Presentaci

1
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
(5116'48"W, 8227'28"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 (29º2629",
80º26166S). 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.
Sampling
Fig,1. Forlidas Pond
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.
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 22C, 12C and 4C 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.
Fig.6. Three morphotypes in the cultures from TM2
Fig.5. TM1 morphotype in culture corresponding to
Leptolyngbya antarctica
Fig.7. Oscillatoria-like morphotype in the
environmental sample and another filamentous
cyanobacterium in the culture from TM3
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).
Molecular analyses
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.
Conclusions
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
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.
(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. 633327-3332. (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. ,
695157-5169 (4)-Saitou, N., Nei, M. (1987) The
neighbor-joining method a new method for
reconstructing phylogenetic trees. Mol. Biol.
Evol. Jul4(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.