Abstract :
[en] Microbial organisms dominate most Antarctic ecosystems and play a crucial role in
their functioning and primary productivity. Compared with temperate and tropical
regions and despite their ecological importance, little is known about Antarctic
microbial diversity and its geographical distribution. This is due the lack of systematic
sampling and geographical coverage, and the problems associated with species
definition, cryptic diversity and cultivability (e.g. Taton et al., 2003). As a result, we
largely lack the „baseline‟ data needed to observe possible future changes in
microbial diversity and taxonomic composition due to ecosystem change and/or
human introductions.
Most of the earlier diversity studies were carried out with traditional methods
such as isolation of bacterial strains and microscopic identifications of cyanobacteria
and protists on the basis of morphological features and „force-fitting‟ of names of
temperate taxa on the Antarctic ones. This approach also lacked stability because of
the plasticity of the morphology. Molecular tools enabled studies based on the SSU
rRNA gene, and have shown a quite different view of the diversity and the existence
of not-yet cultivated genotypes. In contrast to phenotypic markers, the genotypic
based approaches have a more fine-grained taxonomic resolution and reflect the
evolutionary history of the organisms. Molecular-based approaches also have a
considerable potential for the study of the geographical distribution of
microorganisms. This is important, because it is still unclear whether geographic
isolation is present in microorganisms, and hence whether they exhibit a
biogeography at all (Martiny et al. 2006). This „ubiquity hypothesis‟ was first
formulated by Baas-Becking (1934) and states that „everything is everywhere, but the
environment selects‟. It is underlain by the assumption that the vast population sizes
of micro-organisms drive ubiquitous dispersal and make local extinction virtually
impossible (Finlay et al. 2002). However, various recent studies suggest that microorganisms,
do display restricted geographic ranges (Chao et al 2006; Foissner 2006)
and that endemism is possible.
Antarctica is a prime place to investigate microbial biogeography and to
elucidate the roles of historical processes and contemporary environmental conditions
shaping microbial diversity and community structure. This is due to its extreme
isolation with respect to the rest of the world, resulting from its geographic setting and
the nature of ocean and atmospheric currents as well as of the scattered occurrence of
terrestrial oases along the continental margins. Furthermore, organisms inhabiting the
continent need to survive in extreme environmental conditions, such as low and
extremely fluctuating temperatures, dramatically changing light conditions, high
seasonal UV-B loads, and low humidity.Thus, as a whole, the continent bears wide environmental gradients that impose
increasing stresses on the biodiversity and community structures of Antarctic
environments (Gibson et al. 2006a). In addition, certain habitats offer some protection
from the extreme conditions. For example, liquid water in aquatic environments may
act as „thermal buffer‟ (Vincent and Laybourn-Parry. 2008). Moreover, preliminary data
on aerosol diversity in the Antarctic Peninsula showed the potential for wide-range
transport of microbial diversity, though much of the aerobiota found was of local origin
(Hughes et al. 2004).B. Objectives
In the present project, we aimed to extend the baseline information of microbial
diversity through an integrated and standardized analysis of the microbial diversity of
aquatic habitats in terrestrial Antarctic environments. We aimed to use a polyphasic
approach combining morphologic characterization by microscopy with molecular
techniques in order to reveal the diversity of bacteria, cyanobacteria and protists (with
special emphasis on green algae and diatoms), which have been identified as
interesting focal taxa during our earlier studies. To work in parallel on environmental
samples and isolated strains in culture allows us to obtain a more complete image of
the diversity.
C. Conclusions
a) Bacteria diversity
Nine samples were used to study the culturable bacterial diversity by plating on
different types of media and incubation at three relatively low temperatures. A total of
3806 isolates were obtained. They were first characterized by comparison of wholegenome
fingerprints (rep-PCR) and this allowed them to be grouped into about 1400
unique rep-types. Very few of these comprised isolates from more than one sample.
To identify these organisms, the 16S rRNA gene of a representative of each type was
sequenced partially or in full. The diversity recovered belonged to four major phyla,
Actinobacteria, Bacteroidetes, Proteobacteria and Firmicutes. Isolates belonging to
the phylum Deinococcus-Thermus were only recovered from samples BB50, BB115,
PQ1 and SO6. Many potential new species or new genera were documented among
the isolates. Although most genera recovered were reported previously from
Antarctica, for 30 out of the 83 genera, this was not the case. Moreover, several
isolates belonged to genera that at present contain only one species and even one
strain. The additional cultures obtained in this work may give more insight in the
diversity present in these genera. Comparison with sequences from public databases
indicates that an important number (42.2%) of species recovered seem to be
restricted to Antarctica.However, it is known that only about 5% of all bacterial species are currently present
in databases and this number may therefore come down in the future. It does
suggest that in Antarctica both cosmopolitan taxa as well as taxa with limited
dispersal and which evolved in isolation occur.
A selection of Flavobacterium isolates recovered was studied in more detail
using phylogeny of the 16S rRNA gene and the gyrB gene, as well as biochemical and
chemotaxonomic approaches. The data revealed new strains from the Antarctic
Flavobacterium micromati as well as twelve potential new species among our isolates.
These will be studied further to describe and name them. To investigate the
distribution of two of these potential new Antarctic Flavobacterium species, a PCR
test using specific 16S rDNA primers was developed and used to detect these
species in the community DNA of 32 Antarctic samples. This test can be used in the
future to investigate the distribution of these species in environmental samples.
b) Cyanobacterial diversity
Different molecular methods were used to study the cyanobacterial diversity in
strains (5) and environmental samples (95).
Five cyanobacterial strains from four continental samples were isolated.
They belonged to the Oscillatoriaceae. Sequence analysis of these strains allowed
the finding of 2 OTUs, not revealed by other molecular techniques (clone libraries
and DGGE) stressing the importance of a polyphasic approach to unveil the microbial
diversity of an environmental sample.
The uncultivated diversity was studied using clone libraries and DGGE.
Clone libraries gave quite a large range of richness depending on the samples, from
2 to 12 OTUs (OTUs defined at a threshold of 98.5 % 16S rRNA similarity). The
comparison with the sequences in public databases showed the existence of a
majority of cosmopolitan OTUs, but also OTUs restricted to the cold biosphere (Arctic
and/or alpine), and a minority of potentially endemic OTUs.
A Detrended Correspondence Analysis (DCA) was run with data from clone
libraries from 20 samples of Prydz Bay, the Transantarctic Mountains, Shackelton
Range and the Antarctic Peninsula and revealed that the OTU composition is
geographically structured as each region has a more or less unique flora. The
differences might be underlain by several reasons, such as differences in limnological
properties between regions or rather the result from dispersal limitation among
cyanobacteria. We can also observe that saline samples are grouped.
The DGGE band pattern analysis for 95 samples representing the 3
biogeographic provinces showed no clear community structure, probably due to
mixed band classes.The DGGE bands from a subset of 56 samples were sequenced and grouped into
OTUs. We obtained a total of 33 OTUs for which the distribution was investigated.
This showed different patterns of distribution: most of them (60.6%) were
geographically and ecologically widespread. The rest (39.4%) of them seemed to be
restricted to the "cold biosphere" (polar and alpine habitats). Among the latter, 5
OTUs seem to be endemic to Antarctica.
These sequence analyses point towards the existence of environmental and
geographic limitations on the distribution of the cyanobacterial OTUs. Thus, both
cosmopolitan and potentially endemic distributions were observed.
c) Microalgal diversity
The cultivable diversity of coccal green algae was studied in samples from 33
lakes in maritime and continental Antarctica. The 14 distinct chlorophycean and
trebouxiophycean lineages observed were compared with the sequences present in
GenBank and point to a wide phylogenetic diversity of apparently endemic Antarctic
lineages at different taxonomic levels. Two taxa were detected in most regions,
suggesting that they are widely dispersed over Antarctica. Most of the studied taxa (10
out of 14) however were only retrieved from one ice-free region. A molecular clock was
applied and calibrated using absolute ages estimated by setting the split of
Chlorophyta and Streptophyta at 700 and 1500 Ma. On this basis, the majority
(16/26) of the lineages have estimated ages between 17 and 84 Ma and likely
diverged from their closest relatives around the time of the opening of Drake
Passage, while some lineages with longer branch lengths have estimated ages (330
to 708 Ma), that precede the break-up of Gondwana. The variation in branch length
points to several independent but rare colonisation events.
In diatoms, the cultivable diversity was studied in the globally distributed
species complex Pinnularia borealis. The time-calibrated molecular phylogeny based
on concatenated rbcL and LSU (D1-D2 region) sequence data showed a divergence
of the Continental Antarctic lineages from a western European lineage around 7.67
Ma (14.77-1.95 Ma, 95% Highest Posterior Probability interval (HPD)). Combined,
the findings in green algae and diatoms are in agreement with patterns found in
multicellular organisms and they support the „glacial refugia hypothesis‟, which states
that long-term survival took place which resulted in a specific Antarctic flora and
fauna.
d) Geographic patterns in Antarctic microbial diversity
A comparison of the uncultured diversity in 41 samples revealed that conductivity
and variables related to salinity significantly explain differences in the community
structure of diatoms, green algae, and cyanobacteria.The Denaturing Gradient Gel Electrophoresis using a universal prokaryote or
cyanobacterial primers resulted in a relatively large amount of mixed bands, which
prevented a multivariate analysis. A variation partitioning analysis of 41 samples in
which all microbial groups were studied revealed that geographical variables were
more important in the eukaryotic microorganisms compared with the prokaryotes. If
these differences between the different taxonomic groups are real, the contrasting
patterns observed between prokaryotes and eukaryotes are likely related to life cycle
characteristics (e.g. formation of spores, resting stage, sexual versus asexual phase).
Hence, we hypothesize that findings from one particular microbial group cannot be
generalized to microbes as a whole. A 454 pyrosequencing analyses will enable us
to test further this hypothesis. What we already found is that cultivation and cultureindependent
approaches are complementary in exploring the diversity of a
particular habitat, because some cultivated taxa were not detected using the 454
pyrosequencing analysis.
D. Scientific support to a sustainable development policy
Our biodiversity analyses have revealed a considerable diversity. Depending on the
microbial group (bacteria, cyanobacteria or microalgae), and based mostly on SSU
rRNA sequences, a number of species new to science and possibly unique to
Antarctica were identified. These findings demonstrate the large value of Antarctica
as a relatively unexplored territory that represents an immense resource for
biotechnological, biomedical and environmental and applications. The discovery that
Antarctic lakes are dominated by endemic microbial organisms has important
implications for the conservation of these ecosystems. More in particular, the
identification of Antarctic Special Protected Areas (ASPAs) was traditionally based on
the diversity/presence of multicellular organisms. Because microorganisms, together
with a few mosses, lichens, two flowering plants and a number of small invertebrates,
are the only permanent inhabitants, they should be an additional criterion for the
delineation of ASPAs. For example, an endemic, as yet unidentified diatom species
occurs in a few lakes in the Larsemann Hills. The presence of this taxon is likely
related to the fact that some of the lakes acted as glacial refugia during past glacial
maxima (Hodgson et al. 2001). The protection of this region should thus be a priority.
It is also apparent that we have only just caught „the tip of the iceberg‟ of the
biodiversity that inhabits Antarctica. Further studies are needed and will undoubtedly
yield even more novel organisms and insights. In view of the anticipated increased
effects of global warming (such as rising temperature, increased desiccation,
changes in UV radiation and snow/ice cover), it seems urgent to further assess
particularly the impact of global change on Antarctic biota. Indeed, in addition to
endemics, the total microbial biota is important for the ecosystem functioning and
might be impacted by future climate change effects.
