320
targeting plastid genes (Fuller et al. 2006 ; Lepere et al.
2009 ), the use of specifi c primers for photo- synthetic taxa
(Viprey et al. 2008 ) and the construction of clone libraries
from fl ow cytometry-sorted populations (Shi et al. 2009 ;
Marie et al. 2010 ). Finally, the use of 454 pyrosequencing
allowed to obtain a very effi cient sequencing, and phyloge-
netic analyses applied to NGS data allowed to shed light on
clades of freshwater picoeukaryotes rarely or never detected
(including pigmented taxa) with classical molecular ecology
approaches (Debroas et al. 2015 ). For instance Taib et al.
( 2013 ) showed that less than 5 % of the OTUs identifi ed from
massive sequencing data had previously been detected in
lakes.
Anyway, all approaches have led to the common conclu-
sion that photosynthetic picoeukaryotes are more diverse
than previously thought and have highlighted their impor-
tance in aquatic environments. As already highlighted for
non-pigmented picoeukaryotes, community composition
revealed in lakes highlights a difference in the composition
of pigmented picoeukaryotes between these environments
and oceans. Indeed, the main pigmented class identifi ed in
the ocean, the Mamiellophyceae (Marin and Melkonian,
2010 ) with the three genera Ostreoccocus, Bathycoccus , and
Micromonas, have not been detected in lakes in the picoeu-
karyotic fraction so far (Lepère et al. 2008 ; Debroas et al.
2015 ).
The presence of pigmented cells, traditionally considered
as photoautotrophs, in the deepest zone of lakes (Lepere
et al. 2010 , 2016 ) clearly suggests mixotrophic behaviour of
these taxa. Chlorophyta sequences found in lakes were
mostly affi liated to Chlamydomonadales (Lepere et al.
2008 ). Recent experiments by Tittel et al. ( 2009 ) showed that
heterotrophy occurred in Chlamydomonas to exploit dis-
solved organic carbon by osmotrophy. Moreover, Ukeles and
Rose ( 1976 ) showed the mixotrophy of various strains of
Chlorophyta. In ocean environments, Liu et al. ( 2009 )
showed that the phylogenetic position of pico-Haptophyta
implies that they are photophagotrophic, in agreement with
the recent discovery of dominant bacterivory by small
eukaryotic phytoplanktons in oceans (Zubkov and Tarran
2008 ; Hartman et al. 2012 , 2013 ). According to Zubkov and
Tarran ( 2008 ), small picoalgae carry out 40–95 % of the bac-
terivory in the euphotic layer of the temperate North Atlantic
Ocean in summer, suggesting the global signifi cance of mix-
otrophy. This fi nding reveals that even the smallest algae
have less dependence on dissolved inorganic nutrients than
previously thought, obtaining a quarter of their biomass from
bacterivory. Moreover, phagotrophy in photosynthetic
Haptophyta was well described in genus Chrysochromulina
(Legrand et al. 2001 ), a genus observed in various lakes
(Temponeras et al. 2000 ). Mixotrophy may then provide a
competitive advantage over both purely phototrophic micro-
algae and non-pigmented protists (Stickney et al. 2000 ;
Domaizon et al. 2003 ; Troost et al. 2005 ; Kamjunke et al.
2007 ) in oligotrophic systems and/or in situation of phos-
phorous depletion (epilimnion in summer stratifi cation) or
light limitation.
Even more unexpected is the presence of pigmented cells
in the anoxic zone of lake Pavin. Indeed, 454 pyrosequenc-
ing , quantitative PCR as well as microscopic observations
showed the presence of pigmented picoeukaryotes at 80 m in
lake Pavin (Lepère et al. 2016 ). The groups identifi ed are
mainly Haptophyta and Chlorophyta. Anaerobic metabolic
pathways allow unicellular organisms to tolerate or colonize
anoxic environments. Over the past 10 years, genome
sequencing projects have brought a new light on the extent of
anaerobic metabolism in eukaryotes. A surprising develop-
ment has been that free-living unicellular algae capable of
photoautotrophic lifestyle are, in terms of their enzymatic
repertoire, among the best equipped eukaryotes known when
it comes to anaerobic energy metabolism. Among phyto-
plankton, the green algae Chlamydomonas reinhardtii and
Chlorella have the most extended set of fermentative
enzymes reported so far (Atteia et al. 2013 ).19.3 Picoeukaryotic Rare Biosphere
Even though the notion of rare taxa is known for a long time
in ecology, this notion is fairly new in the microbes world.
The recent application of NGS technologies has aided in the
discovery of a tremendous diversity of undescribed microbes
and gave us a way of sequencing the rare microbial biosphere
(Pedros alio 2012 ). Sogin et al. ( 2006 ) provided the fi rst evi-
dence of a myriad of bacterial OTUs found to exist in low
abundance, which has resulted in the ecological signifi cance
of rare microorganisms becoming one of the hottest topics in
microbial ecology in which many questions remain unan-
swered. The studies about the rare microbial biosphere are
mainly focused on bacteria and Archaea and fail to include
the microbial eukaryotes (ie. picoeukaryotes), which are
involved in the main biogeochemical cycles of the earth and
cover all of the functional roles (discussed in the previous
section: phototrophs, parasites, saprotrophs, phagotrophs).
The fi rst attempt to study the dynamics of the rare microbial
eukaryotes was based on the analyses of DNA sequences
obtained by Sanger sequencing method (Caron and Countway
2009 ), demonstrating that it is likely that the rare microbial
biosphere was underestimated. Recently, the NGS approach
demonstrated a stable predominance of a few highly abun-
dant taxa at different temporal scales in lakes (Nolte et al.
2010 ; Mangot et al. 2013 ), while rare microorganisms make
up the majority of the diversity observed within eukaryotic
assemblages. Although the ecological roles of the rare micro-
organisms remain unclear; it is now quite clear that we have
to distinguish different categories of ‘rareness’. Some rareC. Lepère et al.