during its 60 years of existence (Oren, 2010). For instance, only 6 cyanobacterial genera were validated under the bacteriological code in the 2004’ validation list.

The Stanier’s system was the basis for the cyanobacterial taxonomy as described in the Bergey’s Manual of Systematic Bacteriology (Castenholz, 2001). However, the Bergey’s system has the inconvenient that is based only on isolated strains and its resolution stops at the genus level. The actual cyanobacterial taxonomy uses as much information as possible to better identify the species. Therefore, morphological, physiological, chemotaxonomic and genotypic characters are included for an accurate identification (Oren, 2010). The molecular studies are mostly based on the 16S rRNA genes, which are conserved markers. Indeed, the mutation rate of 16S rRNA genes has been estimated as 1% every 50 millions of years (Ochman and Prager, 1987). However other markers are also used in the phylogeny of cyanobacteria, such as rpo C1, cpc BA, asrpo C1, het R, rbc LX, mcy genes, nif H or the Internal Transcribed Spacers (ITS). Several authors pointed to a good correlation between different phylogenetic markers such as 16Sr RNA, rpo C1 or mcy (Rantala et al, 2004). However, Han et al. (2009)

General Introduction suggested a high incongruence between the markers rpo C1, het R, rbc LX and ITS compared with the 16S rRNA-based phylogeny in 7 Nostoc strains. The databases of rpo C1, nif H and other genes with conservation ratios high enough to be used in phylogeny are still fragmentary and therefore, the 16S rRNA genes can provide more information in taxonomic studies. Thus, 16S rRNA analysis remains useful for identification of species from environmental samples. In addition to the 16S rRNA genes, the analysis of the adjacent ITS can provide information at the intra-species level (Taton et al., 2006a).

The increasing number of molecular studies which do not take account the morphologies observed from the environmental samples or do not realise culture efforts, leads to a misidentification of number of sequences in the databases. The lack of sequences from cultured strains cause that in some cases the environmental sequences show very low similarity levels with strain sequences when similitude analyses are performed using the existing databases (i.e. BLAST analysis). In addition, several molecular studies are based on cyanobacterial specific primers and consider that all the obtained sequences are from cyanobacteria. However, these primers can also amplify sequences from plasts (see chapter 6).

Some cyanobacterial lineages are well defined and homogenous, but other lineages are not well delimited and heterogeneous. Genera such as Planktothrix or Arthospira are considered monophyletic while other genera such as Leptolyngbya or Synechococcus are considered polyphyletic. The cyanobacterial taxonomy is still under construction and several groups need a deep revision of their members. For example, Suda et al. (2002) analyzed the water-bloom-forming genus Oscillatoria . Fifty-six Oscillatoria spp. sequences were analyzed and grouped into six clusters, clearly separated from the Oscillatoria type species Oscillatoria princeps Gomont NIVA CYA 150. Therefore, Suda et al. proposed emended (e.g. Planktothrix agardhii , Planktothrix rubescens , Planktothrix mougeotii , Tychonema bourrellyi and Limnothrix redekei ) or new taxonomic descriptions (e.g. Planktothrix pseudagardhii sp. nov. and Planktothricoides raciborskii gen. nov., comb. nov.) for several species.

Leptolyngbya is widely distributed in many ecosystems on Earth, specially dominating polar habitats. The intrageneric taxonomic classification of the genus Leptolyngbya Anagn. et Kom. (1988) is difficult because the small size of the cells and their simple morphology. For instance, several genera from the order Oscillatoriales were previously included within the Leptolyngbya genus. Thus, Phormidium antarcticum was renamed as L. antarctica, Lyngbya erebi as L. erebi , Phormidium glaciale as L. glacialis , etc. (Komarek et al., 2007). Furthermore, Komarek (2010) reviewed the Nostocales using the new molecular data (16S rRNA) and the previously known morphologies and ecologies. For instance, Raphidiopsis was defined as an “unclear and polymorphic genus, containing probably several morpho- and genotypes”. Cuspidothrix , which was originally included within the Aphanizomenon genus and Cylindrospermopsis , which was originally included in the genera Anabaena , were considered as new genera. The

General Introduction genus Aphanizomenon was reduced to eight morphotypes whilst several Anabaena clusters were also reorganized.

In summary, the cyanobacterial taxonomy shows a high degree of complexity and there are several groups which will need further revisions.

In this thesis, we use, in general, a polyphasic approach. The molecular studies are based on the 16S rRNA genes and the sequences compared by grouping them into OTUs. An OTU is a taxonomic unit, formed by sequences which share at least 97.5% of similarity. Because most of the sequences from the databases are short, we used an OTU concept based on the positions 405–780, not taking into account indels and ambiguous bases (Taton et al., 2003). For complete 16S rRNA gene sequences, a binary similarity value less than 97.5% corresponds to a DNA-DNA hybridization value less than 70% (Stackebrandt and Goebel, 1994). This implies that two sequences from different OTUs are surely from different species. In addition, Taton et al. (2003) calculated a good correlation between complete 16S rRNA sequences and shorter partial sequences considering the 97.5% threshold. Indeed, using 53 strain sequences with binary similarity values between 96 and 98%, the average similarities between complete and partial ( E. coli positions 378-784) were calculated as 97.05 and 97.01 respectively, with a standard deviation of 1.24. In 1994, Stackebrandt and Goebel related the DNA- DNA hybridization values and the 16S rRNA similarity levels obtained for the same pairs of bacteria, and suggested that DNA-DNA hybridization should be realised for strains that shared 16S rRNA sequence similarities higher than 97%, for unambiguous identification of species. Later, in 2006, Stackebrandt and Ebers reanalysed the pool of data including new sequences and elevated the threshold to 98.7-99%. Indeed, over 380 data points representing DNA-DNA hybridizations, only 3 strain pairs showed 16S rRNA similarities lower than 99% for DNA reassociation values higher than 70%. This implies that the OTU concept based on 97.5% could be updated using higher similarity percentages. Therefore the 97.5% OTU’s concept could be more conservative than thought before and therefore it really includes several species. Thus, we could be underestimating the richness from environmental samples. However, a less restrictive approach (97.5%) avoids the introduction of bias in the analysis of OTUs due to the e.g. sequencing errors during the PCR amplification and DNA sequencing. Indeed, the generation of misincorporations errors, inherent to the molecular techniques used, together with the consideration of the 99% threshold with more or less short sequences (ca. 400 bp in DGGE), could induce to an erroneous classification.

General Introduction

6.5. Cyanobacterial survival abilities.

6.5.1. Desiccation.

The Antarctic region is characterized by the scarcity of liquid water, which is only available a few weeks to a few months during the summer season. Water removal is lethal for most organisms because it causes serious damages such as membrane pause transition and oxidative damage to proteins, lipids and nucleic acids (Potts, 2001). Reactive oxygen species (ROS) are generated when protein hydration is reduced by lack of water and can affect the enzyme activity resulting in oxidative stress (França et al., 2007). Modification of proteins and nucleic acids, due to the lack of water, such as modifications via the Maillard reaction or metal catalyzed Haber-Weiss and Fenton reactions, can produce damage to macromolecules (Potts, 2001). However some microorganisms can survive with reduced water supply. The exopolysaccharidic sheaths that surround certain cyanobacteria can act as sponges absorbing and retaining liquid water when it is available.

Rothrock and Garcia-Pichel (2005) studied the influence of desiccation frequency on a microbial community structure along a tidal desiccation gradient. Their results suggested that Archaea was the least tolerant group whilst cyanobacteria were the most tolerant to desiccation. Particularly, Chroococcidiopsis shows a special ability to thrive in anhydrobiosis. This cyanobacterium is widespread distributed all around the world, ie. in the hot Negev desert in Israel and the (cold) Ross desert (Antarctica) (Cockell et al., 2005). Billi (2009) showed how Chroococcidiopsis sp. CCMEE 029, stored in a desiccated state for 4 years, limited genome fragmentation and genome covalent modification, preserved intact plasma membranes and phycobiliproteins autofluorescence and exhibited reduced ROS accumulation and dehydrogenase activity upon rewetting. Under stress nutrient conditions, Chroococcidiopsis exhibits cellular morphological changes that lead to the synthesis of thick cell envelopes (Billi and Grilli, 1996). Abundant extracellular polysaccharides play an important role in desiccation tolerance of cells by regulating the loss and uptake of water. It has been reported that Chroococcidiopsis spp. and other cyanobacteria such as Nostoc commune , can produce two non-reducing sugars which replace the structural water of cellular components in anhydrobiosis, thus avoiding damages during drying. Coupling of protection mechanisms with repairing mechanisms should play a crucial role in the tolerance of Chroococcidiopsis to desiccation (Billi, 2001).

Another simpler but effective method to avoid desiccation in cyanobacteria is the hydrotaxis. In 2004, Pringault and Garcia-Pichel showed a motility response to modifications in the water content at the surface of microbial mats almost exclusively composed by Oscillatoria sp. This event occurred also in the dark, indicating that the process was independent of light conditions.

General Introduction

6.5.2. UV resistance.

Cyanobacteria appeared in the Precambrian era, when the atmosphere was still not oxygenated and when the ozone shield was still not formed. In this situation, cyanobacteria endured high UV radiations. UV-A (320-390 nm) and UV-B (280–315 nm) are known to penetrate into the water column up to several dozen meters, causing harmful effects in living organisms. For instance, studies of several lakes in the Larsemann Hills reported that 35-45% of the incident UV radiations can penetrate through the lake-ice (Verleyen et al., 2005). UV-B is more harmful than UV-A by its direct effect on DNA and proteins and indirect effects by producing ROS.

Cyanobacteria use three strategies to prevent the UV damage, which are i) to escape from the UV source by gliding mechanisms, ii) to cope with UV by synthesizing UV- absorbing compounds, antioxidants and extracellular polysaccharides, and iii) repairing the damages induced by UV which includes DNA repair and replacement of proteins damaged by the UV radiation (Ehling-Schulz and Scherer, 1999). i) Terrestrial cyanobacteria can avoid the harmful solar radiation spectra by migrating downwards into mat communities or by sinking deeper into the water column (Quesada and Vincent, 1997). For instance, Microcoleus chthonoplastes seems to be sensitive to UV-B radiation and migrate vertically in mats (Bebout and Garcia-Pichel, 1995). Cyanobacteria can also colonize Antarctic soils as hypolithons. For instance, Cowan et al. (2010) recorded cyanobacterial hypolithons underlying quartz stones in several regions of the Miers Valley (Fig. 25).

Fig. 25. Cyanobacteria inhabiting under quartz stone (picture from Cowan et al., 2010). Notice that the stone has been returned and the green colored part was originally in contact with the soil. ii) Cyanobacteria evolved under high UV conditions and then developed UV protectants to cope with radiation. They therefore synthesize molecules to absorb or screen the UV radiation preventing the photodamage induced by UV. Some of the photoprotectants in cyanobacteria are the mycosporine-like amino acids (MAAs) and the scytonemin pigment (Fig. 23).

General Introduction

Several microorganisms such as bacteria, fungi, cyanobacteria, phytoplankton and macroalgae are able to produce MAAs but not animals which can in some cases, use the MAAs derived from their algal diet. MAAs are small (<400 Da), colorless, water- soluble compounds composed of cyclohexenone or cyclohexenimine chromophores conjugated with amino acids attached to the central ring through imine linkages. Nostoc commune species secrete the MAAs which are linked extracellularly to the glycan, acting as real screening compounds (67% effectiveness in photon absorption) (Böhm et al., 1995). On the contrary, other species such as Gloeocapsa sp. accumulate MAAs in their cytoplasm, conferring lower protection levels (10-20% effectiveness in photon absorption) (Garcia-Pichel and Castenholz, 1993).

Figure 26. Chemical structures of MAAs from cyanobacteria. Picture from Balskus and Walsh (2010).

Scytonemin is a yellow–brown lipid-soluble pigment located in the extracellular polysaccharide sheath, exclusive of some cyanobacterial species (Proteau et al., 1993). This made scytonemin a useful proxy indicative of the presence of cyanobacteria in paleolimnological studies. Scytonemin is a 544 Da dimer composed of indolic and phenolic subunits. Scytonemin plays a role as UV-A screen protector (Ehling-Schulz et al., 1997; Garcia-Pichel et al., 1992). The main scytonemin gene cluster in Nostoc punctiforme ATCC 29133 is a 24.4 Kb operon containing 18 ORFs. Calothrix sp. produces high quantities of scytonemin and it is required to avoid the photosynthesis inhibition under high UV fluxes (Brenowitz and Castenholz, 1997). Scytonemin synthesis is strongly inducible by UV-A irradiation, but only weakly by UV-B irradiation. Soule et al. (2009) showed how the 18 ORFs in N. punctiforme ATCC 29133 were induced by UV-A radiation. UV-A in combination with an increase in temperature and oxidative stress had a synergistic effect on the scytonemin production in Chroococcidiopsis sp. (Sinha and Häder, 2008). The experiments developed by Cockell et al. (2005) suggested a survival ratio for Chroococcidiopsis sp. more than 10 times higher than previously reported for Bacillus subtilis. However, the mechanisms that lead to these increased survival ratios are not yet well known. Fleming and Castenholz (2007) suggested that N. punctiforme and Chroococcidiopsis CCMEE 5056 increased their scytonemin content under UV-A exposure. This induced response was 71

General Introduction enhanced when the strains were simultaneously subjected to desiccation. When water is unavailable, cells are metabolically inactive and the UV radiation became especially dangerous. The production of scytonemin in these conditions could facilitate the reactivation of cyanobacterial cells under optimal conditions as they could allocate more energy to recovery systems other than repairing systems related to UV damage.

UV-A and UV-B can also produce oxidative stress by generation of ROS. Antioxidant molecules such as carotenoids, can remove singlet oxygen, avoid triplet chlorophyll and inhibit lipid peroxidation (Edge et al., 1997). Myxoxanthophyll and echinenone (carotenoids) were suggested to act as UV-B photoprotectors in Nostoc commune (Ehling-Schulz et al., 1997).

Another way to avoid the harmful UV radiation in cyanobacteria is the synthesis of thick polysaccharidic sheaths, as they absorb a wide radiation pathlength, providing a matrix for protectant pigments as well. Interestingly, UV-B radiation seems to stimulate the glycan production in Nostoc commune (Ehling-Schulz et al, 1997). iii- DNA repair mechanisms are universal for all types of cells and have been studied extensively in E. coli (Ehling-Schulz and Scherer, 1999) including photoreactivation, excision repair and postreplication repair. Regarding proteins, Synechocystis sp. PCC 6803 increases the protein turnover of the photosystem II reaction center when exposed to UV-B radiation (Sass et al., 1997). Irradiation of some cyanobacteria to UV-C and of light intensities close to the UV (295-390) has been shown to induce the production of proteins with UV protective functions (Ehling-Schulz and Scherer, 1999).

6.5.3. Freezing tolerance.

Most of the cyanobacteria isolated from Antarctica are not psychrophilic but psychrotolerant, with optimal growth temperatures well above the extreme low temperatures common to the Antarctic environment. Psychrophiles are organisms that grow at 0ºC and have an optimum growth temperature below 15ºC and a maximum growth temperature below 20ºC whilst psychrotolerants are less likely to grow at 0ºC, but will grow at 3-5ºC and have optimum and maximum growth temperatures above 20ºC (Morita, 1975). Nadeau et al. (2001) suggested that Oscillatoriales in both Polar Regions originated in temperate latitudes. This assumption was done on the basis that the psychrotolerant oscillatorian phenotype arises multiple times in the cyanobacteria lineage and that psychrotolerant strains are often most closely related to cyanobacteria from temperate latitudes.

Antarctic cyanobacteria are often exposed to freezing temperatures and repetitive and rapid freeze-thaw cycles, while in spring and summer, the liquid water from melting snow saturates the deglaciated ground areas. For instance, summer temperatures in

General Introduction

Signy Island (Antarctic Peninsula) fluctuate between 8ºC to -5ºC (Davey et al., 1992). In autumn, temperatures are around 0ºC with several daily freeze-thaw cycles. In winter, temperatures descend far under 0ºC, water levels drop and mats desiccated. The presence of mucilage and multilayered walls and high intracellular concentration of solutes protect cyanobacteria against freezing during cold periods. At the cellular level, formation of intracellular ice and cellular dehydration cause important damages. To cope with this, some cyanobacteria produce internal molecules with anti-freezing properties. For instance, Nostoc produces high concentrations of intracellular sugar- phosphates (Becker, 1982).

Another mechanism which leads to the acclimation of cyanobacteria in cold habitats is the desaturation of membrane lipids. The desaturation extent of the membrane lipids influences the fluidity of the membrane. Increase in lipid desaturation is activated by low temperatures. This regulatory mechanism is controlled by a cold-sensing histidine kinase system, which regulates the gene expression responses to low temperature shocks (Singh et al., 2009).

Sabacka and Elster (2006) showed that cyanobacteria from polar wetlands were more resistant to both freezing and desiccation than green algae from similar habitats. During an experiment, Phormidium strains were quickly frozen at -196ºC but also gradually cooled from -40ºC to -100ºC. Cyanobacteria isolated from continental Antarctica were significantly more successful in withstanding low sub-zero temperatures than similar types of cyanobacteria isolated from maritime Antarctica, suggesting special adaptation mechanisms in cyanobacteria from the coldest habitats.

The high desiccation tolerance and the tolerance to freezing stress could be linked as suggested by Lin et al. (2004). The photosynthetic activities and fluorescent emission spectra of several strains ( Nostoc , Synechocystis and Fischerella spp.) were measured under freezing stress. The results related high desiccation tolerance to tolerance for freezing stress, suggesting similar mechanisms for both kinds of resistances.

6.6. Cyanobacterial diversity in the Antarctic continent.

Lakes and soils are the two main habitats in which cyanobacteria establish communities in Antarctica, often as the most abundant primary producers (Fig. 27). Previous studies focused on Antarctic cyanobacterial diversity were carried out based on a traditional approach (microscopy), molecular methods (DGGE, clone library, ARDRA, TGGE, RT- PCR, etc) or a combination of both (polyphasic approach).

General Introduction

The study of 59 strains from 23 lakes from the Prydz Bay and the McMurdo Dry Valleys by a polyphasic approach (Taton, 2005) showed a morphological diversity composed of 12 morphotypes classified into 8 different genera from the Oscillatoriales, the Chroococcales and the Nostocales. In the same study, 56 sequences (16S rRNA) were obtained from the strains and were classified into 21 different OTUs. This study highlighted a high molecular diversity hidden under a more restricted morphological diversity. For instance, the Oscillatoriales showed a rich genotypic diversity (15 OTUs) whilst the morphology was quite simple (7 taxa). Especially, the genus Nostoc showed an important genotypic variability without distinct morphological traits. This is in concordance with other studies (Comte et al., 2007) which analyzed Phormidium strains from Arctic and Antarctic regions. Although the genotypes generally corresponded to the observed morphotypes, the molecular approach revealed a higher degree of biodiversity.

Mosier et al. (2006) studied the cyanobacterial diversity by molecular approach in the perennial ice cover of Lake Vida (McMurdo Dry Valleys). Oscillatoriales related to Limnothrix (95%), Microcoleus (97%) and other unidentified cyanobacterial clones were observed by DGGE and clone library. The presence of cyanobacteria up to 7 m into the ice and the presence of chlorophyll a detected by pigment analysis suggested that prototrophs may photosynthesize below 7 m within the ice core.

The input of aquatic cyanobacteria to the soil communities was analyzed by Wood et al. (2008) by the study of the edaphic cyanobacterial diversity present in the Miers Valley (Dry valleys). ARISA (Automated rRNA Intergenic Spacer Analysis) of the terrestrial samples showed that 29-58% of the soil sequences were also present in the lakes or hydroterrestrial mats, suggesting that lacustrine cyanobacteria could play an important role in the composition of terrestrial cyanobacterial communities. The total carbon content was the principal variable to explain the differences in the structure of the cyanobacterial communities. The mobility of species between near-by terrestrial and aquatic habitats was also suggested by Gordon et al. (2000) which observed that cyanobacteria inhabiting the ice cover from Lake Bonney (McMurdo Dry Valleys) were similar to the cyanobacteria found in the terrestrial mats in the catchment of the lake. The role of lakes and ponds in the cyanobacterial component of the Antarctic soils was postulated by Wood et al. (2008), as they showed an impoverished diversity in soils samples from the Beacon valley (McMurdo Dry Valleys), explained by the total absence of waterbodies in the area.

Although cyanobacteria have been found in multiple habitats within the Antarctic continent, only a few have been observed inhabiting in the permafrost. Gilichinsky et al. (2007) isolated Gloeocapsa and Chroococcidiopsis strains from permafrost samples (sandstone) from the Beacon Valley (McMurdo Dry Valleys) and suggested that cyanobacteria are more developed in Arctic permafrost than in the Antarctic permafrost.

General Introduction

The cyanobacterial communities in the Antarctic Peninsula (AP) have been studied by several authors, mostly based on a traditional approach (microscopy). Only a few papers provide molecular characterizations of microbial mats in maritime Antarctica (Callejas et al., 2010; Comte et al . 2007). For instance, Callejas et al. (2010) observed a low diversity in terrestrial mats from the Fildes Peninsula dominated by Tychonema genotypes related to Tychonema bourrellyi and other T. bornetii isolates. There are numerous papers based on the morphology of cyanobacteria in this area although it is not our purpose to perform an extensive inventory in this introduction. The King George Island ground is almost ice-free in summer, and numerous cyanobacterial mats and bryophytes develop in this area. Komarek and Komarek (2003) observed seepage communities in King George Island containing Phormidium pseudopristleyi , Leptolyngbya vicentii , L. glacialis , or Gloeocapsopsis aurea among others, suggesting a high degree of endemicity in the coastal Antarctic regions in terms of species composition, distinct mat structure and seasonality. Gloeocapsopsis aurea is an endemic Antarctic species widely distributed in coastal ice-free areas in the maritime Antarctic (Mataloni and Komarek, 2004). Similarly, the South Shetland Islands (Antarctic Peninsula) harbors a high diversity of cyanobacterial morphotypes inhabiting wet rocks, coastal pools, stagnant water bodies, soils, streams and seepages with several morphotypes apparently restricted to specific microhabitats. Other authors (Mataloni et al., 2000) defined 15 morphospecies in 18 soil frosted sort-polygons samples at Cierva Point (Antarctic Peninsula) and suggested that “variables describing community development were not significantly correlated with either area of the polygons or the minimum distance between them” but that “biotic interactions could account to a larger extent for community structure”.

In general, filamentous cyanobacteria are more abundant in lacustrine and terrestrial habitats than unicellular cyanobacteria, which are often not observed in the samples (Jungblut et al., 2005, Taton et al., 2003). However, unicellular cyanobacteria can be found in the picoplankton communities of Antarctic lakes. For instance, Synechococcus sp. has been described in the picoplankton of Ace Lake (Powell et al., 2005). Similarly, undetermined picocyanobacteria have been observed in the deep chlorophyll a maximum of Lake Vanda (McMurdo DryValleys) (Vincent and James, 1996). Rankin et al. (1997) observed phycoerythrin-rich picocyanobacteria in the saline lakes of the Vestfold Hills which achieved so high concentrations that rendered the lake water samples pink in color. Although picocyanobacteria (including Synechococcus ) have been detected by several methods as seen before, there are only a few 16S rRNA available sequences (Genbank) from Antarctic Synechococcus spp. from Lake Abraxas, Pendant and Ace (Vestfold Hills) (Vincent et al., 2000b; Coolen et al., 2008) .

The distribution patterns of Oscillatoriales based on water conductivities was suggested by Broady and Kibblewhite (1991) although this relationship is still unclear. Jungblut et al. (2005) observed Oscillatoria priestleyi preferentially in hypersaline environments but also in a freshwater pond (Fresh Pond, McMurdo Ice Shelf). Nostocales such as Nostoc sp. absent in saline lakes (Jungblut et al., 2005; Howard-Williams et al., 1990)

General Introduction although Nodularia sp. was observed in a saline lake (Ace Lake) (Sabbe et al., 2003) as well as in freshwater lakes (Fresh Pond) (Jungblut et al., 2005). Similarly, Jungblut et al. (2005) showed how the hypersaline Salt Pond harbored a lower diversity (5 phylotypes) compared to the freshwater lakes Fresh pond (17 phylotypes) and Orange Pond (9 phylotypes). Hitzfeld et al. (2000) postulated that saline lakes could harbor a greater diversity than freshwater lakes. These results were surely biased by stochasticity, because other analyses on this topic showed the inverse relationship. In another study, Taton et al. (2006a) showed that the saline lakes Ace and Rauer8 possessed a lower diversity compared to freshwater lakes. It was also noticed that the diversity from saline lakes greatly differs from freshwater lakes from the same region. Additionally, these saline lakes shared 2 OTUs, probably OTUs specially adapted to high salinities as their related sequences were also recorded from other saline environments. Similarly, Sabbe et al. (2004) studied the morphological diversity of cyanobacteria from 56 lakes from the Larsemann Hills and the Bollingen Islands related to several physicochemical parameters as salinity. Results showed that salinity did not highly influence the cyanobacterial composition and suggested a strong correlation with the lake depth, pH and calcium and silicate concentrations.

General Introduction

Figure 27. Cyanobacterial mats in lakes and terrestrial locations in Antarctica. a) Blackish cyanobacterial mat from the Utsteinen ridge (Sør Rondane Mountains) (picture from Damien Ertz). b) Colored cyanobacterial mats in Cape Chocolate’s ponds (picture from Larry Coats). c&d) Dried cyanobacterial mats on the Dufek Valley’s ground (pictures from Dominic Hodgson). e) Terrestrial mat from Bratina Island (picture from Taton et al., 2006). f) Cyanobacterial mats from a Bratina Island pond (pictures from Taton et al., 2006). g) Frozen oxygen bubbles from an active mat growing in the shoreline of Forlidas Pond (Transantarctic Mountains) (pictures from Dominic Hodgson). h) Drilling the Lundström Lake (Shackleton range) (picture from Dominic Hodgson).

General Introduction

Research Objetives

PART 2: RESEARCH OBJECTIVES.

Research Objetives

Research objectives.

This thesis aims to approach two main objectives. Firstly, to contribute to a better knowledge of the actual Antarctic cyanobacterial flora in terrestrial and lacustrine habitats. Secondly, to improve the study of the cyanobacteria as a paleolimnological tool in Antarctic studies and relate the changes in past communities to other proxies to understand the factors underlying these changes. Both objectives are cross-linked. Indeed, in order to accurate the identification of ancient cyanobacterial sequences, the most representative databases are needed. Incomplete databases could lead to misinterpret ancient sequences as extinct cyanobacterial flora if no modern analogs are found in the databases.

The first objective aims to increase the knowledge about the terrestrial and lacustrine cyanobacterial diversity in Antarctica. This has been performed by a polyphasic approach in most of the cases, involving microscopic observations and molecular studies. Microscopic observations included the analysis of the environmental samples and cultures obtained from them. The molecular studies implied the study of the genes coding for the 16S rRNA and/or the Internal Transcribed Spacer (ITS) by clone libraries, Amplified Ribosomal DNA Restriction Analysis (ARDRA), Denaturing Gradient Gel Electrophoresis (DGGE) and bioinformatic analysis. Several strains obtained in this study were integrated in the BCCM/ULC Belgian collection of (sub)polar cyanobacteria (http://bccm.belspo.be/about/ulc.php). The results obtained from the first objective of this thesis were obtained in the frame of the BelSPO projects AMBIO (SD/BA/01A) and ANTAR-IMPACT (SD/BA/853) and exposed in the chapters 1 to 5.

The second objective of this thesis was based on the study of two sediment cores from two lakes located in the Beak Island (Antarctic Peninsula). The analysis included light microscopy, DGGE, Whole Genome Amplification (WGA) and High-Performance Liquid Chromatography (HPLC). Previous studies on fossil or ancient DNA (aDNA) from cyanobacteria are almost inexistent, especially concerning Antarctic sediment cores. Only Coolen et al. (2008) studied Antarctic sediment cores for cyanobacterial diversity, but there are no studies about the microfossils and the pigments within Antarctic sediment cores in combination with the analysis of cyanobacterial fossil DNA. The second section of this thesis is integrated within the HOLANT project (BelSPO; SD/CA/01A) and is developed in the chapters 6 to 7 and the Annex I.

In chapter 1, two lakes from inland continental Antarctica were studied for their cyanobacterial communities. These lakes are amongst the most southerly locations where freshwater-related ecosystems are present (Transantarctic Mountains and the Shackleton Range; 80-82ºS). The evaluation of the diversity was assessed by the molecular study of 16S rRNA gene fragments (DGGE) and 16S plus ITS (clone library, ARDRA) as well as by light microscopy and isolation of strains from the environmental samples. The results were linked to the geographical isolation and the environmental

Research Objetives factors to explain the possibility of endemicity in Antarctic cyanobacteria. The lake from the Transantarctic Mountains, Forlidas Pond, is located within the Dufek Valley. The limnology and biology of this valley is described in chapter 2. An inventory of cyanobacteria, together with green algae, cercozoa, metazoa lichens and bacteria, was carried out by a polyphasic approach. These results were analyzed together with environmental measurements and observations to understand the biological interactions with the physical environment. The species diversity and the endemic concepts were discussed in this chapter.

The Utsteinen ridge, where the Princess Elisabeth base was constructed, and the surrounding areas (Utsteinen ridge and a more distant Dry Valley), were sampled before the construction of the base (chapter 3). Thus, this study aims to serve as biological baseline survey and will allow the monitoring of the human disturbances on the biodiversity. The ridge and the nunatak near the station have surely been ice free since at least 39.4 ka B.P (Wand and Hermichen, 2006). Thus, their possible role as biological refuge during the succession of glaciations was outlined in this chapter.

With the test of DNA extraction designed in the chapter 3, several environmental samples from the Utsteinen site were analyzed to improve the yield recovery. This could help to unravel the real biodiversity hidden in terrestrial environmental samples, where thick mucilaginous sheaths protect cyanobacteria from UV radiation, low temperatures and provide a support to attach to the substrate (Kepner et al., 2000; Vincent, 2000). In fact, numerous observations previously reported a mismatch between microscopic and molecular observations (Taton et al., 2003, 2006a&b).

The number of molecular studies based on cyanobacterial 16S rRNA genes from Antarctic lakes has increased significantly in the recent years (Priscu et al., 1998; Vincent et al., 2000; Bowman et al., 2000; Nadeau et al., 2001; Taton et al., 2003; Taton et al., 2006a&b; Mosier et al., 2007; Comte et al., 2007; Verleyen et al., 2010b) but no synthesis was yet carried out. Therefore, we undertook a compilation analysis (chapter 4) to identify the major studied areas and the areas where information was still lacking. One of the latter was the Mc.Robertson and the Kemp Lands, on the western side of the Amery Ice Shelf. We therefore proceeded to study several lakes, by DGGE analysis, to increase the number of lacustrine cyanobacterial sequences in the databases. In addition, we sampled the Lake SH1, from the Stilwell Hills (Kemp Land), at two locations (centre and shoreline) to detect potential differences in the diversity output. In fact, the biodiversity contained in most of the Antarctic lakes has been previously studied by the analysis of a single sample. The results were compared to determine the representativity of the samples.

Regarding the cyanobacterial distribution within the Antarctic continent, chapter 5 discusses the increase in diversity of cyanobacteria inhabiting terrestrial habitats in the areas located between 70º and 80°S. All the Antarctic cyanobacterial sequences recorded from terrestrial habitats were analysed to assess the degree of similarity in Research Objetives cyanobacterial communities across the continent. For that, a methodology based on bioinformatic tools such as BLAST, ARB and DOTUR was developed.

Cyanobacteria are often the dominant phototrophs in Antarctic terrestrial and freshwater ecosystems and especially abundant in lakes, where they can form benthic microbial mats or develop in the water column as part of the picoplankton (Andreoli et al., 1992; Taton et al., 2003). However, despite their prominent role in ecosystem functioning and primary productivity, little is known about their past biodiversity and response to climate and environmental changes. The potential of aDNA in paleolimnological studies is assessed in chapter 6. Fossil pigments and aDNA were analyzed from two sediment cores located at the Beak Island (Antarctic Peninsula) by HPLC and DGGE based on 16S rRNA genes, respectively. The preserved microfossils were observed by light microscopy and all the results were interrelated to the geochronology of the cores to obtain an overview of the past colonization and succession events recorded throughout the lake’s sediment cores.

DNA degradation in fossil cells generally starts immediately after cell death, suffering several damages (Willerslev et al., 2005). Due to this, aDNA is present in low concentrations within sediment core layers. Chapter 7 discusses a methodological approach based on WGA as a possible way to study low concentrated DNA samples or precious samples. We designed two protocols and compared them with one commercial product (REPLI-g kit, Qiagen). The main objectives were i) to amplify aDNA which was present in too low concentrations to be detected by direct PCR and ii) try to improve the cyanobacterial specific amplification.

Research Objetives

General Discussion

PART 4: GENERAL DISCUSSION.

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General Discussion

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General Discussion

We propose an enumeration of the main conclusions extracted from this thesis. The conclusions of the different chapters are summarized and put in a broader context.

Importance of the methodological approaches.

The importance of the methodological approaches has been highlighted in several chapters in this thesis.

Because environmental studies start by the collection of environmental samples, the choice of the sampling strategy is very important. We analyzed the Lake SH1 by sampling two different locations (centre and shoreline) in chapter 4. The results suggested a large difference in diversity composition between both samples. Only one OTU (16ST44) was found in both sampling sites, while 3 OTUs (16ST19, 16ST63 and 16ST90) were found only at the shoreline of the lake. Similarly, the OTU 16ST79 was found only in the centre of the lake. This can be explained by several factors such as the lower light intensities at the bottom of the lakes related to the higher water depth. These factors have been reported to influence the structure, diversity and formation of microbial mat communities in Antarctic lakes based on the pigment composition of benthic microbial communities (Hodgson et al., 2004; Verleyen et al., 2005, 2010b). Similarly, Taton et al. (2006a) showed great differences between samples from Lake Reid (Larsemann Hills) but taken at different years and at different depths. Only 3 out of 17 OTUs were shared between the two samples. There are not other studies analyzing the different cyanobacterial composition related to the sampling sites in Antarctica and therefore, further analysis should be carried out to determine the importance of the sampling methods related to the differences in communities’ composition in Antarctic lakes.

The complementary nature of the polyphasic approach was supported in this thesis. In chapter 1, the isolation of two strains (Phormidium priestleyi and Leptolyngbya cf. foveolarum) allowed the observation of 2 OTUs not retrieved by clone libraries or DGGE. Similarly, a Nostoc sp. strain and a Phormidium priestleyi strain isolated from the Utsteinen ridge (chapter 3), allowed the identification of the OTUs 16ST10 and 16ST34 respectively, which were not found by molecular methods. Several cyanobacterial sequences related to Coleodesmium sp., Leptolyngbya sp. and Phormidium sp. (chapter 3) were detected by molecular methods in some samples but not observed by microscopic observations. This phenomenon has been previously reported (Taton et al., 2006a) and it is surely due to the high sensitivity level of the molecular methods which could amplify DNA from rare morphotypes. In fact, aalthough the DGGE is a less sensitive technique than clone libraries, several OTUs were retrieved by DGGE but not by clone library in this thesis (chapter 1). Indeed, only predominant populations can be detected by DGGE (Nikolausz et al., 2005), when their populations represent at least 1% of the total communities (Muyzer et al., 1993; Murray et al., 1996). Nevertheless, our results can be explained by to the use of a seminested PCR in the DGGE whereas a single PCR was used for the clone library. In addition, the ARDRA analyses used to screen the clone libraries could underestimate the diversity (Cho and Tiedje, 2000).

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General Discussion

For the sediment cores, the polyphasic approach used in chapter 6 was not successful as the microscopic observations did not allow the recognition of cyanobacteria at the species level or even the genus level. This is explained by the bad conservation of cyanobacterial cells and the apparent low number of filaments within the sediment core layers.

The ITS analysis realized in chapter 1 clearly increased the phylogenetic resolution provided by the 16S rRNA genes. Two strains (TM3FOS129 and TM2ULC129) which were at the threshold of the OTU 16ST72 (97.3%) were finally included into a new OTU (16ST80) as the ITS sequences from both strains were identical, but clearly not related to the ITS sequences from OTU 16ST72. We observed a correlation between the ITS subgroups formed in chapter 1 and the different physicochemical parameters of the samples. For instance, the different sub-groups formed within the ITS02 and ITS27 types, suggested a relationship with the different environmental parameters from the lakes from where the sequences were recorded. Finally, lakes sharing similar environmental parameters contained cyanobacterial genotypes with almost identical (99%) ITS sequences (ie. ITS28).

Endemism

The possibility of cyanobacterial endemism in Antarctica was reviewed in chapters 1 and 2. We use the term “potentially endemic” when we made reference to taxa which have been only recorded in the Antarctic continent. Since the mutation rate of 16S rRNA genes has been estimated as 1% every 50 millions of years (Ochman and Prager, 1987), the observed divergence of Antarctic endemic OTUs implies an isolation of at least 125 Ma.

The number of potentially Antarctic OTUs decrease with the appearance of new sequences from other non-Antarctic habitats. Indeed, the volume of the databases is increasing permanently, due to the sampling of new locations which provides new sequences to the databases. It is expected that a certain percentage of these sequences match with sequences from previously defined OTUs. In chapter 1, we showed how the potential endemicity of the OTUs from the Taton et al. (2006 a&b) studies dropped from 57% in 2006 to 28.5% in 2011. Comparison of our OTUs database (2011) with the same database dated from June 2005 shows a decrease in the number of potentially endemic OTUs since 2005 (69%) to 2011 (38%). However, the isolation and endemicity of certain Antarctic taxa has been suggested by several authors (see references in chapter 1). Regarding these two databases (2005 and 20011), 38% of the OTUs described in this thesis did not exist in June 2005. From the other 62% of OTUs already described in 2005, 48% were considered as potentially endemic to Antarctica at that time but changed their status to cosmopolitan in 2011 due to the new sequences deposited into Genbank since then. We also remark that 40 % of the OTUs from the 2005 database were composed of a single sequence whilst this proportion only means 27.5% in the 2011 database. This

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General Discussion indicates that the coverage of cyanobacterial 16S rRNA gene sequences was fragmentary and it is surely still incomplete (see chapter 4).

The potential dispersion of endemic OTUs, at least within Antarctica, seems to be important. In total, 8 out of the 32 OTUs described in this thesis appeared to be potentially endemic to Antarctica (OTUs 16ST02, 16ST42, 16ST73, 16ST84, 16ST85, 16ST86, 16ST87 and 16ST90). From these potentially endemic OTUs, 3 OTUs were recorded for the first time and only present in the Sør Rondane Mountains and one has been only recorded in the Stillwell Hills. The other 4 potentially Antarctic endemic OTUs were retrieved in several Antarctic locations. For instance, sequences belonging to the OTU 16ST02 were recorded in the McMurdo Ice Shelf, the Lundström Lake and the Larsemann Hills (chapter 1). Several sequences belonging to the OTUs from chapter 3 were also recorded in distant Antarctic regions. For example, members of the OTU 16ST42 were recorded in the Sør Rondane Mountains and the Alexander Island (Antarctic Peninsula), sequences from the OTU 16ST73 were found in the Rauer Islands, the Miers Valley and the Sør Rondane Mountains and finally, sequences from the OTU 16ST84 were retrieved in the Ellsworth Mountains, the Alexander Island and the Sør Rondane Mountains (chapter 3).

The highest number of potentially endemic OTUs comes from the Sør Rondane Mountains. We suggested that the Utsteinen nunatak could act as refugee for biota during glaciations (chapter 3). The evidence that some areas in central Dronning Maud Land between 800-1380 m altitude a.s.l. have been ice-free since at least 39.4 ka BP (Wand and Hermichen, 2006) and the persistence of the continental Antarctic mite genus Maudheimia since the Last Glacial Maximum in Dronning Maud Land (Marshall and Coetzee, 2000), could support this proposition. Our results showed that 6 out of 13 OTUs from the Sør Rondane area appeared to be potentially endemic to Antarctica, including 3 OTUs (16ST85, 16ST86 and 16ST87) which were never recorded before. The percentages of similarity with already known sequences (95-99.7%) suggest a long isolation time and thus, support our hypothesis.

The analysis of the bulk of Antarctic terrestrial 16S rRNA sequences (chapter 5) shows a low degree of similarity between terrestrial communities from the different regions studied and thus, supports that the regional populations probably exist.

The selection of different markers (16S rRNA, ITS) can modify the interpretation of the biogeography of the genotypes. We have observed that all ITS types from the Forlidas Pond and Lundström Lake (chapter 1) were potentially endemic to Antarctica whilst, in contrast, the corresponding OTUs (based on 16S rRNA genes) were all cosmopolitan. This is in concordance with other studies, such as the analysis of different markers (16S rRNA, ITS and genomic fingerprints) by Cho and Tiedje (2000), which showed a higher level of endemicity on the basis of ITS sequences than on 16S rRNA sequences. However, this potential endemism could be also related to the incomplete ITS databases, which are still fragmentary.

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General Discussion

Dispersal potential and physical barriers

Four main locations were studied in this thesis: i) the Transantarctic Mountains and the Shackleton Range, ii) the Mac.Robertson and Kemp Lands, iii) the Sør Rondane Mountains and iv) the Beak Island. In general, 92% of the OTUs described in the chapters of this thesis were only recorded in one of these main locations but not in the others.

Cosmopolitan OTUs must possess features enabling the processes of dispersal, colonization and establishment between different continents, and thus, possess high resistance capacities and colonization rates (Hodgson et al., 2010 and references therein). However, Antarctic endemic OTUs should also have well-developed resistance adaptations. Indeed, several Antarctic endemic OTUs have been recorded across distant locations within the Antarctic continent, as commented before.

Regarding the cosmopolitan OTUs, the OTU 16ST11 was shared between the Lundström Lake and the Sør Rondane Mountains whilst the OTUs 16ST49, 16ST63 and 16ST92 were found in the Transantarctic Mountains, the Shackleton Range and the Stilwell Hills. Interestingly, only one OTU (16ST44) was present in all the four main locations of this thesis. In addition, this OTU has been recorded in several locations inside (Lake Fryxell in McMurdo Dry Valleys; Orange and Lunch Ponds in Bratina Island) and outside Antarctica (ie. Douglas river in Australia; Garonne river in France or the Canyonlands National Park, USA). Several strains are included in OTU 16ST44 such as Oscillatoria sp. E17 (AF263338) described as psychrotolerant by Nadeau et al. (2001) or Tychonema sp. K27-Arp (GQ324965), recorded in biofilms on rock surfaces in the Harz Mountains (Germany). This suggests an especially high resistance to transportation and adaptation to a wide range of habitats for this OTU.

Potentially Antarctic endemic OTUs can be found dispersed in the geographical extremes of the continent. The OTU 16ST02 described in chapter 1, has been found in the Larsemann Hills (East Antarctica) (Taton et al., 2006a) and in Bratina Island (Ross Sea region) (Jungblut et al., 2005), ca. 2600 km apart. Other potentially Antarctic endemic OTUs were also not restricted to a unique area but were recorded in distant locations. The OTU 16ST73 has been found in the Rauer Islands, the Vestfold Hills (East Antarctica) and the Sør Rondane Mountains, locations separated by ca. 2000 km. Similarly, the OTU 16ST84 has been recorded in the Sør Rondane Mountains and the Ellsworth Mountains (ca. 2600 km apart) (chapter 3).

Several factors have been proposed to explain the isolation of Antarctica (ie. ocean currents, strong winds and low temperatures). By the contrary, the mechanisms involved in the intercontinental or intra-Antarctic dispersal of cyanobacteria and other microorganisms are still not completely elucidated. These factors include i.e. the transportation by animals and more recently by human mediated activities (humans, transports, etc.), although Pearce et al. (2009) specially suggested the important role of

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General Discussion the aerial environment. Indeed, it has become evident that several microorganisms from remote environments have been transported by air currents. However, the interaction of geophysical barriers such as the Transantarctic Mountains could play an important role in the dispersal limitation by i.e. air currents. It has also been suggested that the most limiting factor in the colonization of microorganisms in Antarctica is not the transportation to the continent or between different parts of the continent, but the environmental conditions found by the microorganisms which arrive to these new habitats (Clarke et al., 2005).

New insights in cyanobacterial detection.

Due to the lack of concordance between the molecular and microscopy approaches, we designed a new DNA extraction method to try to improve the DNA extraction of ensheathed cyanobacteria (chapter 3). Previous molecular studies on cyanobacteria showed a bias between the molecular observations and the morphotypes observed by light microscopy (Taton et al., 2003, 2006a). This was also observed in our studies. In chapter 1, a morphotype related to Phormidum crassior was observed by microscopy although it was not isolated in culture nor by molecular methods and thus, its sequence is not available. The molecular analysis of the sample from the shoreline of the Lundström Lake showed several OTUs related to the Oscillatoriales (16ST80, 16ST92) which could be related to the P. crassior morphotype. Unfortunately, there are no sequences of this morphotype in the databases and, in absence of cultured strains, the identification remains incomplete. In addition, at least 2 out of 10 morphotypes (Asterocapsa sp. and Nostoc sp.) were never detected by DGGE from the environmental samples in chapter 3. Thus, we compared the extraction efficiency of two extraction methods, both developed in our laboratory. However, the diversity output was the same for both protocols. Classical DNA extraction protocols may not be efficient enough to disrupt the complex polysaccharidic matrix often formed by cyanobacterial mats. Mucilaginous sheaths are especially developed in terrestrial habitats, where cyanobacteria must cope with the UV radiation, desiccation, low temperatures, freeze-thaw cycles and winds. We propose new modifications of the extraction method protocols in the “Future research” section. For a better approach, each sample should be preliminarily studied by microscopy to decide which extraction method should be adopted.

When cyanobacterial DNA is present in low concentrations in environmental samples, the traditional primers specific for cyanobacteria could amplify other 16S rRNA genes e.g. from bacteria or plasts, when they are present in much higher number of copies. Thus, we observed that the primer 16S378F, which is commonly used as specific primer for cyanobacteria (Taton et al., 2003, 2006a&b; Verleyen et al., 2010b) showed the most common mismatch (g/g or t/g) with bacterial sequences in the middle (11th base) of the primer sequence. As the discriminating positions should be in the last 3 bases at the 3’end (Bru et al., 2008), we designed the primer 16S369F to place the discriminating position at the 3’end. Therefore, the number of in silico non-specific matches is lower. This primer allowed to amplify cyanobacterial DNA from all the sediment core depths, whilst the

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General Discussion primer 16S378F was less effective and did not lead to PCR amplification from several bands (data not shown in the chapter 6).

Antarctic samples are valuable, because of the difficulties of sampling in these latitudes. The size of these samples is often small because of logistic limitations. Sediment cores layers from Antarctic sediment cores are especially thin (0.5 cm width for the samples studied in chapter 6). In addition, they contain low DNA concentrations because of different degradation processes. We designed an amplification experience based on Multi Displacement Amplification (MDA)-Whole Genome Amplification (WGA) to solve the issue of samples with low DNA quantities (chapter 7). We used random primers and primers based on Highly Iterated Palindromes (HIPs). The HIPs primers were designed on the basis of the HIP1primers of Robinson et al. (1995). MDA is an isothermal amplification reaction which can increase the copy number of DNA molecules and simultaneously dilute inhibitory co-purified substances. Preliminary MDA results showed different results depending of the protocol used. The protocols based on random primers amplified the highest yield of bacterial DNA. By the contrary, the MDA based on HIP primers and the MDA carried out with the REPLI-g kit did not amplify bacterial sequences. However, further studies should be performed to standardize and complete this protocol which could be used as a reference tool in fossil DNA studies.

Mapping of cyanobacterial 16S rRNA Antarctic diversity.

A summary of the distribution of lacustrine Antarctic cyanobacteria was given in chapter 4. The analysis of the bulk of 16S rRNA sequences showed an important bias in the distribution of cyanobacteria in Antarctic lakes. Two regions were highly represented. The first was the East Antarctica, comprising the Rauer Islands, the Larsemann Hills, the Vestfold Hills and the Bølingen Islands. This region represents 55.6% of the total number of sequences in the Genbank database. In particular, the Larsemann Hills ice-free area represents 65 % of the sequences recorded in the ice free areas around the Amery Ice shelf. The South Victoria Land Dry Valleys (McMurdo Dry Valleys, Taylor Valley, Miers Valley) and the ice-free areas around the Ross Ice Shelf (Bratina Island, Ross Island) were moderately studied, representing 35.4% of the sequences. The study also highlighted that the Antarctic Peninsula was poorly represented (2%). In fact, most of the studies on the Antarctic Peninsula focused on soils (ie. Yergeau et al., 2007) or on other organisms as Collembola (Convey et al., 2007) or midges (Allegrucci et al., 2006). The second underrepresented region is the continental inland area, with only 2 lakes (Forlidas Pond and Lundström Lake, studied in chapter 1) and 7% of the bulk of sequences.

Regarding terrestrial habitats, the analysis of a gradient (51-82ºS) suggest that the cyanobacterial diversity is higher between 70 and 80ºS. Further south and further north, the diversity decreases. One explanation could be that cyanobacterial diversity increases from the sub-Antarctic to continental Antarctica due to the disappearance of vegetation cover. Further south of 80ºS, the cyanobacterial diversity should decrease due to the harsher environmental conditions. However, another explanation could be the more

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General Discussion intensive sampling in the areas within this range of latitudes. Indeed, the analysis of sampling efforts and the number of OTUs found in different geographic areas of Antarctica shows that only 3 regions (Southern Victoria Land, Sør Rondane Mountains and Alexander Island) have been particularly well studied, while other areas did not receive enough attention (chapter 5).

It is important to highlight that molecular studies of species distribution and comparison of composition of microbial communities is possible only when the same genes and parts of the genes are analysed. For this, the sequence of 16S rRNA gene between E. coli positions 405-780 which includes the V3 and V4 variable regions, have been shown to well discriminate the different OTUs (chapter 5, Taton et al., 2003). Although in most of the cases the partial 16S rRNA genes from Genbank include these positions, some short sequences are positioned outside these boundaries and therefore do not allow for their alignment and comparison (ie. Smith et al., 2000; Wood et al., 2008).

Cyanobacteria in paleolimnological studies.

The microscopic observations from the sediment core layers from the two Beak Island lakes analysed in chapter 6, showed a low number of structures related to cyanobacteria. From the 3 proxies used in this study (microfossils, fossil pigments and aDNA), the microfossils are the least resistant proxies. The low preservation rates of microfossils within the cores impeded a correct identification of the cyanobacterial morphotypes detected in some layers. However, the fossil pigments and aDNA were better conserved.

The analysis of the fossil pigments allowed the detection of cyanobacteria during the Late Holocene in both lakes. The Beak-1 stratigraphy was dominated by pigments from diatoms, green algae, mosses and cryptophytes, whilst the pigment composition in the Beak-2 core was dominated by cyanobacterial marker pigments, which indicate a well- established benthic cyanobacterial flora during the past ca. 3200 years. The pigment stratigraphy from both lakes agrees with the present-day situation.

Similarly, aDNA from cyanobacteria was detected in all the layers of both cores. The cyanobacterial community structure differed in both lakes. Three OTUs were shared by both lakes (16ST44, 16ST58 and 16ST102), whilst OTU 16ST101 was only detected in Lake Beak-1 and OTUs 16ST33 and 16ST103 were only detected in Lake Beak-2. The different parameters and physicochemical conditions of both lakes seemed to explain the different cyanobacterial composition. Lake Beak-2, the smaller one, receives higher light inputs. This could explain the cyanobacterial dominance in Lake Beak-2 whilst mosses and diatoms dominate Lake Beak-1. In fact, cyanobacteria could need higher light levels than diatoms (Wharton et al., 1983). The presence of mosses in Lake Beak-1 could also explain the increase in cyanobacterial richness in the upper part of the Beak-1 sediment core. Indeed, the mosses present in these layers could be related to decrease in internal nutrient loading and increase in light penetration in the lake.

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General Discussion

The preserved cyanobacterial taxa observed in both lakes suggested that the potential preservation of fossil DNA from cyanobacteria might be genus or species specific. For instance, the OTUS 16ST33 and 16ST103, related to the Nostocales, were recorded in all the depths. Some Nostocales can produce resistant forms called akinetes, which could protect the DNA from degradation. Although no akinetes were detected by light microscopy, they possible were present in the past and could have played a role in the preservation of DNA. Interestingly, the OTU 16ST58 related to Synechococcus sp. was recorded extensively in both cores. Similarly, Synechococcus spp., belonging to a different OTU (16ST59), were also recorded at all the depths of a sediment core from Ace Lake (Coolen et al., 2008). The small cell size of this unicellular cyanobacterium could result in a higher number of DNA copies for the same physical space enhancing the chances of finding well-preserved copies of DNA.

Communities dynamics during the late Holocene and climate warming.

The taxonomic composition of the cyanobacterial communities remained more or less constant throughout the Mid-Late Holocene climate optimum due to their well-developed adaptive capabilities. However, we cannot discard the possibility of bias by the resolution level provided by the 16S rRNA genes. Indeed, in some cases the short 16S sequences obtained by the methods used (i.e. DGGE) do not provide enough information for an accurate identification of the genotypes. The analysis of complete 16S sequences plus ITS could improve this identification. In addition, the conservation ratio of 16S rRNA sequences could be too high, and therefore the diversity underestimated. Recently, Stackebrandt & Ebers (2006) suggested that bacterial DNA-DNA hybridizations values of 70% (which correspond to the species concept) should correspond to 16S rRNA similarities about 98-99%. Thus, this high threshold implies that the marker is maybe, too conserved.

On the contrary in the uppermost sediments of Beak-1, coinciding with the last 2 or 3 decades, the cyanobacterial community structure changed and richness increased. This was in concordance with the increase of tychoplanktonic diatoms. These changes might reflect the first ecological responses of the lakes to the recent period of rapid warming observed in the Antarctic Peninsula (Verleyen et al., 2010a). However, they might also be related to natural lake succession processes. For example, aquatic moss communities become abundant during a period which precedes anthropogenic induced climate warming and slightly precede the shifts in the diatom communities. Hence, it is still uncertain whether these changes are part of the natural lake succession or rather related to climate warming.

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