Cyanobacterial diversity and distribution in the very extreme environments of the Pensacola Mountains (82°S, Antarctica).

(1)

Light microscopy and molecular diversity studies confirmed the low biodiversity expected, with only

1 ,

2 ,

3 and

4 OTUs, depending on the sample site. It is the first time that we observe such a low diversity in our studies concerning

Antarctic coastal lakes and seepages. In the latter regions, the number of OTUs per new sample ranged between 4

(Rauer8 lake) and 12 (ReidJ lake).

Information on the geographic distribution of genotypes show that the four first OTUs obtained here are

cosmopolitan, and were observed in non-polar regions. Our hypothesis is that such cosmopolitan OTUs have to be

very resistant and adaptable to disseminate to Antarctica and especially, to colonise such extreme habitats on the

continent.

OTU 6

seems to represent a new diversity.

References

Thanks for supporting

(1)-Hodgson, D. and Convey, P. (2004) Scientific Report - Sledge Bravo 2003-2004. BAS Signals in Antarctica of Past Global Changes: Dufek Massif – Pensacola Mountains; Mount Gass – Shackleton Mountains. DRAFT. Unpublished BAS Internal Report Ref. R/2003/NT1. British Antarctic Survey, Cambridge; .(2)-Nübel, U., Garcia-Pichel, F., Muyzer, G. (1997) - PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl. Environ. Microb. 63:3327-3332.; (3)-Taton, A., Grubisic, S., Brambilla, E., De Wit, R., Wilmotte, A. (2003) Cyanobacterial diversity in natural and artificial microbial mats of lake Fryxell (McMurdoDry Valleys, Antarctica): A morphological and molecular approach. App. Environ. Microb. , 69:5157-5169; (4)-Saitou, N., Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. Jul;4(4):406-25; (5)-Van de Peer, Y., De Wachter, R. (1997) Construction of evolutionary distance trees with TREECON for windows: accounting for variation in nucleotide substitution rate among sites. Comput. Appl. Biosci. 13: 227-230. Gordon, D.A., Priscu, J., Giovannoni, S (2000) Origin and Phylogeny of Microbes Living in Permanent Antarctic Lake Ice. Microb Ecol. 39 (3):197-202.

Introduction

The evolutionary history and geographical isolation of the Antarctic continent have produced a unique environment, rich in species adapted to its extreme conditions. Despite this harsh environment, cyanobacteria are able to build extensive communities in aquatic and terrestrial biotopes, where they are the most important primary producers.

Forlidas Pond (51°16'48"W, 82°27'28"S) in the Dufek Massif, (Pensacola Mountains) is one of the most southerly freshwater ponds known in Antarctica that contains plant life. It is a perennially frozen, shallow, round lake of 90.3 m diameter (1). We have studied the cyanobacterial diversity in three related sites along a gradient from the lake to the terrestrial biotope in its vicinity in this very isolated place in the middle of the Antarctic continent.

Davis Valley and ‘Forlidas’ Valley are large ice-free ‘dry’ valleys at the northeastern end of the Dufek Massif. There are few comparable areas in this like the western end of the Shackleton Mountains. Shakleton Range is 400 km to the north east of Dufek Massif. Here we find the Lundström lake (29º26´29", 80º26´166¨S). It is perennially ice covered with a seasonal moat that forms during summer from where we studied the last of our samples.

Given the remoteness and harshness, we could expect that the life, if present, should be represented by a low biodiversity and possibly by adapted taxa.

Conclusions

Using cyano-specific PCR primers (CYA359F and 23S30R) (2, 3), fragments of the gene coding for the 16S rRNA and the spacer between the 16S and 23S were amplified by PCR. Sequences were edited with BioEdit software and searched for similarities with BLAST. All sequences were tested for eventual chimeras. We grouped our sequences in OTUs (Operational Taxonomic Units) that are groups of sequences sharing at least 97.5 % of sequence similarity. A DISTANCE TREE was constructed by the neighbor-joining method (4), based on partial 16S rRNA sequences (Fig.9). The tree was

constructed using the TREECON software (5).

All sequences from TM1 are grouped in only one cluster, together with CCR2E7, a clone from a hypersaline lake (lake Rauer 2) of the Rauer Islands, Eastern Antarctica. All TM1 clones form a single OTU, OTU1.

TM3 sequences belong to two OTUs. The sequences of OTU2 are grouped with Phormidium sp. Ant. Orange, and

Phormidium sp. Ant. Lunch, two psychrophilic strains from Mc Murdo Ice Shelf meltwater ponds, with a rare

11-nucleotides insertion, present in all the members of this cluster, except in Oscillatoria sp. E17 and Tychonema bourrellyi. TM3 sequences from OTU3 are grouped with a clone obtained from the lake Fryxell, in the McMurdo Dry Valleys and a strain of Geitlerinema from Loa river in Chile.

Sequences from TM2 are distributed into the three OTUs found in TM1 and TM3, except TM2-D1 that forms a new OTU (OTU4), related (99%) with cyanobacteria sampled in Antarctic Peninsula.

TM4 clones are different from the others. Most of the sequences from TM4 belong to one OTU, except two clones, TM4-C30

(OTU 7) and TM4-G9 (OTU 5). TM4-C30 is a chimera beetween the clone RJ088 (99% similarity), found in lake Reid, and the clones from OTU 6. TM4-G9 has 99% similarity with clone RD069, found in Lake Reid. The most abundant OTU from

TM4, represented by TM4-E5 (OTU 6), seems to represent a new diversity. None of these clones share more than 94% with other sequences in the databases. The most closely related strain is Pseudanabaena tremula UTCC 471 from Canada (94%)

In Fig. 10, we mapped the OTUs present along the spatial gradient existing in and around Forlidas Pond, and observed a continuity of certain OTUs as suggested by Gordon et al. (2000).

Cyanobacterial diversity and distribution in the very extreme environments of the

Pensacola Mountains (82°S, Antarctica)

Fernández-Carazo, R.

1

, Hodgson, D.

2

, Wilmotte, A

1

1-Centre d’Ingénierie des Protéines, B6, Université de Liège, 4000 Liège, Belgique. E-mail: rfcarazo@student.ulg.ac.be

2-BAS, Madlingey Road, Cambridge CB3 0ET, UK

Fig.9. Distance tree based on the partial 16S rRNA sequences of the clones of TM1, TM2, TM3 and TM4. The branches supported by less than 85% of bootstrap are drawn as unresolved. The scale above indicates the number of evolutionary changes per position.

Microscopic analyses

We have cultivated our samples in several specific media for cyanobacteria (BG11 and a range of media, with or without nitrogen, and with different salinities). Cultures were grown at 22°C, 12°C and 4°C for several weeks until a green active biomass became visible. Microscopical observations and pictures were realised directly on the frozen environmental samples and on the cultures.

Molecular analyses

Fig.5. TM1 morphotype in culture corresponding to Leptolyngbya antarctica

Fig.6. Three morphotypes in the cultures from TM2

Fig.7. Oscillatoria-like morphotype in the environmental sample and another filamentous cyanobacterium in the culture from TM3

Fig.8. Two morphotype in the cultures from

TM4

Fig.3. Cyanobacterial mat from Forlidas Pond’s environments (TM3) Fig,1. Forlidas Pond

Sampling

Fig.2 Littoral zone of the lake (TM2)

TM1 is a water sample from the hypersaline layer at the bottom of Forlidas Pond. It is a benthic sample, from a saline slush. The pond was frozen almost completely to its base. The salinity of the bottom-water in Forlidas Pond was four times greater than seawater.

TM3 is a terrestrial cyanobacterial mat from 'Forlidas Valley'. It is a sample of the lush cyanobacterial mats found in the valley ca. 15 -20 m from the shoreline of Forlidas Pond and 1 m above a zone where there was a mineral / salt precipitate.

TM2 is sample of an active cyanobacterial mat growing in the littoral zone of Forlidas Pond under 10 cm of ice and 10 cm of fresh water.

TM4 is a littoral mat sample taken from Lundstrom Lake. In contrast to Forlidas Pomd, this lake is

freshwater with only a moderate increase in conductivity recorded at the base of the water column.

Fig.4. Satelite view of Shackleton Mountains and Lundström lake (TM4) Lundström Lake TM1 TM3 TM2 1 2,3 4

Depth lake littoral

Terrestrial surroundings

Fig.10. Distribution of OTUs in the environments of Forlidas Pond. This seems to indicate that OTUs 2 and 3 are quite resistant to ultraviolet radiations because they are more exposed then OTU1 and OTU2 that are protected under the thick layer of ice. In addition, these OTUs must be more resistant to drying, since the terrestrial mat is exposed to winds and the extremely dry climate of Antarctica. OTU1 seems halotolerant as it was only found in two saline biotopes.