Differential gene retention in plastids of common recent origin.

and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org
Ó The Author 2010. Published by Oxford University Press on behalf of the Society for Molecular Biology

 1

Research Paper:
Differential gene retention in plastids of common recent origin

Adrian Reyes-Prietoa, e*, Hwan Su Yoonb,*, Ahmed Moustafaa, Eun Chan Yangb, Robert A.
Andersenb, Sung Min Booc, Takuro Nakayamad, Ken-ichiro Ishidad, and Debashish
Bhattacharyaa,1

a Department of Ecology, Evolution and Natural Resources and Institute of Marine and Coastal
Sciences, Rutgers University, New Brunswick, New Jersey, USA
b Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine, USA
c Department of Biology, Chungnam National University, Daejeon, Korea
d Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki,
Japan
e Present address. Department of Biology, University of New Brunswick, Fredericton, New
Brunswick, Canada

*These authors contributed equally to this work
1To whom correspondence should be addressed. E-mail: bhattacharya@aesop.rutgers.edu
Key words: endosymbiosis, primary plastids, photosynthesis, Paulinella
Running head: Paulinella plastid evolution
MBE Advance Access published February 1, 2010


 2

Abstract
The cyanobacterium-derived plastids of algae and plants have supported the diversification of
much of extant eukaryotic life. Inferences about early events in plastid evolution must rely on
reconstructing events that occurred over a billion years ago. In contrast, the photosynthetic
amoeba Paulinella chromatophora provides an exceptional model to study organelle evolution in
a prokaryote-eukaryote (primary) endosymbiosis that occurred ca. 60 million years ago. Here we
sequenced the plastid genome (0.977 Mb) from the recently described Paulinella FK01 and
compared the sequence to the existing data from the sister taxon Paulinella M0880/a. Alignment
of the two plastid genomes shows significant conservation of gene order and only a handful of
minor gene rearrangements. Analysis of gene content reveals 66 differential gene losses that
appear to be outright gene deletions rather than endosymbiotic gene transfers (EGTs) to the host
nuclear genome. Phylogenomic analysis validates the plastid ancestor as a member of the
Synechococcus-Prochlorococus group and the cyanobacterial provenance of all plastid genes
suggests these organelles were not targets of interphylum gene transfers after endosymbiosis.
Inspection of 681 DNA alignments of protein-encoding genes shows that the vast majority have
dN/dS ratios <<1, providing evidence for purifying selection. Our study demonstrates that plastid
genomes in sister taxa are strongly constrained by selection but follow distinct trajectories during
the earlier phases of organelle evolution.


 3

Introduction
The ancient origins of mitochondria and plastids explains the fundamentally chimeric nature of
eukaryotes (Sagan 1967). Photosynthesis entered the eukaryotic domain via primary
endosymbiosis, whereby a cyanobacterium was captured by a heterotrophic protist and converted
into a photosynthetic organelle. This pivotal event occurred more than a billion years ago and
laid the foundation for many food webs on our planet (Falkowski et al. 2004; Reyes-Prieto,
Weber, and Bhattacharya 2007). The host lineage for the endosymbiosis is the putative ancestor
of the Plantae that subsequently split into the glaucophyte, red, and green algae (including land
plants, Cavalier-Smith 1992; Bhattacharya and Medlin 1995; Palmer 2003). The canonical
Plantae plastid spread via secondary and tertiary endosymbiosis to other lineages such as
chromalveolates and euglenids (Palmer 2003; Bhattacharya, Yoon, and Hackett 2004; Reyes-
Prieto, Weber, and Bhattacharya 2007). All extant plastids, whether of primary, secondary, or
tertiary origin are specialized organelles with highly reduced genomes (100-200 Kb) leaving us
to speculate about the pattern and process of gene loss early in their evolution. Given this
situation, there is much interest in identifying a more recent case of organelle establishment via
primary endosymbiosis. This need appears to have been recently fulfilled with molecular studies
of the thecate amoeba Paulinella chromatophora M0880/a (Marin, Nowack, and Melkonian
2005; Yoon et al. 2006; Marin et al. 2007; Nowack, Melkonian, and Glockner 2008). Paulinella
is a member of the supergroup Rhizaria, yet it contains two blue-green “chromatophores” (Figs.
1A, 1B) that resulted from a novel plastid acquisition (Marin, Nowack, and Melkonian 2005;
Yoon et al. 2006; Nowack, Melkonian, and Glockner 2008) about 60 million years (My) ago
(Nowack, Melkonian, and Glockner 2008). Several lines of evidence support the hypothesis that
Paulinella contains bona fide photosynthetic organelles. These include a constant plastid number


 4

(i.e., two per cell) following each round of coordinated cell division (Kies 1974), a plastid
genome that is 1/3 the size of chromosomal DNA in putative free-living cyanobacterial donors
(Nowack, Melkonian, and Glockner 2008), and evidence for gene transfer to the amoeba nuclear
genome via EGT (i.e., the cyanobacterium-derived psaE gene; Nakayama and Ishida 2009).
Here we generated the plastid genome sequence from a sister taxon of Paulinella M0880/a
that was recently isolated in Japan (Yoon et al. 2009). This second isolate, Paulinella FK01,
provides an ideal tool to understand plastid genome evolution using a homologous organelle of
recent origin. A strategy utilizing fluorescence-activated cell sorting (FACS) to isolate organelles
was followed by single-cell genomics and ‘454’ pyrosequencing to generate a draft genome of
FK01 that was closed using targeted PCR.

Materials and Methods
Cell isolates
Paulinella FK01 was established from a single cell collected at Daigo-machi, Ibaraki prefecture,
Japan (Yoon et al. 2009). Paulinella chromatophora M0880/a was kindly provided by Michael
Melkonian (University of Cologne, Germany). Both isolates are maintained at the Bigelow
Laboratory for Ocean Sciences using DY-V medium at 20°C with a 14/10 hr light/dark cycle.

Plastid isolation and whole genome amplification
After cell disruption with glass-bead beating, single plastids were isolated using FACS (see Fig.
S1, Supplementary Material online). DNA derived from 50 isolated plastids was used for
genome amplification using the Repli-G mini kit (Qiagen), which applies multiple displacement
amplification (MDA) methods (Stepanauskas and Sieracki 2007). This resulted in ca. 10 µg of


 5

DNA per reaction with an A260/280 ratio of 1.85. After the de-branching step with S1 nuclease
to reduce chimeric sequences during MDA, a PCR survey was done using several gene markers
for nuclear and plastid (pt)DNA (16S rDNA, 18S rDNA, rbcL, ftsZ, beta-tubulin) to validate the
source of the nucleic acids; i.e., the nuclear gene amplifications acted as negative controls for
this procedure. The PCR products were sent to the DNA Facility at the University of Iowa for
Sanger sequencing, whereas the amplified plastid DNA was sent to Macrogen (Seoul, Korea)
and to the University of Iowa for 454 (Roche) pyrosequencing.

Genome sequencing, assembly, and annotation
Combination of two separate runs of a ¼ plate each from Macrogen and the University of Iowa
using the GS-FLX standard chemistry generated a total of ~2.4 million sequences with an
average length of 230 bases, providing >55X theoretical coverage of the FK01 plastid genome.
The filtered reads were assembled using the GS De Novo Assembler software (Roche
Diagnostics Corporation), resulting in 11 contigs. Using as reference both the M0880/a plastid
genome and the termini of the FK01 contigs, we designed specific PCR primers to complete the
sequence of the missing regions in FK01. The final assembly was done using Sequencher. A
total of 888 protein-coding genes were predicted in FK01 ptDNA using GeneMarkS (Besemer,
Lomsadze, and Borodovsky 2001) and 912 proteins, using RAST (Aziz et al. 2008). Visual
inspection and manual refinement substantiated a total of 841 likely protein-encoding gene
models. After annotation, alignment of the Paulinella M0880/a and FK01 ptDNA was done
under Mauve (Darling et al. 2004) using the progressive algorithm with default parameters.

Phylogenomic analysis