Current Biology Vol 16 No 17
R670
first individual to scan was the
first to cross the small road in
100% of cases, compared to
70% for the large road. On the
large road the second-ranking
male sometimes continued
scanning while the elderly third
male and alpha female took
up the lead on the large-road
progressions. The alpha male
increased his rearward presence
on the large road, whereas the
alpha female showed a dramatic
reduction in frequency of being
last; in other words when the
degree of risk increased she
took up a more forward position.
Additionally, when the alpha
male was present in mixedgroup progressions containing
one other adult male (N = 6,
mean group size: 6.7), he was
first to scan and cross in 50% of
large road- crossings and last in
only 33%. This suggests that his
rearward position at other times
was not due to fear.
Modern Bossou chimpanzees
encounter predators infrequently
[6], and although humans
themselves are not ‘predators’
of these chimpanzees, we
propose that road-crossing,
a human- created challenge,
presents a new situation that
calls for flexibility of responses
by chimpanzees to variations in
perceived risk.
Crossing the large road and
leaving forest for open areas
are potentially risky situations
for chimpanzees, reflected
in increased waiting time.
During dangerous excursions
certain positions may be more
advantageous than others,
depending upon age and sex
[4]. Adult males, less fearful and
more physically imposing than
other group members, take up
forward and rearward positions,
with adult females and young
occupying the more protected
middle positions.
As hypothesised, the
Bossou chimpanzees employ a
phylogenetically-old mechanism
to adapt to a more recent
dangerous situation. However,
the positioning of dominant
and bolder individuals, in
particular the alpha male,
changed depending on both the
degree of risk and number of
adult males present; dominant
individuals act cooperatively
with a high level of flexibility to
maximise group protection. At a
proximate level each individual
may have preferred and
recognised positions; however, it
is unknown whether positioning
is individual- or rank-specific.
Data on progression orders of
other great ape populations are
required, and would help shape
hypotheses about emergence of
this aspect of hominoid social
organisation.
Acknowledgments
We are grateful to the Direction
­Nationale de la Recherche ­Scientifique
et Technique, the Republic of Guinea,
for granting us permission to carry
out this research. We would like to
thank all the guides who helped ­during
this ­research period. This work was
­supported by a Stirling University
­studentship, MEXT grant #16002001
and JSPS-HOPE.
Supplemental data
Supplemental data, with a video-clip of
the Bossou chimpanzees crossing the
large road, are available at http://www.
current-biology.com/cgi/content/
full/16/17/R668/DC1/
References
1.Altmann, S.A. (1979). Baboon
progressions: order or chaos? A study of
one-dimensional group geometry. Anim.
Behav. 27, 46–80.
2.DeVore, I., and Washburn, S.L. (1963).
Baboon ecology and human evolution.
In African ecology and Human evolution,
F.C. Howell and F. Bourliere eds.
(Chicago: Aldine), pp. 335–367.
3.Rhine, R.J., and Westlund, B.J. (1981).
Adult male positioning in baboon
progressions: order and chaos revisited.
Folia. Primatol. 35, 77–116.
4.Rhine, R.J., and Tilson, R. (1987).
Reactions to fear as a proximate factor in
the sociospatial organization of baboon
progressions. Am. J. Primatol. 13,
119–128.
5.Matsuzawa, T. (2006). Sociocognitive
development in chimpanzees: A synthesis
of laboratory work and fieldwork. In
Cognitive Development in Chimpanzees,
T. Matsuzawa, M. Tomonaga and M.
Tanaka, eds. (Tokyo: Springer), pp. 3–33.
6.Sugiyama, Y. (2003). Demographic
parameters and life history of
chimpanzees at Bossou, Guinea. Am. J.
Phys. Anthropol. 124, 154–165.
1Department
of Psychology, University
of Stirling, Stirling FK9 4LA, Scotland,
UK. 2Primate Research Institute,
Kyoto University, Inuyama-city, Aichi,
484- 8506, Japan.
E-mail: k.j.hockings@stir.ac.uk
Minimal plastid
genome evolution
in the Paulinella
endosymbiont
Hwan Su Yoon1, Adrian
Reyes-Prieto1, Michael
Melkonian2 and Debashish
Bhattacharya1
It is an enduring mystery how
organelles were first established
in eukaryotes. A key player in
this saga is the thecate amoeba
Paulinella chromatophora
which over 100 years ago [1]
showed naturalists that once
free-living cells could exist as
endosymbionts [2]. This species
has the honor of being the only
known case of an independent
primary (cyanobacterial) plastid
acquisition [3,4] and is a model
for understanding plastid
establishment. The Paulinella
plastid, often referred to as
the cyanelle, retains typical
cyanobacterial features such as
peptidoglycan and phycobilisomes,
but is considered to be a bona
fide endosymbiont because it is
no longer bound by a vacuolar
membrane but lies free in the
cytoplasm, its number is regulated,
suggesting genetic integration, and
it cannot be cultured outside the
host [5–7]. Paulinella is, however,
difficult to culture, and so it
has resisted detailed molecular
biological investigation. Here
we took advantage of a Lambda
DASH II phage library made from
limited amounts of Paulinella total
genomic DNA to reconstruct the
evolutionary history of its recently
established plastid [3]. Our data
show the Paulinella plastid genome
to have characteristics typical
of cyanobacterial, not plastid
genomes.
The Paulinella library was
screened with the highly conserved
psbA, psbC and 16S rDNA plastid
genes from the glaucophyte
Glaucocystis nostochinearum.
Two plastid inserts of 9.4 kb and
4.3 kb were obtained by this
approach; a third, 5 kb fragment
has already been described [3].
Because the P. chromatophora
culture is not axenic (see [3]), we
Magazine
R671
isolated small subunit rDNA from
the phage library to establish the
identities of the different culture
inhabitants. Sequence analysis
revealed 20 clones from two
classes of prokaryote-derived
rDNA genes (see Figure S1 in the
Supplemental data available on­ line
with this issue). An additional
PCR product was sequenced that
encodes the previously reported
nuclear rDNA [8] from Paulinella
that has Euglyphidae homologs.
The first class of prokaryotic
genes, from two clones, is identical
to a sequence from isolated,
washed Paulinella plastids [3],
and is of cyanobacterial origin.
The second class is rDNA from
bacterial lineages representing
18 distinct, putative culture
inhabitants (Figure S1). One is a
Pseudomonas-like sequence (e.g.
PC9, 11 distinct clones found);
one is Agrobacterium- like (PC3);
two are related to Solibacter (PC1
and, PC7, two distinct clones
found); and two are related to
low G + C Gram-positive bacteria
(e.g. PC18 and PC22, four
distinct clones found). Clearly,
the Paulinella culture contained
a wide variety of bacteria that we
have likely incompletely sampled
in our PCR screen. Despite the
contaminants, the identity of
the cyanobacterial- like small
subunit rDNA found in our
work with the gene Marin et al.
[3] sequenced from isolated
Paulinella plastids assures us
that the culture contains only a
single cyanobacterial-derived
rDNA sequence, derived from the
plastid genome. The Paulinella
plastid rDNA is most closely
related to those from a clade of
Synechococcus species defined
by strains WH5701, BS4, and
kpr27rc (Bayesian posterior
probability = 1.0) within a larger
clade of Prochlorococcus and
Synechococcus (PS, Figure S1)
cyanobacteria [3].
Alignment of the 9.4 kb
plastid genome fragment with
the homologous regions in PS
cyanobacteria is shown in Figure
1A. This comparison reveals strong
conservation of plastid gene order
to Synechococcus sp. WH5701,
with the level of conservation to
closely related cyanobacteria
approximately related to the
A
0
5
10 (kb)
70
50 (%)
30
GC = 40.0 ± 7.6 %
Paulinella
chromatophora
Plastid
Synechococcus
sp. WH5701
(3,044 kb)
Synechococcus
sp. CC9605
(2,511 kb)
psb O
fts H
psb O
cysD
fts H
eda
psb O
(9,398 nt)
psb A
aro C
Na-T
psb A
nif B
308
cysD
fts H
eda hp
(225)
aro C hp
hp
psb A
nif B
220
psb O cysD
fts H
eda
aro C
1329
psb O
psb A
1324
cysD
0
C
5 (kb)
psb A
gst C mp
hp2
70
50 (%)
30
(4,294 nt)
ABC transporter
696
1 kb
701
psb A
ast E
gst C mp ABC transporter
hp1hp2 hp3
1 kb
fts H
hp
GC = 39.7 ± 7.7 %
Synechococcus
sp. WH5701
aro C
1262
303
Synechococcus
elongatus
PCC6301
(2,696 kb)
Paulinella
chromatophora
Plastid
eda
nifB
1267
Prochlorococcus
marinus
CCMP1986
(1,658 kb)
B
cysD
hp1hp2
Gene Plastid Nucleus
psb O
cysD
fts H
eda
aro C
psb A
nif B
gst C
mp
ABC
+
+
-
+
+
+
+
+
+
+
+
Current Biology
Figure 1. The Paulinella chromatophora plastid genome.
Alignment of the (A) 9.4 kb and (B) 4.3 kb plastid genome fragments from Paulinella with
homologous regions in closely related cyanobacteria. The GC-content from a sliding
window analysis is shown above the plastid regions. The genes are for: photosystem
II manganese-stabilizing polypeptide, PSBO; ATP-sulfurylase, CYSD; cell division protein, FTSH; KDPG and KHG aldolase, EDA; chorismate synthase, AROC; photosystem
II reaction center protein D1, PSBA; elongator protein 3/MiaB/NifB, NIFB; possible sodium dependent transporter, Na-T; conserved hypothetical protein, HP, HP1, HP2, HP3;
putative glutathione S-transferase, GSTC; conserved hypothetical membrane protein,
MP; aspartoacylase, ASTE; ABC transporter ATP binding protein, ABC transporter. Only
HP2 is homologous in Paulinella and Synechococcus WH5701. (C) Location of plastid
genes in the plastid or the nucleus of other photosynthetic eukaryotes. Presence is (+)
and absence is (–).
phylogenetic distance. Additional
evidence that the Paulinella plastid
is a recent acquisition comes
from the gene distribution among
cyanobacteria and eukaryotes. The
9.4 kb fragment contains a number
of genes that have been transferred
to the nucleus in photosynthetic
eukaryotes — for example, psbO
is nuclear in all algae and plants
(Figure 1C) — whereas only psbA
and ftsH are maintained in plastid
genomes. Equally significant is
the elongator protein 3/miaB/nifB
gene that is shared by the plastid
and two Synechococcus species
(Figure 1A). Unlike psbO, which is
essential for photosynthesis, nifB
is required for the biosynthesis of
the iron–molybdenum (or iron–
vanadium) cofactor used by the
nitrogen-fixing enzyme nitrogenase.
One would expect this gene to
be lost early in plastid evolution
because of the high energetic costs
of nitrogen fixation, and it is absent
from photosynthetic eukaryotes.
The remaining 4.3 kb fragment
that we found, and the 5 kb
fragment reported by Marin et al.
[3], encode the psbA-HP-gstC-HP
ABC transporter protein (Figure 1B)
and 16S rRNA-ITS1-tRNA(Ile)- tRNA
(Ala)-23S rRNA, respectively. The
gene order in these regions is also
typical of the PS clade. These
Current Biology Vol 16 No 17
R672
Figure 2. Maximum likelihood (ML) tree for four
­concatenated
proteins,
(FTSH, PSBA, PSBO and
­elongation factor EF-Tu,
TUFA).
49
Data from all available cy57
anobacterial genome se100
100
quences are included in
90
77
this analysis. ML bootstrap
100
51
100
support values are shown
Synechococcus elongatus PCC 7942
92
Synechococcus elongatus PCC 6301
on top and maximum parsi65
Trichodesmium erythraeum IMS101
98
mony bootstrap values are
98
Crocosphaera watsonii WH 8501
below. The thick branches
Synechocystis sp. PCC 6803
98
62
100
Nostoc sp. PCC 7120
65
have >0.95 posterior probAnabaena variabilis ATCC 29413
ability in a Bayesian inferThermosynechococcus elongatus BP1
99
100
ence. Gloeobacter viol93
Synechococcus sp. JA-3-3Ab
99
Synechococcus sp. JA-2-3Ba
aceus and the Yellowstone
Gloeobacter violaceus PCC 7421
Synechococcus
strains
0.01 substitution/site
Current Biology
(JA-3-3Ab, JA-2-3Ba) are
used to root the tree. The
PS clade is defined by Prochlorococcus–Synechococcus strains and is sister to the
Paulinella plastid.
99
100
63
80
data demonstrate the essential
‘cyanobacterial’ nature of the
Paulinella endosymbiont, and
if our data are typical then the
plastid genome is likely to be of
cyanobacterial proportions rather
than characteristic of a plastid
genome. A multi-gene phylogeny
using a concatenated data set of
FTSH, PSBA, PSBO, and TUFA
(isolated from Paulinella by PCR)
confirms that the plastid originated
from a member of the PS-clade
(Figure 2), consistent with results of
the rDNA analysis (Figure S1) and
the gene order data (Figure 1). The
individual protein trees are shown
in Figure S2.
The finding of large-scale
morphological changes associated
with a recent green algal
engulfment in the katablepharid
protist ‘Hatena’ underlines the
extent to which endosymbiosis
may modify organisms [9]. In
the case of Paulinella, additional
analyses of the plastid and the
nuclear genome will help us
better understand this primary
endosymbiosis. A prediction is
that genes required for organelle
division (such as ftsZ) have been
transferred to the nucleus and are
now under host control. In addition,
we would expect Paulinella has
evolved a highly regulated transport
system for directly connecting
carbon metabolism of the host cell
and the endosymbiont. A recent
analysis shows that the main
primary plastid-containing group
(i.e., red, green [including plants],
and glaucophyte algae) achieved
PS - clade
Prochlorococcus marinus MIT 9312
Prochlorococcus marinus CCMP1986
Prochlorococcus marinus NATL2A
100
Prochlorococcus
marinus CCMP1375
100
83
85
Prochlorococcus marinus MIT 9211
Prochlorococcus marinus MIT 9313
100
96
100
96
Synechococcus sp. CC9902
100
100
Synechococcus sp. CC9605
90
Synechococcus sp. WH 8102
99
Synechococcus sp. WH 7805
82
90
Synechococcus sp. RS 9917
Synechococcus sp. WH 5701
Paulinella chromatophora - Plastid
100
100
this function through the co- option
and retargeting of existing
endomembane transporters to the
plastid [10].
We suggest that the major
insights into primary plastid
establishment such as control
of organelle division and carbon
translocation [10] may come from
analysis of the Paulinella nuclear
genome rather than that of its
recent endosymbiont. This makes
Paulinella an ideal model for a
complete genome sequencing
project that could also include
its closely related sister species
P. ovalis, a heterotroph which
feeds actively on cyanobacteria
and other prey that have been
identified in food vacuoles in
its cytoplasm [7]. Comparison
of these amoebal genomes will
allow us to potentially identify the
genetic inventions that underlie the
critical transition from heterotrophy
to photoautotrophy.
des Süßwassers mit blaugrünen
chromatophorenartigen Einschlüssen.
Z. Wiss. Zool. 59, 537–544.
2.Melkonian, M., and Mollenhauer, D.
(2005). Robert Lauterborn (1869–1952)
and his Paulinella chromatophora. Protist
156, 253–262.
3.Marin, B., Nowack, E.C., and Melkonian,
M. (2005). A plastid in the making:
evidence for a second primary
endosymbiosis. Protist 156, 425–432.
4.Rodriguez-Ezpeleta, N., and Philippe, H.
(2006). Plastid origin: replaying the tape.
Curr. Biol. 16, R53–R56.
5.Kies, L., and Kremer, B.P. (1979). Function
of cyanelles in the thecamoeba Paulinella
chromatophora. Naturwissenschaften 66,
578.
6.Kies, L. (1974). Elektronenmikroskopische
Untersuchungen an Paulinella
chromatophora Lauterborn, einer
Thekamobe mit blau-grünen
Endosymbionten (Cyanellen).
Protoplasma 80, 69–89.
7.Johnson, P.W., Hargraves, P.E., and
Sieburth, J.M. (1988). Ultrastructure
and ecology of Calycomonas ovalis
Wulff, 1919, (Chrysophyceae) and its
redescription as a testate rhizopod,
Paulinella ovalis n. comb. (Filosea:
Euglyphina). J. Protozool. 35, 618–626.
8.Bhattacharya, D., Helmchen,
T., and Melkonian, M. (1995).
Molecular evolutionary analyses
of nuclear-encoded small subunit
ribosomal RNA identify an
independent rhizopod lineage
containing the Euglyphina and the
Chlorarachniophyta. J. Eukaryot.
Microbiol. 42, 65–69.
9.Okamoto, N., and Inouye, I. (2005).
A secondary symbiosis in progress?
Science 310, 287.
10.Weber, A.P., Linka, M., and Bhattacharya,
D. (2006). Single, ancient origin of a
plastid metabolite translocator family in
Plantae from an endomembrane-derived
ancestor. Eukaryot. Cell 5, 609–612.
1Department
of Biological Sciences and
Roy J. Carver Center for Comparative
Genomics, University of Iowa, 446
Biology Building, Iowa City, Iowa
52242-1324, US. 2Botanisches Institut,
Lehrstuhl I, Universität zu Köln,
Gyrhofstr. 15, 50931 Köln, Germany.
E-mail: Debashi-Bhattacharya@uiowa.
edu; Michael.Melkonian@uni-koeln.de
The editors of Current Biology
Supplemental data
Supplemental data are available at
http://www.current-biology.com/cgi/
content/full/16/17/R670/DC1/
welcome correspondence on
any article in the journal, but
reserve the right to reduce
the length of any letter to be
Acknowledgments
This research was supported by grants
from the NSF and NASA awarded to
D.B. (EF 04-31117, NNG04GM17G).
We acknowledge technical help from
L. Pingel and M. Wu (University of Iowa).
published. All Correspondence
References
should be sent by e-mail to
1.Lauterborn, R. (1895). Protozoenstudien
II. Paulinella chromatophora nov. gen.,
nov. spec., ein beschalter Rhizopode
cbiol@current-biology.com
containing data or scientific
argument will be refereed.
Queries about articles for
consideration in this format
Supplemental Data: Minimal plastid genome evolution in the
Paulinella endosymbiont
Hwan Su Yoon, Adrian Reyes-Prieto, Michael Melkonian and Debashish Bhattacharya
Supplemental Experimental Procedures
Library Construction
Total genomic DNA was isolated from pooled cultures of Paulinella chromatophora as described in
Bhattacharya et al. [S1] and digested with EcoRI. Due to the small amount of total DNA that was
recovered, we cloned directly the inserts without size-fractionation. In addition, the Paulinella cells
grew with bacteria therefore the majority of the DNA was prokaryotic. About 100 ng of the digested
DNA was used to prepare a phage library using EcoRI-digested, dephosphorylated vector LambdaDash II (9 – 23 kb cloning capacity [Stratagene, La Jolla, CA, USA]). Ligation and packaging of the
vector and DNA were done according to the manufacturer’s instructions. The library was amplified
(ca. 106,000 original PFU) and stored in SM buffer (10 mM NaCl, 10 mM MgCl2, 50 mM Tris-HCl
[pH 7.5], 2% w/v gelatin) with 5% chloroform.
The genomic bank of Paulinella was screened with a probe cocktail containing sequences of
the highly conserved psbA, psbC, and 16S rDNA plastid genes from the glaucophyte Glaucocystis
nostochinearum using standard methods (e.g., [ S2]). The Glaucocystis DNA fragments were isolated
using PCR (as in [S3-S5]). Filters were washed two times in 2X SSC, 0.1% SDS at 40°C prior to
autoradiography. Secondary and tertiary screens were used to isolate single plaques containing the
target genes. The phage inserts (9.4 kb and 4.3 kb) were amplified using long-range PCR (Qiagen,
Valencia, CA, USA) that were directly sequenced using primer walking and the BigDyeTM Terminator
Cycle Sequencing Kit (PE-Applied Biosystems, Norwalk, CT, USA), and an ABI-3700 at the Roy J.
Carver Center for Comparative Genomics at the University of Iowa. The genes encoded by the two
inserts, tufA, and small subunit rDNA have been released to GenBank under the accession numbers,
DQ789029 – DQ789040.
PCR Isolation of Small Subunit rDNA
To identify the inhabitants in the Paulinella culture, we isolated prokaryote-derived small subunit
rDNA sequences using specific primers that are complementary to the 5’- and 3’-termini of these
genes (SG1, SG2; see [S3, S6]). For the nuclear rDNA, we used the primers described in
Bhattacharya et al. [S1]. PCR products were cleaned using the QIAquick gel Purification kit (Qiagen)
and directly sequenced (nuclear rDNA) or cloned into the pGEM-T vector (Promega). A total of 20
clones encoding prokaryote-derived rDNA were picked and the inserts were sequenced as described
above. Plasmid (T3, T7), external PCR, and internal genic (e.g., p651F, p16SR) primers were used to
sequence the inserts [S1, S3, S6].
1
Phylogenomic Analysis
We used a phylogenomic pipeline (as in [S7, S8]) with 6 genes (psbO, cysD, ftsH, eda, aroC, psbA)
from the 9.4 kb and 4 genes (psbA, gstC, hypothetical membrane protein [mp], ABC transporter) from
the 4.3 kb Paulinella cyanelle fragment and a tufA fragment that was isolated from the phage library
using PCR as a query against a local data base of 23 complete or nearly-complete cyanobacterial
genomes and complete genome data from 21 other bacteria, 10 plastids, and 6 eukaryotes. All
significant hits (BLAST e-value < 10-10) to each of the Paulinella sequences were identified and
stored. The resulting alignments were manually refined and submitted to protein phylogenetic
analyses.
Phylogenetic Analysis
The phylogenies of 7 individual Paulinella proteins and of a concatenated data set of 4 highly
conserved sequences (psbO, ftsH, psbA, tufA) were reconstructed under maximum likelihood (ML)
using proml in the PHYLIP V3.6b program package [S9]. The trees were inferred with the JTT + Γ
evolutionary model and global rearrangements with 4 random addition replicates. The alpha values for
the gamma distribution for the different data sets were calculated using PHYML V2.4.3 [S10] and the
same evolutionary model. To assess the stability of monophyletic groups in the ML trees, we
calculated bootstrap support values using PROML (100 replicates). For the 4-protein data set we also
used unweighted maximum parsimony (MP) using PAUP*V4.0b10 [S11] and calculated Bayesian
posterior probabilities (BPP) using MrBayes (V3.0b4, [S12]. In the Bayesian inference of the amino
acid data, we used the WAG + Γ + I model with Metropolis-coupled Markov chain Monte Carlo from
a random starting tree. These analyses were run for 1,000,000 generations with trees sampled each
200 cycles. Four chains were run simultaneously of which 3 were heated and one was cold, with the
initial 20,000 cycles (200 trees) being discarded as the “burn in”. A consensus tree was made with the
remaining 1,800 phylogenies to determine the posterior probabilities at the different nodes. In the MP
analyses, 1000 bootstrap replicates were analyzed with ten heuristic searches with random-additionsequence starting trees and tree bisection-reconnection (TBR) branch rearrangements. For the rDNA
tree of the different genes isolated from the Paulinella library, we inferred a PHYML tree using the
GTR + Γ + I model and tested support for clades in this phylogeny with PHYML analysis of 100
bootstrap replicates. In addition, we did MP bootstrap and Bayesian analyses with the rDNA data as
described above.
GC-Content, Gene Order, and Gene Presence/Absence Analyses
We used the SWAAP (V1.0.2 http://www.bacteriamuseum.org/SWAAP/SwaapPage.htm) computer
program to calculate GC-content across the Paulinella cyanelle fragments using a sliding window
approach. Gene order was compared between the Paulinella 9.4 kb and 4.3 kb fragments with
homologous regions in sequenced genomes of closely related cyanobacteria using the NCBI microbial
genome database (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). The results of this analysis were
2
manually prepared for presentation (see Fig. 1 in text). Gene presence/absence in plastid versus
nuclear genomes for the coding regions in both cyanelle fragments was done as follows. First,
phylogenomics using the local database described above was used to identify all orthologs in the
organellar and nuclear genomes to identify their cellular location. To verify these results, we then did
a BLAST search against the NCBI non-redundant database to search for all available orthologs. The
results of these two analyses were used to infer the putative cellular location of the Paulinella
cyanelle genes across photosynthetic eukaryotes.
Supplemental References
S1. Bhattacharya, D., Helmchen, T., and Melkonian, M. (1995). Molecular evolutionary analyses of
nuclear-encoded small subunit ribosomal RNA identify an independent rhizopod lineage
containing the Euglyphina and the Chlorarachniophyta. J. Eukaryot. Microbiol. 42, 65-69.
S2. Bhattacharya, D., and Weber, K. (1997). The actin gene of the glaucocystophyte Cyanophora
paradoxa: analysis of the coding region and introns, and an actin phylogeny of eukaryotes. Curr.
Genet. 31, 439-446.
S3. Yoon, H.S., Hackett, J.D., Pinto, G., and Bhattacharya, D. (2002). The single, ancient origin of
chromist plastids. Proc. Natl. Acad. Sci. USA 99, 15507-15512.
S4. Yoon, H.S., Hackett, J.D., Ciniglia, C., Pinto, G., and Bhattacharya, D. (2004). A molecular
timeline for the origin of photosynthetic eukaryotes. Mol. Biol. Evol. 21, 809-818.
S5. Yoon, H.S., Hackett, J.D., and Bhattacharya, D. (2002). A single origin of the peridinin- and
fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis. Proc. Natl.
Acad. Sci. USA 99, 11724-11729.
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Figure S1. Small subunit rDNA tree of plastid and nuclear encoded genes showing the origins of the
20 prokaryote-derived and the nuclear sequence found in the Paulinella chromatophora culture. This
is a maximum likelihood (ML) phylogeny with ML bootstraps shown at the branches on the left of the
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slash marks and maximum parsimony (MP) bootstrap values on the right. The thick branches have
>95% posterior probability in a Bayesian inference. The branch connecting the nuclear (18S) and
plastid/bacterial (16S) small subunit rDNAs has been truncated to focus attention on the branching
order within the two clades.
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Figure S2. ML trees of all proteins identified on the 9.4 kb Paulinella cyanelle fragment and TUFA.
Data from all sequenced (at the time of writing) cyanobacterial genomes are included in this analysis.
ML bootstrap support values are shown above the branches. Gloeobacter violaceus and/or the
Yellowstone Synechococcus strains (JA-3-3Ab, JA-2-3Ba) are used to root the tree.
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