319are a mixotrophic class characterized by plastidic and color-
less cells. On average, in lake environment, the class of
Chrysophyceae represents about 18 % of the diversity
described in the epilimnion by molecular techniques.
Sequences of Chrysophyceae obtained in lakes allowed
phylogenetic analysis of these environmental clades
(Richards et al. 2005 ; Lepère et al. 2008 ; Tarbe et al. 2011 ).
The majority of these Chrysophyceae sequences are close to
non- pigmented species such as Spumella , Oïkomonas ,
Paraphysomonas or Poterioochromonas (Lefranc et al. 2005 ;
Richard et al. 2005 ). However, some sequences phylogeneti-
cally close to photosynthetic cells (eg Mallomonas ) are also
found (Tarbe et al. 2011 ). Although ciliates are generally
known to have a size superior to 5 μm (Bourrelly 1970 ), their
presence in this size fraction of freshwater lakes could repre-
sent the detection of previously unisolated groups or the
results of cell damages during fi ltration steps. On average, in
lake environment, 8 % of the OTUs characterized in the epi-
limnion are affi liated to ciliates whatever the lake and the
trophic level (Lepère et al. 2008 ).
Results obtained with molecular and microscopic
approaches in lakes showed differences with oceanic ecosys-
tems. Indeed, parasites also seem to be important in marine
environments, but most of them are affi liated with Syndiniales
(Alveolates) (Guillou et al. 2008 ). The exclusively marine
provenance of this group is confi rmed by the fact that their
18S rRNA gene sequences have not been retrieved from lake
PCR surveys using eukaryotic general primers.
19.2.2 Pigmented Picoeukaryotes
Studies conducted in lakes using the cloning-sequencing
method with generalist primers targeting gene coding for
18S SSU rRNA encoding gene highlighted that putatively
pigmented picoeukaryotes sequences were always present at
a very low proportion in clone libraries (Richards et al. 2005 ;
Chen et al. 2009 ; Lefèvre et al. 2008 ; Lepere et al. 2008 ).
Most of the sequences affi liated to pigmented eukaryotes
were retrieved from epilimnic samples and were represented
by Cryptophyta, Chlorophyta, and Haptophyta. However,
using FISH method, Mangot et al. ( 2009 ) showed during
temporal study (1-year) that Chlorophyta were well repre-
sented, accounting for 17.9 % of small eukaryotes abundance
in lake Bourget. Surprisingly, Chlorophyta are present all
along the water column, even in the deepest points out of the
euphotic zone; up to 950 cells ml −1 were counted at 50 m in
lake Pavin (Lepère et al. 2010 ). Similarly, Chen et al. ( 2009 )
observed, by morphological observations, that some pig-
mented taxa, such as Tetraedron sp., were abundant in the
Meiliang Bay, but were not detected by molecular analysis.
FISH data also showed the presence of members of
Prymnesiophyceae (Haptophyta) in lakes (Lepère et al.
2010 ) although they were present at very low proportions in
18S sequences bank (Lefranc et al. 2005 ; Lepère et al. 2008 ;
lefèvre et al. 2008 ; Richards et al. 2005 ) which is interesting,
since, described species of Prymnesiophyceae are generally
considered to be larger than 5 μm (Vaulot et al. 2008 ).
Recently, this group was recognized as a major component
of the eukaryotic picoplankton in marine water and particu-
larly in oligotrophic waters (Liu et al. 2009 ; Lepère et al.
2009 ; Cuvelier et al. 2010 ). Haptophyta are well known in
marine systems (Moon-van der Staay et al. 2000 ; Iglesias-
Rodriguez et al. 2002 ), especially because of their ability to
form toxic blooms (Gjøsæter et al. 2000 ; Baker et al. 2007 ),
only a dozen Haptophyta species have been previously
described from freshwater or terrestrial habitats (John et al.
2002 ). However, some haptophyte blooms have been previ-
ously recorded in different lacustrine systems (Nicholls et al.
1982 ; Hansen et al. 1994 ), suggesting an importance of this
group in other freshwater ecosystems. Statistic analysis
revealed an opposite distribution between Chlorophyta and
Haptophyta groups (Lepère et al. 2010 ). Haptophyta seems
to be confi ned to surface waters, 0–20 m, but they can also be
detected below the photic zone in lake Pavin and other lakes
(Lepère et al. 2010 , unpublished data). On average,
Haptophyta contribution to the total diversity seems less
important than chlorophytes in all the lakes studied, and
sequence contribution seems to decrease with the trophic
level.
According to these methods (microscopy, SSU rDNA
PCR and FISH), Cryptophyta is an important group among
the pigmented lacustrine picoeukaryotes. For example,
Mangot et al. ( 2009 ) showed by the TSA–FISH method that
cryptophytes accounted on average for 9.5 % of the total
picoeukaryotes (<5 μm) and they could represent up to 30 %
of the sequences in 18S libraries (Lepère et al. 2008 ; Lefèvre
et al. 2008 ). These sequences revealed four clades where
some of them seem to be restricted to oligo- and mesotrophic
systems (Lepère et al. 2008 ).
Although the use of molecular approaches, especially
sequencing of the 18S rRNA gene, has greatly improved our
understanding of the diversity and distribution of aquatic
picoeukaryotes, all these data suggest that 18S rRNA clone
libraries construction have underestimated pigmented cells,
and because of their limitations, molecular approaches based
on PCR may not refl ect the real diversity. Quantitative
approach by the TSA–FISH method confi rmed these poten-
tial PCR biases, revealing the relative importance of
Chlorophyta and Haptophyta in lakes. Indeed, nuclear rDNA
PCR-based studies of eukaryotic communities are subject to
selective amplifi cation biases due to GC content (Liu et al.
2009 ). Gene copy number is another factor that must be
taken into account when considering the bias of clone librar-
ies (Zhu et al. 2005 ). In order to focus on phototrophs, sev-
eral strategies have been developed, including studies19 Diversity and Biogeography of Lacustrine Picoeukaryotes