1. DIVERSITY OF CYANOBACTERIA (BLUE GREEN ALGAE) IN THE MICROBIAL MATS OF ANTARCTIC LAKES A. TATON 1, S. Grubisic 1, D. Hodgson 2, L. Hoffmann 1, A. Wilmotte 1 1 - Laboratory of Algology, Mycology and Experimental Systematic, Institute of Plant biology B22, University of Liège, B-4000 Liège, Belgium. 2 - B.A.S. High Cross, Madingley Road, GB-CB3 0ET Cambridge, United Kingdom. Chroococcales Aphanothece spp. (4 species) Synechocystis sp. Gloeocapsa spp. : Gl. cf. rupestris Gl. cf. compacta Gl. cf. alpina Gl. cf. sanguinea Chondrocystis cf. dermochroa Chroococcus sp. Pleurocapsa cf. aurantiaca Chamaesiphon cf. subglobosus Oscillatoriales Pseudanabaena sp. Schizothrix spp. : Sch. cf. tenuis Sch. cf. braunii or cf. heufleuri Leptolyngbya spp. (6 species) Oscillatoria spp. : O. cf. simplissima O. sp. Nostocales Nostoc spp. (3 species) Calothrix spp. (2 species) Petalonema cf. involvens Coleodesmium cf. scottianum or cf. swazilandicum Dichothrix sp. Culture and isolation To isolate a maximum of strains, we have used different methods : 1 - using a dissecting needle under the binocular. 2 - homogenising the mats with a potter tube and spreading out 500 µl on solid media. 3 - for the mats rich in sediments, aliquoting in different culture media, vortexing and letting of the mineral sediments at the bottom of the tubes, transfering of 1 ml of the upper phase in other tubes with different liquid media or spreading out 200 µl on solid media. To obtain clonal strains without fungi or eukaryotic microalgae, only one filament or some cells were transferred on a media with cyclohexamide. In total, 17 culture media were tested of which we have created 8 media with chemical compositions similar to those observed in the original lakes. In addition, three incubation temperatures were used: 5, 12, and 22°C. Introduction Research on microbial biodiversity in Antarctica is still in a starting phase though it is a very promising area of research. Antarctica is characterised by its geographical isolation and its extreme climate. This has led to the evolution of novel biochemical adaptations to severe low temperatures and hypersalinity in lakes (Fritsen & Priscu 1998), and possibly also of endemic species. In addition, most of the continent has experienced little or no anthropogenic influence. This offers a unique opportunity to study diversity of pristine biotopes. The microbial mats appear as fibrous matrixes formed of interlocking filaments and produced by a microbial community generally associated to the sediments (Wharton et al. 1983) and generally dominated by cyanobacteria (Castenholz 1994). In the Antarctic lakes, the benthic microbial mats have accumulated since about ten of thousand of years on the bottom of lakes. The absence of predation and disturbance by eukaryotes, as well as the low temperatures slow down the decomposition of the deep layers (Ellis-Evans 1996). The primary production of these mats is higher that the planktonic one (Ellis-Evans 1996). Therefore they play a major role in the dynamics of these lakes but also for the production of the continent. Conclusion These are the first steps of the study of the biodiversity of cyanophyceae in the microbial mats from Antarctic lakes. We have now a precise idea of this biodiversity, as it appears in light microscopy. In addition, 65 strains have been isolated. With the help of the molecular tools, we will study the genetic biodiversity of these mats in culture and in situ. The comparison of results obtained with these two approaches will allow us to define their advantages and pitfalls. Schizothrix sp. & Leptolyngbya sp. Leptolyngbya sp. Calothrix sp. Obtention of clonal strains without fungi or eukaryotic micro-algae. Examples of manipulations. We have presently obtained 37 cyanobacteria strains at 25°C, 20 strains at 12°C and 8 strains at 5°C. The strains are distributed in the following genera : Leptolyngbya, Schizothrix, Nostoc, C alothrix, Petalonema, Gloeocapsa and Chamaesiphon and come from 20 different lakes. A small saline lake (24.4 mS/cm) in the Rauer Islands, near Larsemann Hills. BIOTECH Life Sciences and Technologies Biotechnology Programme (1994-1998) http://europa.eu.int/comm/dg12/biot1.html Contact person in Biotech Unit: stephane.hogan@dg12.cec.be stephane.hogan@dg12.cec.be Biotechnology unit - DG XII European Commission rue de la Loi, 200 / wetstraat, 200 B-1049 Bruxelles / Brussel Belgique / België Fax number: +32-2-299.18.60 E-mail: life-biotech@dg12.cec.belife-biotech@dg12.cec.be List of samples and handling of studied samples For the identification, the observations were realised in light micro- scopy and the measures were taken with an ocular micrometer. Gloeocapsa sp. from lake Gentner, Larsemann Hills. Floristic inventory Molecular study A molecular approach will be carried out on selected samples to study the genetic biodiversity of these mats in culture and in situ. The techniques used will be based on the use of rDNA sequences and include DGGE/TTGE and constructions of clone libraries. Bibliography Castenholz R.W. (1994) - Microbial mat research: The recent past and new perspectives. In: Microbial Mats, Structure, Development and Environmental Significance (Stal L.J., Caumette P. eds), pp. 3-18. Springer Verlag, Berlin. Ellis-Evans J.C. (1996) - Microbial diversity and function in Antarctic freshwater ecosystems. Biodiversity and Conservation 5:1395-1431. Fritsen C.H. & Priscu J.C. (1998) - Cyanobacterial assemblages in permanent ice-covers on Antarctic lakes: distribution, growth rate, and temperature response of photosynthesis. J. Phycol. 34:587-597. Wharton R.A., Parker C.B. & Simmons Jr. G.M. (1983) - Distribution, species composition and morphology of algal mats in Antarctic dry valley lakes. Phycologia 22:355-365. The samples have been collected in 3 different parts of the continent. In the Larsemann Hills, benthic microbial mats were collected from 73 lakes. We received also samples from the Dry Valleys (Lake Fryxell and Hoare) and from Vestfold Hills (6 different lakes). A physico-chemical characterisation of the lake water was carried out and the lakes were selected to cover a wide range of chemical environments (pH ranging from 5.5 to 9.4, salinity from 0.01 to 31 %) to maximise the potential for microbial diversity in the sampling program. When we received the samples, they were processed immediately. parts of the samples were fixed in 4% formaldehyde for microscopic observation, transferred in liquid culture media and stored at – 20°C for molecular study. Ecology In waters with a salinity lower than 0,1% as well as in hypersaline waters, there is a low diversity ( Leptolyngbya spp. and Oscillatoria spp.). The highest diversity occurs in salinities ranging from 0,1 to 0,5%. In these waters, we have observed all the genera. Whereas the genera Calothrix, Synechocystis and Gloeocapsa seem to be limited to lakes with weak salinity (from 0,1 to 0,5%), the genera Leptolyngbya, Oscillatoria, Schizothrix, Nostoc and Aphanothece have a larger distribution. We observed them in brackish waters and in waters with a salinity close to the the sea water. A surface sediment core containing finely-layered microbial mats from lake Nella in the Larsemann Hills. Nostoc sp. From Big lake, Larsemann Hills. Calothrix sp. From Firelight lake, Bolingen island near Larsemann Hills. 500 ul Vortex Original samples 5°C, 12°C, 22°C BG11 GANXBG11 o GOX 11 NP 33 NP 1 ml200 ul 1 2 3 Coleodesmium sp. From lake n° LH61, Larsemann Hills. 10µm 20µm 10µm