, 253 (2007);
et al.Fabrice Not,
Group with Unknown Affinities to Other Eukaryotes
Picobiliphytes: A Marine Picoplanktonic Algal
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on March 10, 2007
Algal Group with Unknown Affinities
to Other Eukaryotes
Fabrice Not,1*‡§ Klaus Valentin,2‡ Khadidja Romari,1† Connie Lovejoy,3
Ramon Massana,4Kerstin Töbe,2Daniel Vaulot,1Linda K. Medlin2§
Environmental sequencing has revealed unimagined diversity among eukaryotic picoplankton. A
distinctpicoplanktonic algal group, initially detectedfrom18S ribosomal DNA(rDNA) sequences,was
hybridized with rRNA l-targeted (rRNA-targeted) probes, detected by tyramide signal amplification–
fluorescent in situ hybridization, and showed an organelle-like body with orange fluorescence
indicative of phycobilins. Using this fluorescence signal, cells were sorted by flow cytometry and
probed.Hybridized cellscontaineda 4´,6´-diamidino-2-phenylindole–stainedorganelleresembling a
plastid with a nucleomorph. This suggests that they may be secondary endosymbiotic algae. Pending
the isolation of living cellsand their formal description, these algaehave been termed picobiliphytes.
among the smallest eukaryotic cells (1–3),
paralleling that found among marine prokary-
otes. Together with a high taxonomic diversity,
the finding of many sequences unrelated to
those of known organisms was an additional
striking feature of these first studies. Clone
libraries for the eukaryotic 18S ribosomal RNA
(rRNA)genewere constructed atdifferenttimes
from fractionated water samples (using a filter
poresize of 3 mm)from three coastalsites (4–6),
and additional libraries were established from
three more open-water sites (7, 8) (table S2). A
particular group of sequences was recovered
irregularly throughout the year (8) (table S2)
and referred to as the “Rosko II” group from
partial 18S sequence phylogenies from these
sites (4–6). Analyses of full-length sequences
(8) reveal that they form an independent
phylogenetic group among major eukaryotic
taxa (Fig. 1), (9, 10), which we have tentatively
called picobiliphytes. Our complex iterative
Bayesian analyses (8) indicate that the picobili-
phytes are an independent lineage, possibly
having a weak sister relationship with the
cryptophyte/katabletablepharid clade, although
its true sister group is difficult to assign using a
single gene phylogeny. The inability to assign
an affinity of the picobiliphytes to any other
major eukaryotic group (table S1) in the
olecular tools applied to DNA re-
trieved from marine microorganisms
have revealed considerable diversity
eukaryotic 18S rDNA tree was confirmed with
the Kashino-Hasagawa test (8) (table S3). Their
deep branching suggests that they probably
deserve ataxonomic rank ofdivisionorphylum.
Picobiliphytes consist of at least three dif-
ferent clades (Fig. 1), for which we were able to
identify two signature sequences: PICOBI01
which target most picobiliphytes (tables S4 and
S5).They have two or more mismatcheswith all
available GenBank sequences from cultivated
protists (tables S4 and S5) and do not display
any fluorescence when hybridized to a variety
of algal strains from the Roscoff Culture Col-
lection (8, 11) (table S6). In addition, they
match a set of five additional environmental 18S
rDNA partial sequences: four from the western
North Atlantic (12) and one from a mid-Atlantic
estuary (Barnegat Bay, New Jersey), extending
the possible distribution of the picobiliphytes.
These probes enabled us to determine, by mi-
croscopy after tyramide signal amplification–
fluorescent in situ hybridization (TSA-FISH)
(13), the gross morphology of fixed cells from
the Roscoff coastal site (Fig. 1 and fig. S1). The
morphology of other unknown marine protist
groups was also determined by Massana et al.
(14), using probe methods.
Picobiliphytes are unicellular, slightly ob-
long, and approximately 2 × 6 mm (n = 9 cells)
and were recovered in the picoplankton size
fraction of our water samples because they
probably passed though the 3-mm pores in
the filter by way of their smallest dimension.
Thus, we have referred to them as picoplank-
ton. One remarkable feature is the presence of
an organelle-like structure having orange auto-
fluorescence when excited with blue light under
epifluorescence microscopy (Fig. 1), a structure
similar to that of phycobiliprotein-containing
rhodophytes and cryptomonads (fig. S1). These
pigments, in contrast to chlorophylls, are water-
soluble (15) and thus not removed by the TSA-
FISH alcohol dehydration steps. Moreover, any
chlorophyll remaining after alcohol dehydration
fluoresces yellow, not orange, under blue light
(fig. S1). Thus, picobiliphytes probably have a
phycobiliprotein-containing organelle, most
probably a plastid. Another distinctive feature
is a small body that is stainable with the nucleic
acid–specific dye DAPI (4', 6-diamidino-2-
phenylindole), distinct from the main nucleus
and consistently seen in close proximity to the
presumed plastid (Fig. 1, fig. S1).
Picobiliphyte sequences have been found in
a variety of marine systems, including the
European coast (8), the North Atlantic (from
GenBank Blast searches), and the Arctic Ocean
TSA-FISH in size-fractionated (<3 mm) sea-
water samples from the English Channel, re-
vealed that picobiliphytes occurred mostly in
summer, although their sequences were occa-
sionally detected in summer clone libraries
(tables S2 and S8). Their concentration, up to
80 cells ml−1, accounted for about 1.6% of the
total picoeukaryote cell counts at one coastal
to a major proportion (33 to 81%) of orange-
fluorescing picoeukaryotic cells detected by
blue laser flow cytometry (tables S7 and S8).
In one particular sample, cells exhibiting this
fluorescence were sorted by flow cytometry and
subsequentlyhybridized by TSA-FISHwithour
two probes (8) (table S7). We found that 48 to
PICOBI01 and PICOBI02, suggesting that the
picobiliphytes may constitute a substantial pro-
portion of the orange-fluorescing eukaryotic
picoplankton previously thought to be crypto-
phytes (16). The fact that our cells could have
been sorted and enriched with a phycobilin
pigment signature detected with flow cytometry
possess such pigments (15, 16).
The inferred presence of a phycobiliprotein-
containing plastid in picobiliphytes is in good
agreement with their putative sister relationship
to cryptophytes and katablepharids, the first of
which containphycobiliproteins. Whereascryp-
tophytes are common in the marine nanophyto-
plankton, pico-sized cryptophytes are not as
abundant, as judged by their relative frequency
in clone libraries; and where found, their 18S
rDNA sequence places them as an independent
lineage within the nano-sized cryptomonads
our group does not belong to the rhodophytes,
based on our phylogenetic analysis. Crypto-
phytes are a well-known example of a second-
ary endosymbiosis of a rhodophyte, which
brings phycobilin pigments to the new host cell.
Because picobiliphytes are sister to the crypto-
phyte/katabletablepharid clade in most of our
be most parsimonious to assume that our group
is a secondary endosymbiotic alga. The small
1Station Biologique de Roscoff, UMR 7144 CNRS and Université
Pierre et Marie Curie, Boîte Postale 74, 29682 Roscoff Cedex,
France.2AlfredWegenerInstitute forPolar andMarine Research,
Ocean, Département de Biologie, Université Laval, Quebec, QC
Canada G1K 7P4.4Institut de Ciències del Mar, Passeig Marítim
de la Barceloneta 37-49, 08003 Barcelona, Spain.
*Present address: Institut de Ciències del Mar, Passeig
Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain.
†Present address: Albany Molecular Research, BRC 18804
North Creek Parkway, Bothell, WA 98011–8012, USA.
‡These authors contributed equally to this work.
§To whom correspondence should be addressed. E-mail:
VOL 315 12 JANUARY 2007
on March 10, 2007
body stainable with the nucleic acid–specific
dye DAPI (Fig. 1) may be a DNA-containing
nucleomorph, similar to that found in crypto-
phytes and chlorarachniophytes (17), support-
ing the idea that picobiliphytes are another
secondary endosymbiotic algal group (18).
Kleptoplastidy is another possibility, such as
in the katablepharids (19, 20), which along with
the cryptophytes are the picobiliphytes’ pur-
ported sister group. However, kleptoplastidy is
unlikelyinsuch smallorganisms.In theabsence
screened filtered 3-mm–fractioned water for
cells that hybridized with our probes, using a
ChemScan solid-phase cytometer (8) (fig. S2).
We never encountered positive cells without a
plastid on the filters scanned by the laser,
which implies that the cells are predominately
pigmented, so kleptoplastidy does not seem
Are the picobiliphytes representatives of
another red algal secondary endosymbiosis,
such as chromo-alveolates, in the broad sense,
or do they have kleptoplastids? Without living
cells, the status of their endosymbiosis and a
formal description will remain unresolved.
Nevertheless, picobiliphytes are pigmented and
thus contribute to primary production. Molecu-
lar analysis confirms that they are a eukaryotic
group that should be recognized at the phylum
or division level, without any real indication of
their sister group. We found that they are well
represented in polar and cold temperate coastal
marine ecosystems, as judged from their ap-
body in the purported plastid places them in an
intriguing position in the study of plastid re-
duction to organelles.
Within the past 15 years, four algal classes
have been described from the picoplankton [see
(5) for details], and picobiliphytes represent
another division or phylum. The phylogenetic
analysis indicates that they are a highly diverse
group, composed of atleastthree distinct clades.
The temporal and spatial scales at which they
occur, as inferred from molecular data, indicate
that they could make up a substantial picoplank-
ton fraction under certain conditions. The ex-
istence of small, sometimes rare, organisms is
only now being recognized, and their role in
ecosystem function is unknown, but they prob-
ably act as reservoirs of genetic capacity that are
activated under specific conditions. The discov-
ery of picobiliphytes and their apparent wide-
spread distribution and contribution to marine
protist assemblages highlight the imperative of
understanding biodiversity before its loss on a
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Fig. 1. Phylogenetic trees were reconstructed from full-length 18S rRNA sequence data listed in table
tree is the 50% majority-rule tree of the last 100 trees saved from one of the parallel runs. Support for
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experiments. This work was supported by the European
Union project PICODIV and by the following sources of
funds: PICMANCHE (Region Bretagne), CNRS-Aventis
Foundation, and PICOCEAN (Gis-Génomique). Arctic
sampling was made possible with support from the
Canadian Climate Change Action Fund, Fisheries and
Oceans; the Natural Sciences and Engineering Research
Council, Canada; and funds from the ARTIC program,
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in situ hybridization work. Both F.N. and K.V. wrote
earlier versions of this paper. K.V. and L.M. provided the
sequences from the Helgoland site, K.R. and D.V. those
from the Roscoff sampling site, R.M. those from Blanes,
and C.L. those from the Arctic. K.T. performed the
ChemScan analyses. L.M. performed the phylogenetic
analyses and owes thanks to A. Culham for useful
discussions about appropriate analytical methods and to
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implementing the complex Bayesian analyses. All authors
discussed the results and commented on the manuscript.
Full-length sequences have been deposited at GenBank with
DQ222800, DQ060523, and DQ0605236. The authors
declare no competing financial interests.
Supporting Online Material
Materials and Methods
Figs. S1 and S2
Tables S1 to S8
12 October 2006; accepted 27 November 2006
VOL 31512 JANUARY 2007
on March 10, 2007