Transcriptome Analysis of Foraminiferan Elphidium
margaritaceum Questions the Role of Gene Transfer in
Kleptoplastidy
Loı̈c Pillet*,1 and Jan Pawlowski1
1
Department of Genetics and Evolution, University of Geneva, Geneva 4, Switzerland
*Corresponding author: E-mail: [email protected].
Associate editor: Charles Delwiche
Abstract
Key words: kleptoplastidy, Foraminifera, diatom, EST, plastid, photosynthesis.
Letter
The process of kleptoplastidy defined as the ability of some
heterotrophic organisms to sequester chloroplasts from their
algal preys is astonishing in many ways. The variability of
eukaryotic species in which it was reported is remarkable
and ranges from metazoans to protists, such as ciliates, dinoflagellates, and foraminiferans (fig. 1). At least eight foraminiferal genera are known to perform kleptoplastidy, but the
process was especially well studied for the genus Elphidium,
which exclusively retains chloroplasts of diatom origin (Pillet
et al. 2011).
A particularly striking feature of kleptoplastidy is the longevity of the plastids inside the host cell. Starved individuals
having no possibility of renewing their stock of stolen chloroplasts can remain photosynthetically active for several
months. The foraminiferans are no exception to that rule
and a previous study showed that, in starved individuals of
Elphidium excavatum, the chloroplasts had half lives of up to 9
weeks (Correia and Lee 2002).
As a consequence of the evolutionary history of endosymbiosis, most of the required genes to sustain plastid activity
and maintenance are encoded in the algal or plant nuclei
(Lane and Archibald 2008). For example, the chloroplast
genome of diatoms only contains about 160 genes
(Oudot-Le Secq et al. 2007) of the predicted 1,000–5,000
that are required (Martin et al. 2002; Richly and Leister
2004). Furthermore, because of the activity of proteases and
fast protein turnover in the chloroplasts (Sakamoto 2006),
efficient renewal of the photosynthetic machinery and housekeeping proteins is crucial. However, during kleptoplastidy,
the mechanism leading to this protein repair remains unclear,
as the genetic information contained in the nuclear
component of the algal prey is not present anymore within
the kleptoplastidic host cell.
To explain the longevity of the chloroplasts within their
host cell, it has been proposed that horizontal gene transfer
(HGT) occurred between the prey and host nuclei. The study
of the sacoglossan molluscs showed the presence of at least
11 algal nuclear genes (Pierce et al. 2012). An elegant experiment (Rumpho et al. 2008) showed the expression of psbO,
a nuclear gene coding for a subunit of the photosystem II
complex, in the sea slug Elysia chlorotica. Based on these
studies, it has been suggested that deeper sequencing of
kleptoplastidic organisms would lead to the discovery of
more algal genes inherited via HGT.
Recently, several transcriptome sequencing projects were
conducted on different sea slug species but surprisingly conclusions were not univocal. The analyses of EST data sets
from E. chlorotica (Pelletreau et al. 2011), E. timida, and
Plakobranchus ocellatus (Wägele et al. 2011) were unable to
recover nuclear-encoded chloroplastic proteins. Although
these studies contradicted years of research supporting
HGT hypothesis, another study came out and draw completely different conclusions (Pierce et al. 2012). Indeed,
using a larger E. chlorotica EST data set in parallel with a
comprehensive database of the native algal sequences,
Pierce et al. revealed 52 nuclear genes from the algal chloroplast source that were putatively transferred to the sea slug
nuclear genome.
In comparison to the sacoglossan molluscs, information
about maintenance of plastids in other kleptoplastidic organisms is limited. Until now, the only other attempt to highlight
algal nuclear genes in the host genome was performed on the
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Mol. Biol. Evol. 30(1):66–69 doi:10.1093/molbev/mss226 Advance Access publication September 19, 2012
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Foraminifera from the genus Elphidium are heterotrophic protists that graze on diatoms and sequester chloroplasts from their
algal preys, while digesting the rest of the diatom cell. During that process, known as kleptoplastidy, the acquired plastids remain
active inside the foraminiferan cell for several months. As most of the genes required to sustain the activity of the chloroplasts are
encoded in the diatom nucleus, it is unknown how the host cell can maintain the photosynthetic activity without this information. It has been proposed that maintenance of kleptoplastids could be explained by horizontal gene transfer (HGT). To test
this hypothesis we obtained 17,125 EST sequences of Elphidium margaritaceum, and we screened this data set for diatom
nuclear-encoded proteins having a function in photosynthetic activity or plastid maintenance. Our analyses show no evidence for
the presence of such transcriptionally active genes and suggest that HGT hypothesis alone cannot explain the chloroplast’s
longevity in Elphidium.
Plastid Maintenance in Foraminifera . doi:10.1093/molbev/mss226
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Table 1. Summary of Sampling and Sequencing Data for Elphidium
margaritaceum.
Sampling
Coordinates
Depth/habitat
No. of cells
Sequencing
Raw reads
Contigs
Average length
Largest length
N50
Total unitigs
48 43’39" N; 3 59’29" W
Intertidal zone/epiphytic
3,000
17,125
5,401
1,189
2,835
1,379
12,638
FIG. 2. NADPH quinone oxidoreductase phylogeny. Maximum likelihood and statistical support inference evaluated with 100 bootstrap
replicates under the PROTCATLGF model using RAxML (Stamatakis
et al. 2008). Black circles indicate node support higher than 95%.
dinoflagellate Dinophysis acuminata (Wisecaver and Hackett
2010). In this study, the authors screened the dinoflagellate
EST data set and found five nuclear genes that were plastid
related. However, the origin of these genes was not clear, as
some of them could have been inherited from a photosynthetic ancestor, instead of horizontally transferred from the
prey to the host nucleus.
Our study represents the first transcriptomic analysis carried on a foraminiferal species: E. margaritaceum. The samples
were collected from the English Channel, and EST library was
prepared using poly-A selection, as described in supplementary material S1, Supplementary Material online. Sanger
sequencing of this library generated 17,125 raw reads.
Useful information about this data set is summarized in
table 1. The 12,638 unique contigs plus singletons obtained
from the raw reads were then annotated using Blast2GO
(Conesa et al. 2005), where 46 sequences were identified as
putatively plastid related (supplementary table S1,
Supplementary Material online). These sequences were
then compared with our comprehensive prokaryotic and eukaryotic database, and rigorous phylogenetic inferences were
conducted on each gene separately to identify its origin. As
elphidiids were shown to retain exclusively chloroplasts from
diatom donors (Pillet et al. 2011), we expected the putative
horizontally transferred genes to be of diatom origin.
Unexpectedly, we identified only one sequence having a
strong phylogenetic affinity to the diatom Fragilariopsis cylindrus and corresponding to the NADPH quinone oxidoreductase. As shown in the phylogenetic tree (fig. 2), this sequence
was also found in the ESTs of another foraminiferan genus,
Ammonia. Although this genus is closely related to Elphidium
(Schweizer et al. 2008) and also grazes on diatoms, kleptoplastidy has been never observed in Ammonia. The NADPH
sequence could represent a good candidate for a putative
HGT between a diatom and the ancestor of Ammonia and
Elphidium, but as this protein was also found in other cellular
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FIG. 1. Tree of life representing the main eukaryotic supergroups. Adapted from Burki et al. (2012). Branch color indicates autotrophic (green) and
heterotrophic (black) lineages. Bold line indicates lineage that includes kleptoplastidic organisms.
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Pillet and Pawlowski . doi:10.1093/molbev/mss226
68
Assuming that the HGT hypothesis was correct, it has been
proposed that kleptoplastidy could be considered as a secondary or tertiary endosymbiotic event in progress (Gast et al.
2007; Johnson 2011). But if the HGT hypothesis is wrong and
the chloroplast stability hypothesis is right, then kleptoplastidy appears more like a temporary plastid usage. In that case,
there is no reason for the stolen plastids to be permanently
integrated inside the host cell as in secondary or tertiary
endosymbiosis. At present, the most important question to
answer would be what mechanism protects chloroplasts from
digestion? Is it the same mechanism that allows various endosymbiotic algae, including diatoms to enter the foraminiferal
cells (Lee and Anderson 1991) and to live there? Is kleptoplastidy another form of symbiotic association developed by
some heterotrophic protists? Further testing of alternative
hypotheses regarding kleptoplastidy in Foraminifera could
help answering these questions.
Supplementary Material
Supplementary material S1 and table S1 are available at
Molecular Biology and Evolution online (http://www.mbe
.oxfordjournals.org/).
Acknowledgments
The authors thank Colomban de Vargas and the members of
the team “Evolution of Plankton and PaleoOcean” in Roscoff
for their help during the collection of the samples. They also
thank Fabien Burki for helping with the bioinformatic and
phylogenetic pipeline and for helpful comments and discussions, and Julie Poulain and Corinne Da Silva for the sequencing as part of the Genoscope Rhizarian Genomics project.
This work was supported by the Swiss National Science
Foundation (grant number 31003A_140766) and by a
G.&A. Claraz Donation.
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