246
experiments were based on sequencing after cloning a DNA
fragment (ranging from 30 to 100 kb) in Escherichia coli.
This method tends to disappear in favour of high-throughput
sequencing (pyrosequencing and Illumina methods) without
cloning. High throughput sequencing technologies used after
PCR amplifi cation of marker gene amplicons (such as
16S/18S rRNA) refered as metabarcoding or metagenetics,
has also emerged as a powerful and straightforward means to
analyze richness, diversity and composition of microbial
communities. In contrast to shotgun metagenomics, metage-
netics enable the detection of rare taxa. Metagenomics and
metagenetics have been used in lake Pavin to decipher the
microbial and viral diversities. These approaches are illus-
trated here through the analysis of three taxonomic groups of
peculiar interest as they correspond to dominant
( Actinobacteria ), minor also referred to as rare ( Archaea )
and uncharacterized (viruses) groups in lacustrine freshwater
ecosystems.
The phylum Actinobacteria is defi ned as Gram-positive
bacteria with a high mol% GC DNA composition. These
bacteria are conventionally found in soils. However, 16S
rRNA gene- and FISH-based studies revealed that
Actinobacteria are also common and often dominant in a
variety of freshwater habitats (Newton et al. 2011 ).
Likewise, in a 2 year study of the epilimnion of the Pavin
Lake, Actinobacteria dominated the main bacterial group,
followed by Betaproteobacteria (Boucher et al. 2006 ). The
16S rRNA genes of these freshwater bacteria are distinct
from the soil ones and can be considered as typical from
lacustrine ecosystem, while the latest review on freshwater
bacteria shows that Actinobacteria can be divided in nine
lineages (Newton et al. 2011 ). However, the physiology of
these bacteria is still poorly understood and the lineages
were delineated from a single gene ( i.e. 16S rRNA). The
clades therefore defi ned may not correspond to distinct
ecological populations ( i.e. ecotypes). However, the isola-
tion and sequencing of DNA fragments with phylogenetic
markers among these groups can highlight relations
between phylogenetic position and physiological
properties.
Another enigmatic lineage in the oxic zone of aquatic
ecosystems is the Archaea that have originally been isolated
from environments considered as extreme with high tem-
peratures, very high salt concentrations or without oxygen
(DeLong 1998 ). First “non extreme” Archaea were discov-
ered as free-living mesophilic in oxic zone of ocean by
DeLong ( 1992 ). Ammonia oxidation by Archaea was fi rst
shown through metagenomic studies (Treusch et al. 2005 )
and then through the cultivation of isolates like
Nitrosopumilus maritimus (Konneke et al. 2005 ) or
Nitrososphaera gargensis (Hatzenpichler et al. 2008 ).
Lacustrine freshwater habitats have also emerged as an
unsuspected reservoir of archaeal diversity (Galand et al.
2006 ; Llirós et al. 2010 ; Hugoni et al. 2013a , b ) and abun-
dance, ranging between 1 and 20 % of the total bacterio-
plankton (Pernthaler et al. 1998 ). However, the activity of
some Archaea , like Euryarchaota , in the oxic zones is still
discussed. Metagenetics can be then used for sequencing
transcripts allowing determining the activity of these less
abundant taxa.
Finally, viruses, the most abundant biological entities
on earth, act in the regulation of microbial community
composition by establishing specifi c interactions with
their host. However, if the marine viruses were extensively
studied, the freshwater ones were less known until the
study of (Roux et al. 2012a ) conducted in the lake Pavin.
In addition, the presence of specifi c clades among hosts in
a given type of ecosystem ( e.g. saline or freshwater sys-
tems) suggests the existence of specifi c viral clades. The
viral diversity of freshwater systems was thus investigated
from several lakes through metagenomics analyses to eval-
uate this hypothesis.
These three case studies illustrate the power of “omic”
approaches in the analysis of microbial and viral communi-
ties in lacustrine ecosystems.15.2 Phylogeny and Ecology
of Actinobacteria by a Targeted
Metagenomic Approach15.2.1 Phylogeny of the 16S rRNA Gene
and Ecology of ActinobacteriaIn a recent synthesis (Newton et al. 2011 ), freshwater
Actinobacteria diversity was described through 9 clades
defi ned as monophyletic groups of 95 % identity sequences
and 32 tribes (97 % identity). Most clades (acI, acIII, acTH1,
acTH2, acSTL, Luna1, Luna3) belong to the Actinomycetales ,
while clades acIV and acV were affi liated to the
Acidimicrobiales. The acI lineage is more abundant in the
free-living fraction (Allgaier et al. 2007 ), and the acIV lin-
eage exhibits more localized dominance (Wu et al. 2007 ).
Clade acI represents about 66 % of the freshwater actinobac-
terial 16S rRNA gene sequences available in databases, with
tribes acI-B1 and acI-A6 representing 28 % and 18 % of this
clade, respectively. The occurrence of freshwater actinobac-
terial clades may depend on some environmental parameters
such as pH (acidic for acI-A1, B2 and B3, more basic for
clades acI-A2, A6 et B1) (Lindström et al. 2005 ) or tempera-
ture (adaptation to different thermal niches within the Luna2
tribe from calde acIII) (Hahn and Pöckl 2005 ), while actino-
bacterial abundance decreases with decreasing oxygen con-
centration (Garcia et al. 2013 ). From a metagenomics studyG. Bronner et al.