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Phosphoribulokinase (PRK) is an essential enzyme of photosynthetic eukaryotes which is active in the plastid-located Calvin cycle and regenerates the substrate for ribulose-bisphosphate carboxylase/oxygenase (Rubisco). Rhodophytes and chlorophytes (red and green algae) recruited their nuclear-encoded PRK from the cyanobacterial ancestor of plastids. The plastids of these organisms can be traced back to a single primary endosymbiosis, whereas, for example, haptophytes, dinoflagellates, and euglenophytes obtained their "complex" plastids through secondary endosymbioses, comprising the engulfment of a unicellular red or green alga by a eukaryotic host cell. We have cloned eight new PRK sequences from complex algae as well as a rhodophyte in order to investigate their evolutionary origin. All available PRK sequences were used for phylogenetic analyses and the significance of alternative topologies was estimated by the approximately unbiased test. Our analyses led to several astonishing findings. First, the close relationship of PRK genes of haptophytes, heterokontophytes, cryptophytes, and dinophytes (complex red lineage) supports a monophyletic origin of their sequences and hence their plastids. Second, based on PRK genes the complex red lineage forms a highly supported assemblage together with chlorophytes and land plants, to the exclusion of the rhodophytes. This green affinity is in striking contrast to the expected red algal origin and our analyses suggest that the PRK gene was acquired once via lateral transfer from a green alga. Third, surprisingly the complex green lineages leading to Bigelowiella and Euglena probably also obtained their PRK genes via lateral gene transfers from a red alga and a complex alga with red plastids, respectively.
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A ‘‘Green’’ Phosphoribulokinase in Complex Algae with Red Plastids: Evidence
for a Single Secondary Endosymbiosis Leading to Haptophytes, Cryptophytes,
Heterokonts, and Dinoflagellates
Jo
¨
rn Petersen,
1
Rene
´
Teich,
1
Henner Brinkmann,
2
Ru
¨
diger Cerff
1
1
Institut fu
¨
r Genetik, Technische Universita
¨
t Braunschweig, D-38106 Braunschweig, Germany
2
De
´
partement de Biochimie, Universite
´
de Montre
´
al C.P. 6128, Montre
´
al, Quebec, Canada
Received: 21 October 2004 / Accepted: 24 May 2005 [Reviewing Editor: Dr. Patrick Keeling]
Abstract. Phosphoribulokinase (PRK) is an essen-
tial enzyme of photosynthetic eukaryotes which is
active in the plastid-located Calvin cycle and regen-
erates the substrate for ribulose-bisphosphate car-
boxylase/oxygenase (Rubisco). Rhodophytes and
chlorophytes (red and green algae) recruited their
nuclear-encoded PRK from the cyanobacterial
ancestor of plastids. The plastids of these organisms
can be traced back to a single primary endosymbiosis,
whereas, for example, haptophytes, dinoflagellates,
and euglenophytes obtained their ‘‘complex’ plastids
through secondary endosymbioses, comprising the
engulfment of a unicellular red or green alga by a
eukaryotic host cell. We have cloned eight new PRK
sequences from complex algae as well as a rhodo-
phyte in order to investigate their evolutionary origin.
All available PRK sequences were used for phyloge-
netic analyses and the significance of alternative
topologies was estimated by the approximately
unbiased test. Our analyses led to several astonishing
findings. First, the close relationship of PRK genes of
haptophytes, heterokontophytes, cryptophytes, and
dinophytes (complex red lineage) supports a mono-
phyletic origin of their sequences and hence their
plastids. Second, based on PRK genes the complex
red lineage forms a highly supported assemblage to-
gether with chlorophytes and land plants, to the
exclusion of the rhodophytes. This green affinity is in
striking contrast to the expected red algal origin and
our analyses suggest that the PRK gene was acquired
once via lateral transfer from a green alga. Third,
surprisingly the complex green lineages leading to
Bigelowiella and Euglena probably also obtained their
PRK genes via lateral gene transfers from a red alga
and a complex alga with red plastids, respectively.
Key words: Secondary endosymbiosis Gene
transfer Plastid Nuclear genes Calvin cycle
— Phosphoribulokinase — Complex algae — Red
algae
Introduction
The fascinating global biodiversity of photosynthetic
eukaryotes, which includes such disparate organisms
as huge sequoia trees, fields of seaweed, diatoms, and
bioluminescent dinoflagellates, is to a large extent the
result of consecutive endosymbioses. It is widely ac-
cepted that the origin of all plastids can be traced
back to a single primary endosymbiosis between a
The nucleotide sequence data will appear in the DDBJ/EMBL/
GenBank International Nucleotide Sequence Database under the
following accession numbers. cDNA clones: AY772245 (Pavlova
lutheri); AY772246 (Guillardia theta); AY772247 (Lingulodinium
polyedrum); AY772248 and AY772249 (Pyrocystis lunula);
AY772250 (Euglena gracilis); AY772251 (Chondrus crispus).
Genomic clone: AY772252 (Prymnesium parvum). Genomic PCR
clone: AY772253 (Bigelowiella natans).
Correspondence to: Jo
¨
rn Petersen email: j.petersen@tu-bs.de
J Mol Evol (2006) 62:143–157
DOI: 10.1007/s00239-004-0305-3
cyanobacterium and a nonphotosynthetic eukaryote
(Delwiche and Palmer 1997; Douglas 1998; Stoebe
and Kowallik 1999). The direct descendants of this
event are the three lineages of primary photosynthetic
eukaryotes, green plants (chlorophytes and land
plants), red algae, and glaucocystophytes, which
possess primary plastids surrounded by two mem-
branes. The presence of a cyanobacterium-like plas-
tidial peptidoglycan layer suggests an ancestral
position of the glaucocystophytes (Kies and Kremer
1990). However, the relative branching order of the
three primary photosynthetic lineages is still contro-
versial (Martin et al. 1998; Nozaki et al. 2003). It is
generally accepted that chlorophytes and rhodo-
phytes were involved in several independent second-
ary endosymbioses through engulfment by a
eukaryotic host cell. The subsequent reduction of the
former photosynthetic eukaryotes generated complex
plastids surrounded by three or four membranes. A
vestigial eukaryotic nucleus of the endosymbiont,
designated as nucleomorph, is still present in the
plastids of chlorarachniophytes and cryptophytes
(Gilson and McFadden 1997; Douglas et al. 2001).
As indicated by the green pigmentation (chloro-
phyll b), chlorarachniophytes and euglenophytes
originated via the recruitment of chlorophytes. Their
independent origin is proven by molecular, bio-
chemical, and ultrastructural analyses of the host
cells, which exhibit a relationship between
Chlorarachnion and cercomonads (McFadden et al.
1994; Bhattacharya et al. 1995), whereas Euglena
clearly groups together with kinetoplastids and dip-
lonemids (Maslov et al. 1999; Dooijes et al. 2000). In
contrast to this clear picture emerging for the com-
plex green lineage, molecular phylogenies to date al-
low no clear-cut conclusion for the complex red
lineage, comprising haptophytes, heterokontophytes,
and cryptophytes, designated chromists (Cavalier-
Smith 1986), and peridinin containing dinophytes
(Delwiche 1999). In addition to secondary endosym-
bioses, there is increasing evidence for a tertiary ori-
gin of plastids from at least several dinophycean
lineages, which recruited complex algae such as
haptophytes (Tengs et al. 2000; Yoon et al. 2002a;
Inagaki et al. 2004).
The number of independent endosymbiotic events
leading to the different groups of complex algae is still
undetermined (Van de Peer et al. 2000; Medlin et al.
1997; Daugbjerg and Andersen 1997). According to
rRNA phylogenies, the host cells of apicomplexa,
dinophytes, and ciliates share a common origin.
These organisms have therefore been combined in the
superensemble alveolata (Van de Peer and De
Wachter 1997). A second rRNA-based superensem-
ble containing species with complex red plastids
(rhodoplasts) is the stramenopiles. It unites aplasti-
dial oomycetes and the morphologically very diverse
but phylogenetically closely related photosynthetic
heterokontophytes, including diatoms (Bacillario-
phyceae) and brown (Phaeophyceae) and yellow-
green algae (Xanthophyceae). The phylogenetic
position of the remaining two orders with complex
rhodoplasts, the haptophytes, and the cryptophytes
is, however, completely unresolved.
Evidence of a possible monophyletic origin of the
complex red lineage comes from phylogenetic analy-
ses of the nuclear-encoded plastidial glyceraldehyde-
3-phosphate dehydrogenase (GAPDH). This gene,
named GapCI, was originally discovered in crypto-
phytes (Liaud et al. 1997) and subsequently also
found in all other orders of the complex red lineage
(Fagan et al. 1998; Liaud et al. 2000; Fast et al. 2001;
Harper and Keeling 2003). Its presence seems to be
exclusively restricted to complex algae with red
plastids, where it replaced the typical Calvin cycle
GAPDH of cyanobacterial origin (GapA) present in
rhodophytes. The finding of a GapCI gene in the
apicomplexan parasite Toxoplasma gondii therefore
strongly argues in favor of a red alga-related origin of
the reduced apicomplexan plastid (apicoplast), de-
spite putative and controversially discussed green
traits in these protists (Ko
¨
hler et al. 1997; Funes et al.
2002; Cai et al. 2003; Funes et al. 2003; Waller et al.
2003; Hackett et al. 2004).
In the present study we analyzed the phylogenetic
origin of a nuclear-encoded plastidial enzyme, phos-
phoribulokinase (PRK; EC 2.7.1.19), in order to
investigate the evolutionary relationship of photo-
synthetic organisms. PRK is essential for the photo-
synthetic Calvin cycle, the exclusive pathway of
phototrophic CO
2
fixation in eukaryotes, which
serves as a spinning wheel for the creation of all new
biomass (Calvin 1956). PRK catalyzes the final step
in the regeneration of ribulose-1,5-bisphosphate via
ATP-dependent phosphorylation of ribulose-5-phos-
phate and recycles the substrate for the CO
2
fixing
enzyme ribulose-bisphosphate carboxylase/oxygenase
(Rubisco; EC 4.1.1.39). PRKs are divided into two
distantly related classes, which share only 20% amino
acid identity (Martin and Schnarrenberger 1997).
Proteobacterial class I enzymes are octamers, whereas
class II enzymes from cyanobacteria and plants occur
as tetramers and dimers, respectively (Harrison et al.
1998). Nuclear-encoded PRK clones from
Chlamydomonas, higher land plants, and several
complex algae have been isolated (Milanez and
Mural 1988; Raines et al. 1989; Roesler and Ogren
1990; Horsnell and Raines 1991; Archibald et al.
2003). Their close relationship to cyanobacterial PRK
genes reflects a typical example of endosymbiotic
gene transfer to the host cell nucleus (Martin and
Schnarrenberger 1997). In contrast to many isoen-
zymes, e.g., the GAPDHs, which are essential for the
plastidial reductive pentose phosphate cycle as well as
144
for cytosolic glycolysis/gluconeogenesis, both PRK
and Rubisco are unique to the Calvin cycle and have
no cytosolic equivalent. Their essential function for
photoautotrophic growth ensures that both enzymes
are maintained after secondary endosymbioses. Un-
like the plastid-encoded large subunit of Rubisco
(rbcL), which has been studied in detail due to easy
availability via PCR, the nuclear-encoded PRK has
been insufficiently investigated.
Here we present eight PRK sequences from a
rhodophyte (Chondrus crispus), Euglena, and five
complex algae with red plastids. The phylogenetic
analyses support a monophyletic origin of PRK se-
quences from haptophyes, cryptophytes, heterokonts,
and dinoflagellates. Moreover, the results reveal an
unexpectedly close relationship of PRK sequences of
complex algae with red plastids and green plants, to
the exclusion of rhodophytes.
Materials and Methods
Algal Material
The haptophytes Prymnesium parvum (strain 127.79) and Pavlova
lutheri (strain 926-1) were obtained from the ‘‘Sammlung von Al-
genkulturen’’ at the University of Go
¨
ttingen (SAG), Germany.
Culturing was performed in brackish water medium (Schlo
¨
sser
1994) under greenhouse conditions in Erlenmeyer flasks under
vigorous shaking at 22C on a 14.5-h light/9.5-h dark cycle. The
plant material of the rhodophyte Chondrus crispus was field-col-
lected on the North Sea island Helgoland.
Isolation of Poly(A)
+
mRNA and Construction of
cDNA Libraries
Total RNA from Chondrus crispus, Pavlova lutheri, and Prymne-
sium parvum was isolated according to Meyer-Gauen et al. (1998)
from 3.5 to 5.5 g fresh weight. Poly(A)
+
mRNA was prepared as
described by Henze et al. (1995). The cDNA libraries of the rho-
dophyte Chondrus crispus and the haptophyte Pavlova lutheri were
constructed from 5 lg poly(A)
+
mRNA using the kZAP-cDNA
synthesis kit (Stratagene). We used SizeSep 400 Spun Columns
(Pharmacia) for size separation and obtained libraries with 6.0 ·
10
5
and 1.5 · 10
6
recombinant clones.
Both mRNA and the kZAPII cDNA library from Euglena
gracilis were provided by William Martin (University of Du
¨
ssel-
dorf), the kZAPII libraries from Lingulodinium polyedrum and
Pyrocystis lunula were donated from Woodland Hastings (Harvard
University), and the mRNA and kNM1149 cDNA library from the
cryptophyte Guillardia theta were given by Marie Francoise Liaud
from our group (Liaud et al. 1997).
Construction of a Genomic Library
DNA from Prymnesium parvum was obtained from the supernatant
of a CsCl purification step (RNA isolation) as described (Schwarz-
Sommer et al. 1987). Sau3AI fragmens of 15–20 kb were purified
on a sucrose gradient (Sambrock et al. 1989), ligated into BamHI
kEMBL3 arms, and packaged with Gigapack III Gold extracts
(Stratagene). We obtained a library comprising 8.0 · 10
5
re-
combinant clones.
RT-PCR Amplification
Homologous probes were amplified via RT-PCR using the Ther-
moscript RT-PCR System (Invitrogen). Degenerated primers for
PCR amplification were designed based on the universally con-
served N- and C-terminal PRK motifs TVICLDDYH (5Õ-ACS-
GTSATCTGCCTSGACGAYTAYC-3Õ) and WKIQRDMAE (5Õ -
TCSGCCATGTCSCGCTGGATYTTCC-3Õ). One microgram
poly(A)
+
RNA was reverse transcribed using oligo(dT)
20
primers
following the manufacturerÕs instructions for 60 min at 55C. For
PCR amplification of PRK cDNAs, 1 ll of first strand reaction and
1 ll of each primer (100 pmol) were used under the following
conditions: 96C for 2 min; 30 cycles at 96C for 30 s, 54C for 30 s,
72C for 1.0 min; and, finally, 72C for 5 min. PCR products were
extracted from the gel (GENECLEAN Turbo kit; QBIOgene) and
subsequently cloned into the plasmid pCR 2.1 (TA-Cloning kit;
Invitrogen). The different PRK clones were identified by sequencing
using radioactive techniques.
Homologous probes from Lingulodinum polyedrum and
Pyrocystis lunula were directly amplified from the cDNA clones.
Therefore, we mass excised 1.0 · 10
6
clones from each kZAPII
library and isolated plasmid DNA by maxipreparation. Under the
assumption that all cDNAs including the PRK clones are repre-
sented in this sample, we used 10 ng of the plasmid DNA instead of
reverse-transcribed cDNA for PCR amplification. All further steps
were performed as described above.
Isolation and Sequencing of cDNA and Genomic
Clones
All libraries were screened using
32
P-labeled homologous probes
under the same conditions. Hybridization of the filters was per-
formed at 60Cin3· SSPE, 0.1% sodium dodecyl sulfate (SDS; w/
v), 0.02% polyvinylpyrrolidone (PVP) 90, 0.02% Ficoll 400, and 50
lg/ml denatured salmon sperm DNA; for washing we reduced the
SSPE concentration to 0.3 · SSPE. The cDNA clones from kZAPII
cDNA libraries were subcloned in pBluescript II SK(+) by single
clone excision. cDNAs from the kNM1149 library of Guillardia
theta and genomic clones from Prymnesium parvum were subcloned
into the EcoRI, EcoRV, or HindIII site of pBluescript II SK(+).
All clones were sequenced on both strands using pBluescript or
gene-specific primers.
Sequence Handling and Phylogenetic Analyses
The deduced amino acid sequence of Arabidopsis phosphoribulo-
kinase (P25697) was used as a query sequence in a BLAST search,
using the program BLASTP either against the nonredundant pro-
tein database at NCBI or, alternatively, against nucleotide data-
bases with the program TBLASTN. The sequences were retrieved
from Genbank and the initial alignment, obtained with CLUSTAL
X (Thompson et al. 1997), was manually refined using the ED
option of the MUST program package (Philippe 1993). Four
slightly different data sets were created; the largest contained 31
PRK sequences and 292 unambigously aligned amino acid posi-
tions. In the second, the very fast-evolving sequences of the dino-
phytes were eliminated, thus allowing inclusion of more positions
in the analysis (28 sequences and 312 positions). Subsequently, the
sequence of the basal rhodophyte Cyanidioschyzon (27 sequences,
312 positions) and the fast-evolving sequence of Bigelowiella (26
sequences, 312 positions) were also eliminated. All data sets were
analyzed by all four standard methods of phylogenetic
reconstruction, including bootstrap analyses (or equivalent meth-
ods), to estimate the support for internal nodes of the phylogenies.
Two likelihood methods (maximum likelihood and Bayesian
inference) were used with a model based on the WAG matrix of
145
amino acid replacements assuming a proportion of invariant
positions and gamma-distributed rates. All phylogenetic trees
presented are MrBayes consensus trees; in the corresponding trees
the posterior probabilities are displayed as percentages. Bayesian
inference was performed with the program MrBayes version 3.0B4
(Huelsenbeck and Ronquist 2001) using the WAG+F+I+G4
model. Usually 200,000 generations were completed with trees
collected each tenth generation; the number of generations needed
until the likelihood values converged (burn-in) was typically less
than 5% of the total. Maximum likelihood (ML) bootstrap values
(100 replicates) were calculated with the program PHYML version
2.4 using a WAG+F+I+G8 model (Guindon and Gascuel 2003).
Furthermore, a distance-based approach (ML distances using
WAG+F+I+G8 model) was done using the program TREE-
PUZZLE version 5.2 together with the unix shell script PUZ-
ZLEBOOT (http://hades.biochem.dal.ca/Rogerlab/Software/soft-
ware.html), the trees were obtained using the program BIONJ
(Gascuel 1997), and the bootstrap consensus tree (1000 replicates)
was subsequently generated by the CONSENSE option of the
PHYLIP package (Felsenstein 2004). The maximum parsimony
analyses were done with PAUP* version 40b10 for unix (Swofford
1999), using 1000 replicates with TBR heuristic search and 10 times
random addition of sequences.
Alternative topologies were analyzed with the approximately
unbiased (AU) test using the consel package (Shimodaira and
Hasegawa 2001). The branch lengths for the user-defined topolo-
gies were calculated using TREE-PUZZLE version 5.2 under a
WAG+F+I+G8 model (Schmidt et al. 2002). Topology-depen-
dent sitewise likelihood values were established using the CO-
DEML option of the PAML package version 3.14 (Yang 1997).
Probability values (p) <0.05 are considered significant. The
resampling estimated log-likelihood (RELL) bootstrap support
values were obtained as a part of the results of the AU test.
Results
Isolation and Characterization of PRK Sequences
Here we present eight new PRK sequences, one each
from the red alga Chondrus crispus and from the
‘‘secondary green alga’’ Euglena gracilis and six
from the following complex algae of the secondary
red lineage: the haptophytes Prymnesium parvum and
Pavlova lutheri, the cryptophyte Guillardia theta,
and the dinophytes Lingulodinium polyedrum and
Pyrocystis lunula (two sequences). All PRK sequences
stem from cDNA clones, except that of Prymnesium
parvum, which is derived from a genomic clone (see
below). For all clones screening was performed using
previously amplified homologous RT-PCR probes
(see Materials and Methods). The 5¢ ends of at least
three independent cDNA clones were determined for
each sequence and the largest clones were chosen for
complete sequencing (see also the list of accession
numbers in the title-page footnote). All seven new
PRK cDNAs contain putative ATG start codons, and
the comparison of the deduced amino acid (aa) se-
quences with cyanobacterial and plant homologs
suggests that the plastid specific transit sequences of
all clones are probably full-length (an alignment with
all sequences is available on request). Several addi-
tional PRK sequences were retrieved from the public
databases by data mining via BLAST searches. The
coding sequence from Lotus corniculatus was ob-
tained by comparisons of the genomic sequence of
chromosome 2 with available EST data (AV428286)
as well as with our alignments. Two PRK genes were
identified in the rice genome; our analysis showed
that one of the genes is wrongly annotated, indicating
a nonexisting intron in position 382-0 (for numbering
see below). The assembled raw genome sequence of
the diatom Thalassiosira pseudonana, which was
established at the JGI (http://www.jgi.doe.gov/),
shows that this alga contains only one PRK gene
(Scaffold_36).
Characterization of Genomic PRK Sequences
The precise intron positions in different PRK genes
were determined by comparison of genomic and
cDNA sequences. Intron positions were numbered
using the amino acid sequence of the PRK from
Arabidopsis as a reference (P25697; single-copy gene).
The positions and length of the three introns in the
genomic clone of Prymnesium were obtained by
comparison with RT-PCR amplificates. Moreover,
the structures of the PRK genes from Bigelowiella
and Chondrus were determined. All genes were
amplified from genomic DNA using homologous
primers; the PCR products were cloned and se-
quenced. The PRK gene of the haptophyte
Prymnesium contains three introns (positions 194-0,
244-0, and 330-0), the chlorarachniophyte Bigelowi-
ella harbors four introns (–49-1, 9-0, 81-0, 116-0), and
the gene from the rhodophyte Chondrus is intron-free
(Fig. 3).
Furthermore, all genomic PRK sequences available
from the databases were analyzed. Intron borders
were determined in comparison with cDNA/EST data
or with our alignments (Fig. 3). The whole-genome
sequences of the diatom Thalassiosira pseudonana
(JGI) and the rhodophyte Cyanidioschyzon merolae
reveal that the respective PRK gene of both algae is
lacking introns. All land plant genes from Arabidopsis
thaliana, Triticum aestivum, Lotus corniculatus, and
Oryza sativa (two clones) contain four GT–AG
introns that are absolutely conserved with respect to
both position and phase (175-2, 204-0, 232-1, 314-0).
The PRK gene from Chlamydomonas has seven
introns (25-2, 37-0, 60-1, 83-1, 244-0, 299-1, 375-0), six
of them at unique positions. Intron 244-0 was also
found in the PRK of the haptophyte Prymnesium at
the same position.
Phylogenetic Analyses of PRK Sequences
Figure 1 shows a phylogenetic MrBayes consensus
tree of all available PRK sequences including 25
146
eukaryotic ingroup and 6 cyanobacterial outgroup
sequences. The data set comprises red algae
(Chondrus, Galdieria, and Cyanidioschyzon), green
plants (Chlamydomonas and land plants), and repre-
sentatives from all orders of complex algae including
haptophytes (Pavlova, Prymnesium), dinophytes
(Pyrocystis, Lingulodinium), cryptophytes (Guillar-
dia), heterokontophytes (Odontella, Thalassiosira,
Vaucheria), euglenophytes (Euglena), and chlor-
arachniophytes (Bigelowiella).
Eukaryotic PRK sequences can be divided into
three distinct subtrees. With respect to PRK, red al-
gae surprisingly group together with Bigelowiella
(chlorarachniophyte), which contains a green plastid
(subtree I), and branch basal to the complex red
lineage plus Euglena (subtree II) and the green plants
(subtree III). The unexpected sister group relation-
ship between the latter two groups is well supported
by bootstrap values or Bayesian posterior probabili-
ties between 66 and 100% for the four analyses.
Within the second assemblage, sequences of hapto-
phytes, dinophytes, and diatoms (Thalassiosira,
Odontella) form separate branches. The PRK se-
quences of heterokontophytes are not monophyletic
because the diatoms are closely associated with the
cryptophyte Guillardia (53 to 99%), to the exclusion
of the xanthophyte Vaucheria (Figs. 1 and 2). The
dinophytes contain by far the most divergent se-
quences, with several unique insertions and deletions.
They have an extraordinary long branch within the
phylogenetic tree and a common branching together
with Euglena is only found in the MrBayes analysis.
The very fast-evolving sequences were subse-
quently excluded from the analyses, because they are
expected to disturb the phylogenetic reconstruction
and probably create long branch attraction (LBA)
artifacts (Felsenstein 1978; Figge et al. 1999;
Brinkmann and Philippe 1999; Philippe et al. 2000).
The elimination of the dinophycean sequences did
not result in any topological changes, but the statis-
tical support significantly increased (data not shown;
see supplementary Figure S1 at http://www.tu-
braunschweig.de/ifg/ag/cerff/petersen/download/
PRK). The moderate values for, e.g., a common
branch uniting the complex red lineage plus Euglena
(subtree II) increased from 60–83% (Fig. 1) to 74–
100% (supplementary Fig. S1). The additional re-
moval of the PRK sequence of the basal-branching
rhodophyte Cyanidioschyzon (supplementary Fig. S2)
led to a further increase in the statistical support,
especially for a common branching of rhodophytes
and Bigelowiella (Fig. S1, 47–92%, versus Fig. S2, 74–
100%). Finally, we have also withdrawn the fast-
evolving PRK sequence of Bigelowiella for the last set
of phylogenetic analyses (Fig. 2). The exclusion of
divergent PRK sequences causes an increment in the
statistical support, reaching nearly maximum values
for subtree II (Fig. 2; 96–100%). A conspicuous
exception is the support values for Chlamydomonas
together with land plants, which continuously de-
crease after the successive removal of divergent se-
quences (73–100% [Fig. 1], 52–95% [Fig. S2], 35–75
% [Fig. 2]).
In order to test the statistical significance of
various alternative topologies, we created altogether
54 ‘‘user-defined trees’’ and analyzed them in three
series (A, B, and C) with the AU test (Table 1). The
RELL-bootstrap support values, which add up to
1.0, are indicated for each series of ‘‘user-defined
trees,’’ thus allowing an additional comparison and
a relative weighting of the given topologies (Ta-
ble 1). First, we investigated all alternative positions
of the dinoflagellates by using the complete data set
in Figure 1; here we present the tests of 20 biological
meaningful topologies (A1 to A21). Due to their
extreme sequence divergence only a placement to-
gether with diatoms (A2), land plants (A11), and
Chlamydomonas (A12) or together with red algae
(A15 to A19) is significantly rejected. However, the
RELL bootstrap values (Table 1; A1 to A9 versus
A10 to A21) clearly favor a positioning of the di-
noflagellates within subtree II (Fig. 1), which is in
accordance with the phylogenetic analyses (60 to
83% support values). Second, we added Bigelowiella
to all possible positions and chose 18 topologies to
test the significance of its grouping together with
rhodophytes (alignment without dinophytes; B1 to
B19). A position of the chlorarachniophyte either
together with Chondrus (BI), together with Cyani-
dioschyzon (MP), or basal to Chondrus and Galdieria
(ML, NJ) is weakly supported by the different
phylogenetic analyses. Despite the fact that the exact
position in subtree I is not resolved, all ‘‘user-de-
fined trees’’ which place Bigelowiella outside the
rhodophytes are significantly rejected by the AU test
(B6– B19). Finally, we had three questions regarding
the topology of the phylogenetic tree shown in
Figure 2 (C1), which was constructed without the
sequences from dinophytes, Bigelowiella, and Cy-
anidioschyzon. First, we tested the position of Eu-
glena (C2 to C12) and all topologies where Euglena
was placed outside the complex algae with red
plastids were significantly rejected (subtree II; C8 to
C12). Second, the proposed common branching of
stramenopiles, comprising Vaucheria and diatoms, is
significantly rejected (C17), possibly due to their
strikingly different evolutionary rates (Fig. 2). Third,
we investigated additional user-defined topologies in
order to determine the most probable relationship
between the complex red lineage and green plants
(Chlamydomonas and land plants) (C13 to C16). An
association of subtree II with rhodophytes (C15) or
a position basal to rhodophytes and green plants
(C16) is significantly rejected, thus supporting a
147
Fig. 1. Phylogenetic MrBayes consensus tree (WAG+F+G8+I
model) based on 31 PRK sequences and 292 amino acid positions.
Sequences established in this study are in boldface. The statistical
support for internal nodes was determined by bootstrap analyses or
posterior probabilities (BI; given as % values) and is indicated at
the corresponding branches. Support values 30% are shown. BI,
Bayesian inference; ML, maximum likelihood; NJ, distance; MP,
maximum parsimony. Capital letters at nodes specify the following
statistical support values (BI|ML|NJ|MP): (A) 100|97|98|96; (B)
80|–|–|–; (C) 82|–|–|–; (D) 100|90|97|95; (E) 50|–|–|–; (F) 75|–|–|–;
(G) 62|–|–|–; (H) 74|–|–|–; (I) 43|49|65|63; (J) 100|100|100|100; (K)
99|70|60|–; (L) 53|–|–|–; (M) 78|60|76|–.
148
common branching of subtrees II and III. In con-
trast, placement of subtree II together with Chla-
mydomonas (C13) is not rejected and this topology
obtained a likelihood virtually equivalent to that of
the best tree (C1). Moreover, its RELL bootstrap
support is surprisingly even higher than that for the
Fig. 2. Phylogenetic MrBayes consensus tree (WAG+F+G8+I
model) based on 26 PRK sequences and 312 amino acid posi-
tions. For more details see the legend to Fig. 1. Capital letters
at nodes specify the following statistical support values
(BI|ML|NJ|MP): (A) 91|85|72|70; (B) 86|53|70|51; (C) 66|49|43|–;
(D) 74|47|55|–; (E) 100|98|99|96; (F) 89|30|–|–; (G) 55|–|–|30; (H)
100|89|96|96; (I) 100|100|100|100; (J) 87|64|50|39; (K)
55|54|73|47; (L) 76|58|78|32.
149
best tree (bp = 0.4670 vs. bp = 0.2610), providing
evidence for a possible chlorophycean origin of the
PRK genes from the complex red lineage. A com-
parison of the two crucial topologies C1 and C13
(Table 1) is shown in Figure 4. Even though the
most divergent sequences including those of dino-
flagellates and Bigelowiella were previously excluded
from the analyses, the average evolutionary rate of
the complex red lineage is clearly accelerated in
comparison with that of streptophytes and rhodo-
phytes. This observation generally argues in favor of
an artificial placement of the divergent subtree due
to LBA (Fig. 4), an explanation that may also apply
to the complex red lineage (Table 1).
Discussion
Monophyletic Origin of the PRK Genes from Complex
Algae with Red Plastids
Recent findings primarily based on photosynthetic
enzymes such as Calvin cycle glyceraldehyde-3-
phosphate dehydrogenase (GapCI [Liaud et al. 1997,
(B) User-defined PRK trees including Bigelowiella (for reference topology B1, see supplementary Fig. S1)
Topology Likelihood Dli ±SE p
AU
RELL
B1 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7321.51 0.00 0.00 0.6480 0.4290
B2 Cyano((Cm((Bn+Gs)Cc))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7322.00 0.50 3.88 0.5450 0.3670
B3 Cyano((Cm((Bn(Gs+Cc)))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7322.91 1.41 3.41 0.4400 0.0920
B4 Cyano(((Bn+Cm)(Gs+Cc))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7324.74 3.23 4.55 0.3140 0.0600
B5 Cyano((Bn(Cm(Gs+Cc)))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7324.75 3.25 4.55 0.3420 0.0380
B6 (Bn+Cyano)((Cm(Gs+Cc))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7330.43 8.93 6.21 0.0270 0.0030
B7 Cyano((Cm(Gs+Cc))(Bn((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom))))))) )7330.43 8.93 6.21 0.0280 0.0100
B8 Cyano((Cm(Gs+Cc))((Bn(Cr+Land))(Hapto(Vl(Eg(Gt+Diatom)))))) )7356.00 34.49 9.97 0.0030 0.0000
B9 Cyano((Cm(Gs+Cc))(((Bn+Cr)Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7365.82 44.32 11.73 0.0010 0.0000
B10 Cyano((Cm(Gs+Cc))((Cr(Bn+Land))(Hapto(Vl(Eg(Gt+Diatom)))))) )7365.82 44.32 11.73 0.0010 0.0000
B11 Cyano((Cm(Gs+Cc))((Cr+Land)(Bn(Hapto(Vl(Eg(Gt+Diatom))))))) )7355.22 33.71 10.25 0.0010 0.0000
B12 Cyano((Cm(Gs+Cc))((Cr+Land)((Bn+Hapto)(Vl(Eg(Gt+Diatom)))))) )7356.18 34.68 11.29 0.0010 0.0000
B13 Cyano((Cm(Gs+Cc))((Cr+Land)(Hapto(Bn(Vl(Eg(Gt+Diatom))))))) )7357.97 36.46 10.98 0.0010 0.0000
B14 Cyano((Cm(Gs+Cc))((Cr+Land)(Hapto((Bn+Vl)(Eg(Gt+Diatom)))))) )7369.87 48.36 13.15 0.0000 0.0000
B15 Cyano((Cm(Gs+Cc))((Cr+Land)(Hapto(Vl(Bn(Eg(Gt+Diatom))))))) )7367.14 45.64 13.67 0.0001 0.0000
B16 Cyano((Cm(Gs+Cc))((Cr+Land)(Hapto(Vl((Bn+Eg)(Gt+Diatom)))))) )7362.99 41.48 14.27 0.0040 0.0010
B17 Cyano((Cm(Gs+Cc))((Cr+Land)(Hapto(Vl(Eg(Bn(Gt+Diatom))))))) )7367.14 45.64 13.67 0.0000 0.0000
B18 Cyano((Cm(Gs+Cc))((Cr+Land)(Hapto(Vl(Eg((Bn+Gt)Diatom)))))) )7376.36 54.86 15.19 0.0000 0.0000
B19 Cyano((Cm(Gs+Cc))((Cr+Land)(Hapto(Vl(Eg(Gt(Bn+Diatom))))))) )7376.88 55.38 15.00 0.0000 0.0000
Table 1. Approximately unbiased (AU) tests
(A) User-defined PRK trees including Dinophytes and Bigelowiella (for reference topology A1, see Fig.1)
Topology Likelihood Dli ±SE p
AU
RELL
A1 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto(Vl((Eg+Dino)(Gt+Diatom)))))) )7410.44 0.00 0.00 0.7770 0.4990
A2 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto(Vl(Eg(Gt(Dino+Diatom))))))) )7422.75 12.32 7.91 0.0150 0.0010
A3 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto(Vl(Eg((Gt+Dino)Diatom)))))) )7419.10 8.67 8.96 0.2220 0.0920
A4 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto(Vl(Eg(Dino(Gt+Diatom))))))) )7418.51 8.07 6.25 0.1510 0.0050
A5 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto(Vl(Dino(Eg(Gt+Diatom))))))) )7418.77 8.33 6.21 0.0520 0.0010
A6 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto(Dino(Vl(Eg(Gt+Diatom))))))) )7415.63 5.20 7.30 0.3940 0.0340
A7 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto((Dino+Vl)(Eg(Gt+Diatom)))))) )7418.91 8.47 6.30 0.0600 0.0010
A8 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)((Dino+Hapto)(Vl(Eg(Gt+Diatom)))))) )7416.52 6.08 8.85 0.2960 0.0570
A9 Cyano((Cm(Gs(Cc+Bn)))((Cr+Land)(Dino(Hapto(Vl(Eg(Gt+Diatom))))))) )7415.16 4.72 9.27 0.4970 0.0490
A10 Cyano((Cm(Gs(Cc+Bn)))((Dino(Cr+Land))(Hapto(Vl(Eg(Gt+Diatom)))))) )7417.48 7.04 10.55 0.0990 0.0130
A11 Cyano((Cm(Gs(Cc+Bn)))((Cr(Dino+Land))(Hapto(Vl(Eg(Gt+Diatom)))))) )7431.75 21.31 12.51 0.0150 0.0020
A12 Cyano((Cm(Gs(Cc+Bn)))(((Cr+Dino)Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7432.11 21.67 12.30 0.0030 0.0004
A13 Cyano((Cm(Gs(Cc+Bn)))(Dino((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom))))))) )7415.07 4.63 11.12 0.6300 0.2060
A14 Cyano((Dino(Cm(Gs(Cc+Bn))))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7425.69 15.25 13.47 0.1030 0.0040
A15 Cyano(((Dino+Cm)(Gs(Cc+Bn)))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7427.11 16.67 13.65 0.0480 0.0020
A16 Cyano((Cm(Dino(Gs(Cc+Bn))))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7427.11 16.67 13.65 0.0490 0.0020
A17 Cyano((Cm((Dino+Gs)(Cc+Bn)))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7432.64 22.21 14.61 0.0090 0.0000
A18 Cyano((Cm(Gs((Dino(Cc+Bn))))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7431.40 20.97 14.74 0.0150 0.0010
A19 Cyano((Cm(Gs((Dino+Cc)Bn)))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7432.42 20.98 14.74 0.0330 0.0010
A20 Cyano((Cm(Gs(Cc(Dino+Bn))))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7429.13 18.69 15.16 0.0810 0.0240
A21 (Dino+Cyano)((Cm(Gs(Cc+Bn)))((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )7425.75 15.31 13.41 0.0600 0.0060
150
2000; Fast et al. 2001; Harper and Keeling 2003]) and
aldolase (Patron et al. 2004) support the idea that all
complex plastids derived from rhodophytes origi-
nated via a single secondary endosymbiosis (chrom-
alveolate hypothesis [Cavalier-Smith 1999]). To
further test this hypothesis we chose phosphoribulo-
kinase (PRK) as a phylogenetic marker, since PRK
enzymes have an exclusively photosynthetic function
and are present in all plastids of primary or second-
ary origin (primary and complex plastids, respec-
tively). We analyzed PRK sequences from all orders
of algae containing complex plastids (haptophytes,
Table 1. Continued
(C) User-defined PRK trees (for reference topology C1, see Fig. 2)
Topology Likelihood Dli ±SE p
AU
RELL
C1 Cyano((Cc+Gs)((Cr+Land)(Hapto(Vl(Eg(Gt+Diatom)))))) )6549.30 0.00 0.00 0.6610 0.2610
C2 Cyano((Cc+Gs)((Cr+Land)(Hapto(Eg(Vl(Gt+Diatom)))))) )6552.18 2.88 2.80 0.0870 0.0090
C3 Cyano((Cc+Gs)((Cr+Land)(Hapto((Eg+Vl)(Gt+Diatom))))) )6550.10 0.80 4.10 0.4300 0.2290
C4 Cyano((Cc+Gs)((Cr+Land)(Hapto(Vl((Eg+Gt)Diatom))))) )6559.00 9.70 6.18 0.0490 0.0090
C5 Cyano((Cc+Gs)((Cr+Land)(Hapto(Vl(Gt(Eg+Diatom)))))) )6559.07 9.77 6.10 0.0620 0.0060
C6 Cyano((Cc+Gs)((Cr+Land)((Eg+Hapto)(Vl(Gt+Diatom))))) )6558.58 9.28 6.19 0.0290 0.0090
C7 Cyano((Cc+Gs)((Cr+Land)(Eg(Hapto(Vl(Gt+Diatom)))))) )6559.39 10.09 5.80 0.0120 0.0010
C8 Cyano((Cc+Gs)((Eg(Cr+Land))(Hapto(Vl(Gt+Diatom))))) )6582.73 33.43 11.26 0.0001 0.0000
C9 Cyano((Cc+Gs)(((Eg+Cr)Land)(Hapto(Vl(Gt+Diatom))))) )6589.06 39.76 12.05 0.0010 0.0001
C10 Cyano((Cc+Gs)((Cr(Eg+Land))(Hapto(Vl(Gt+Diatom))))) )6590.71 41.41 11.83 0.0001 0.0000
C11 Cyano((Cc+Gs)(Eg((Cr+Land)(Hapto(Vl(Gt+Diatom)))))) )6582.70 33.40 11.30 0.0002 0.0001
C12 Cyano(Eg((Cc+Gs)((Cr+Land)(Hapto(Vl(Gt+Diatom)))))) )6608.04 58.74 14.76 0.0010 0.0000
C13 Cyano((Cc+Gs)(Land(Cr(Hapto(Vl(Eg(Gt+Diatom))))))) )6549.43 0.13 5.62 0.6240 0.4670
C14 Cyano((Cc+Gs)(Cr(Land(Hapto(Vl(Eg(Gt+Diatom))))))) )6553.44 4.14 3.64 0.0310 0.0060
C15 Cyano(((Hapto(Vl(Eg(Gt+Diatom))))(Cc+Gs))(Cr+Land)) )6585.25 35.95 12.73 0.0020 0.0010
C16 Cyano((Hapto(Vl(Eg(Gt+Diatom))))((Cc+Gs)(Cr+Land))) )6586.77 37.47 12.20 0.0001 0.0000
C17 Cyano((Cc+Gs)((Cr+Land)(Hapto(Eg(Gt(Vl+Diatom)))))) )6566.28 16.98 7.85 0.0060 0.0010
Cyano: cyanobacterial subtree. Land: subtree of land plants. Hapto: Pavlova and Prymnesium. Dino: Pyrocystis and Lingulodinium. Diatom:
Thalassiosira and Odontella. Cm, Gs, Cc, Bn, Cr, Vl, Eg, and Gt: sequences from Cyanidioschyzon, Galdieria, Chondrus, Bigelowiella,
Chlamydomonas, Vaucheria, Euglena, and Guillardia. Dli: difference in the likelihood value versus the ML tree. p
AU
: probability
aproximately unbiased test. RELL: resampling estimated log-likelihood bootstrap support. Topologies which are rejected at the 5%
significance level in p
AU
test are in boldface.
Fig. 3. Intron positions in PRK genes. Positions are numbered using the amino acid sequence of Arabidopsis thaliana (P25697) as a
reference. Intron positions with the extension ‘‘–0’’ indicate that the intron is inserted between two codons, ‘‘–1’’ is after the first nucleotide,
and ‘‘–2’’ is after the second nucleotide of the codon.
151
heterokonts, cryptophytes, dinophytes, eugleno-
phytes and chlorarachniophytes) including eight new
ones established in our laboratory. Our phylogenetic
analyses show that, with respect to PRK, all complex
algae with rhodoplasts (red lineage) and Euglena
form a monophyletic group which is, however, only
moderately supported by statistical support values
(60 to 83%; see subtree II in Fig. 1). After elimination
of the fast-evolving sequences from dinophytes and
Bigelowiella and the early-branching sequence from
the simple unicellular rhodophyte Cyanidioschyzon,
the statistical support reaches nearly maximum val-
ues (Fig. 2; 95 to 100%), thereby providing evidence
in favor of a monophyletic origin of subtree II. The
unexpected presence of Euglena in this group prob-
ably reflects a lateral gene transfer (LGT) from a
secondary red lineage (see below).
The fast-evolving PRK sequences from dinophytes
merit special attention. Dinophytes have by far the
strangest genetic composition among all photosyn-
thetic eukaryotes (Morse et al. 1995; Zhang et al. 1999;
Bachvaroff et al. 2004; Hackett et al. 2004), and their
phylogenetic position in the PRK topology is rather
uncertain. In order to determine their precise position,
we tested 20 alternative topologies, but the AU tests
only indicated significant rejection of a placement to-
gether with rhodophytes, Chlamydomonas, and land
plants (Table 1; A11, A12, and A15– A19). However,
the RELL bootstrap values (Table 1; A1 to A9 versus
A10 to A21) clearly favor a positioning of the dino-
phyte PRK sequences within subtree II (Fig. 1) and,
hence, a monophyletic origin of complex rhodoplasts.
Complex Algae with Red Plastids Possess a PRK Gene
Related to Green Plants
The most surprising outcome of our study is the
observation that PRK genes from complex algae with
red plastids are not related to those of rhodophytes,
as would be expected (Fig. 5), but exhibit a sister
group relationship to those of green plants (chloro-
phytes and land plants, subtree III). This relationship
seems rather stable (66 to 100 %; see Fig. 1) and
receives maximum support after elimination of the
divergent sequences (98 to 100%; see Fig. 2). The
increase in support after elimination of divergent se-
quences is also observed for other assemblages such
as branches bearing rhodophytes (subtree I) and
complex algae with red plastids (subtree II; see
above). However, there is one conspicuous exception
concerning Chlamydomonas, whose affiliation with
land plants becomes considerably weakened (35 to
75%; see Fig. 2). Therefore, we tested alternative
topologies where subtree II was combined with
Chlamydomonas as well as with red algae (Table 1;
C13 and C15), respectively. In agreement with our
Fig. 4. Comparison of evolutionary rates of cyanobacterial and
eukaryotic PRK sequences (26 sequences and 312 amino acid posi-
tions). The left image corresponds to the best ML tree obtained in the
MrBayes analysis (see Fig. 2 and Table 1: C1), whereas the right
image represents the user-defined topology C13 (Table 1). Average
branch lengths of the different subtrees are displayed by triangles. A
possible artificial placement of complex algae with red plastids
(complex red lineage) due to long branch attraction (LBA) is indi-
cated by an arrow.li, likelihood values of the given topologies; RELL,
resampling estimated log-likelihood bootstrap support values.
152
phylogenetic analyses, only the association with red
algae was rejected, while that with Chlamydomonas
gave a likelihood value equivalent to that of our best
tree (Fig. 2; C1 in Table 1). Moreover, the RELL
bootstrap support, which allows a relative weighting
of the tested topologies, is even higher for topology
C13 than for the best tree (bp = 0.4670 versus
bp = 0.2610). The results of these AU tests suggest
that in terms of PRK genes, complex algae with red
plastids represent an in-group rather than a sister
group of green plants and seem to be specifically re-
lated to chlorophytes. The apparent sister group
affiliation is probably due to a LBA artifact (Fig. 4),
since subtree II bears mainly fast-evolving sequences.
Independent evidence for a green algal origin of
PRK genes from complex algae with red plastids
comes from an identical intron position (244-0)
exclusively found in Chlamydomonas and the hapto-
phyte Prymnesium (Fig. 3). Shared intron positions
generally argue in favor of a common ancestry of the
respective genes, since independent intron gains at the
same position are expected to be extremely unlikely
events (Long and Cerff 2003). The exclusive presence
of intron 244-0 in Chlamydomonas among the pri-
mary lineages can be explained either by several
parallel losses in rhodophytes as well as in land plants
or by a gain in chlorophytes. Even if the former
assumption is true, its absence in the early-branching
red alga Cyanidioschyzon and in Chondrus argues in
favor of an ancient loss in rhodophytes. Taken to-
gether, irrespective of whether multiple losses or a
single gain occurred in the primary lineage, the intron
distribution is compatible with our phylogenetic
analyses and indicates that a common ancestor of
complex algae with red plastids recruited a chloro-
phycean PRK gene including intron 244-0. Thus,
present-day haptophytes and diatoms subsequently
inherited the ‘‘green’’ PRK gene and the latter sec-
ondarily lost all introns. Conspicuously, the intron
pattern of the PRK gene in Chlamydomonas differs
greatly from that in land plants (Fig. 3), a phenom-
enon previously also observed for different GAPDH
genes (Kersanach et al. 1994; Petersen et al. 2003). It
remains to be clarified whether or not there was an
active phase of intron rearrangement in the chloro-
phycean lineage that generated the intron positions so
far exclusively found in Chlamydomonas.
Bigelowiella and Euglena Recruited Their PRK Genes
via Lateral Gene Transfer (LGT)
The PRK gene of the chlorophyll b containing
complex alga Bigelowiella natans surprisingly groups
together with the rhodophyte sequences (Fig. 1).
The green descendance of the complex plastid from
this protist formerly designated Chlorarachnion
sp. CCMP621 has been proven by multiple molec-
ular phylogenies of chloroplast-encoded genes
(McFadden et al. 1995 and 1997; Van de Peer et al.
1996; Ishida et al. 1997 and 1999; Durnford et al.
1999). A recent study (Archibald et al. 2003) has
shown that the majority of nuclear-encoded and
plastid-targeted proteins of Bigelowiella have a chlo-
rophycean origin, however, in this study the prove-
nance of PRK remained unclear. Our phylogenetic
analyses of PRK genes, including the rhodophyte se-
quences from Chondrus crispus (this survey), Galdieria
sulphuraria, and Cyanidioschyzon merolae (genome
project), solidly support a close relationship between
Bigelowiella and red algae (Fig. 1; supplementary
Figs. S1 and S2). This affiliation is significantly sub-
stantiated by AU tests (Table 1; series B), which reject
all other topologies including an association with
green plants (B8 to B10). Hence, Bigelowiella acquired
its red algal PRK gene probably via LGT in a non-
endosymbiotic context, an explanation that was al-
ready proposed for several other genes of this
mixotrophic amoeboflagellate (Archibald et al. 2003).
Fig. 5. Origin of plastids through primary endosymbiosis and distribution via subsequent endosymbioses involving eukaryotes. ES:
endosymbiosis.
153
Also, Euglena, which represents a second but
unrelated lineage of complex algae with green plastids
(Douglas 1998; Martin et al. 1998; Figge et al. 1999),
has probably received its PRK gene via LGT, since it
groups together with complex algae harboring red
plastids (subtree II; Fig. 1). This unusual association
is more or less solidly supported whether or not
dinophycean sequences are included in the phyloge-
netic analyses (Figs. 1 and 2). The positioning of
Euglena within subtree II was confirmed by the AU
test (Table 1; series C), and all alternative PRK
topologies (C8 to C12), including the placement to-
gether with Chlamydomonas or land plants (C9; C10),
were significantly rejected. In conclusion, the rela-
tionships of PRK genes in Bigelowiella and Euglena
with those from primary and complex red lineages,
respectively, probably represent analogous examples
of horizontal gene recruitment after secondary en-
dosymbioses.
PRK Genes Support a Common Ancestry of Complex
Algae with Red Plastids
Organisms and their corresponding genes evolve
quite constantly over large periods of time. In con-
trast, evolutionary leaps and dramatic changes in
genetic composition are caused by endosymbioses,
the amalgamation of two unrelated organisms. An
exhaustive comparison of completely sequenced ge-
nomes revealed that 18% of the nuclear-encoded
genes of Arabidopsis (about 4300) have a cyanobac-
terial origin and that the majority of the corre-
sponding proteins (about 3000) are not redirected to
the chloroplast (Martin et al. 2002; Leister and
Schneider 2004). On the other hand, inside the plastid
some of the cyanobacterial enzymes were replaced by
nuclear-encoded host cell counterparts, e.g., the
Calvin cycle enzymes triosephosphate isomerase,
aldolase, and fructose bisphosphatase (Martin and
Herrmann 1998). In contrast to prokaryote-to-
eukaryote gene transfer after primary endosymbioses,
gene flux after secondary endosymbioses mainly oc-
curs between the two different nuclei of the endo-
symbiont (donor) and the host cell (recipient).
However, in complex algae numerous cases of LGT
have been observed which presumably occurred in a
nonendosymbiotic context (see above and Archibald
et al. 2003; Takishita et al. 2003; Rogers and Keeling
2004). These obervations suggest that the origin of
genes for plastid functions especially in complex algae
is not strictly predictable.
Here we demonstrate a close relationship between
PRKs of haptophytes, crytophytes, heterokonts, and
dinoflagellates, on the one hand, and those of green
plants, on the other (Figs. 1 and 2), indicating that
none of the former orders has recruited its phos-
phoribulokinase from a rhodophycean endosymbiont
ancestor. Under the assumption that the scheme of
plastid evolution shown in Figure 5 is correct, three
scenarios can, in principle, explain this unexpected
affiliation. First, a gene duplication may have oc-
curred in a common ancestor of rhodo- and chloro-
phytes, the complex algae with red plastids
exclusively recruited the ‘‘green’’ PRK gene, and the
two copies were differentially lost in the primary
lineages (red and green algae). The observation that
all species analyzed so far, including those with
completely sequenced genomes, harbor a single PRK
gene implies that the predicted losses presumably
occurred in common ancestors of their respective
lineages. This scenario is extremely unlikely, because
even under the assumption that complex algae with
rhodoplasts have a common origin, at least four
independent events (one duplication, one transfer,
and two differential losses) would have been required.
The second and by far more likely scenario requires
only one single LGT from an ancestral green alga to
an ancestral complex alga with a rhodoplast, fol-
lowed by the replacement of the original red copy.
The exact context of the recruitment of the PRK
gene(s) remains unclear, but the most simple expla-
nation comprises a LGT in a nonendosymbiotic
context analogous to Bigelowiella and Euglena, fol-
lowing DoolittleÕs (1998) concept of ‘‘you are what
you eat.’’ The third alternative is the possibility that
the host cell for the secondary endosymbiont was a
green alga that subsequently lost its primary plastid
(Ha
¨
uber et al. 1994; Nozaki et al. 2003), but so far no
specific association between green algae and either
stramenopiles or alveolates was detected. However, if
the last hypothesis is true, many more nuclear genes
of complex algae with red plastids are expected to be
of green origin, a prediction which can be tested in
the future when sufficient whole-genome sequences
are available.
Whichever scenario is correct, the exclusive
presence of green PRK genes in haptophytes, cryp-
tophytes, heterokontophytes, and dinophytes
strongly supports their monophyletic origin regard-
less of the context of recruitment (see also our
phylogenetic analyses above). PRK, as well as
GapCI (Harper and Keeling 2003) and aldolase
(Patron et al. 2004), favors this common origin, and
these genes are phylogenetic markers of exceptional
diagnostic value (‘‘lucky genes’ [Bapteste et al.
2002]). They encode indispensable plastidial proteins
of the Calvin cycle and exhibit striking similarities in
that they were all fixed in the host cell nucleus after
secondary endosymbiosis and were obviously not
acquired from the red algal endosymbiont. In con-
sequence, PRK, GapCI, and aldolase are ancestral
components of a common secondary host cell and,
as a corollary, argue in favor of a monophyletic
origin of the complex red plastids via a single sec-
154
ondary endosymbiosis. These data are compatible
with the chromalveolate hypothesis (Cavalier-Smith
1999), which postulates that present-day chromists
(Cavalier-Smith 1986; Yoon et al. 2002b) and alve-
olates (Van de Peer and De Wachter 1997) can be
traced back to a common secondary host cell with a
complex red plastid. However, trees based on single-
gene analyses such as 18S rDNA (van de Peer and
De Wachter 1997) and mitochondrial genes (San-
chez Puerta et al. 2004) indicate that the hosts of the
different orders are probably unrelated. Apart from
lacking phylogenetic support, which may simply
reflect the missing resolution of the available
markers, the different morphology of host cell
mitochondria, which can be assigned to three well-
defined types (Gray et al. 1998), also argues against
a common origin of the complex red lineage. How
can these contradictory data be reconciled? The
occurrence of tertiary endosymbioses would be a
plausible explanation, as shown, for example, for
fucoxanthin containing dinoflagellates (Tengs et al.
2000; Takishita et al. 2004) In this scenario a ter-
tiary host cell engulfed a secondary alga with red
plastids and recycled the preexisting ‘‘green’’ PRK
by integration in its own tertiary nucleus. Taking all
present data together, the chromalveolate hypothesis
seems to tell only part of the story and probably
needs to be modified to accommodate tertiary en-
dosymbioses. Therefore, additional careful analyses
of plastid- and host cell-related markers including
gene-by-gene comparisons of whole metabolic
pathways are necessary to reveal the true evolu-
tionary history of complex phototrophic eukaryotes,
apicomplexa, and ciliates.
Acknowledgments. We thank Woodland Hastings (Harvard) for
the cDNA libraries from Pyrocystis lunula and Lingulodinium
polyedrum, William Martin (Du
¨
sseldorf) for the cDNA library
from Euglena gracilis, Geoff McFadden (Melbourne) for provision
of genomic DNA from Bigelowiella natans, Sarina Scharbatke
(Hannover) for excellent technical assistance, and Carina Grau-
vogel for critical discussions. We are grateful to Naiara Rodriguez-
Ezpeleta for helpful comments on an earlier version of the manu-
script. The associate editor and four anonymous reviewers pro-
vided very helpful suggestions. Major financial support, including a
Ph.D. stipend for R.T., was received from the Deutsche Fors-
chungsgemeinschaft (CE 1/27-1).
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Fig. S1.
Supplementary Material.
Fig. S2.
... En effet, toutes les espèces photosynthétiques des CASH contiennent de la chlorophylle c, qui est absente chez toutes les autres algues (y compris les rouges). De plus, dans les phylogénies basées sur les gènes plastidiques (Yoon et al., 2002b), ainsi que sur certaines protéines codées dans le noyau et impliquées dans la fonction du plaste (Fast et al., 2001;Patron et al., 2004;Petersen et al., 2006), la monophylie des CASH est retrouvée. ...
... En conséquence, la monophylie observée lors de l'analyse des protéines adressées au plaste mais codées dans le noyau ne serait pas due à l'héritage vertical. Au lieu de cela, ces gènes seraient relocalisés dans le noyau du nouvel hôte à partir du nucléomorphe, par coévolution avec les plastes transférés en série (Petersen et al., 2006). Ces dernières années, la plausibilité des hypothèses "en série" a augmenté avec des exemples confirmés d'EEE d'ordre supérieur et indépendantes (c'est-à-dire résultant de l'endosymbiose d'un cryptophyte, d'un haptophyte ou encore d'un hétérokonte selon les lignées) dans les dinoflagellés (Hackett et al., 2004;Inagaki et al., 2000;Tengs et al., 2000). ...
Thesis
Retracer l'histoire évolutive des eucaryotes est une question majeure en évolution et fait l'objet de nombreux débats. Le développement de techniques à haut débit, en particulier en protéomique et en génomique, a permis d'obtenir de nombreuses données pouvant être exploitées lors d'analyses évolutives. Dans ce contexte, les structures multiprotéiques eucaryotes (SME) constituent des objets d'intérêt. En effet, ces gros complexes protéiques sont impliqués dans de nombreux processus fondamentaux de la cellule eucaryote, et n'ont pas d'homologues chez les procaryotes (même si les fonctions dans lesquelles ils sont impliqués peuvent exister). Ils ont donc certainement joué un rôle prépondérant dans l'eucaryogénèse. L'analyse phylogénomique de deux SME impliquées dans la division cellulaire (le midbody et l'APC/C) montre que ces systèmes ont une origine évolutive ancienne et étaient déjà présents chez le dernier ancêtre commun des eucaryotes (LECA), tout en étant issus d'innovations eucaryotes. Ceci implique que l'émergence de ces deux SME s'est faite après la divergence de la lignée eucaryote et avant la diversification ayant donné naissance aux lignées actuelles. Au-delà de ces considérations évolutives, l'analyse de ces SME ouvre des pistes sur certains aspects de la biologie de ces systèmes. En effet, si ces systèmes ont été globalement bien conservés au cours de la diversification des eucaryotes, leur analyse révèle une grande plasticité de composition dans certaines lignées de protistes. Ceci suggère des changements récents concernant certaines étapes du cycle cellulaire de ces organismes qu'il serait intéressant d'explorerexpérimentalement.En parallèle, ce travail a montré que, bien qu'étant des protéines opérationnelles, lescomposants de ces SME portent un signal phylogénétique exploitable pour inférer les relations de parentés entre lignées eucaryotes. La construction de supermatrices à partir de ces protéines a permis l'inférence de phylogénies de qualité, même si non totalement résolues, dans lesquelles, par exemple, la monophylie des Excavata ou encore le placement des microsporidies au sein des Fungi est bien supporté. La combinaison de ces données avec celles issues d'analyses basées sur des protéines informationnelles montrent des avancées significatives concernant la résolution des arbres inférés. Ces résultats ouvrent le champ des possibles quant à la recherche d'autres marqueurs encore inexploités parmi les protéines opérationnelles. L'intégration de ces nouveaux marqueurs associée à l'augmentation de l'échantillonnage taxonomique représente une piste prometteuse pour l'avenir.Ce travail illustre l'intérêt de généraliser les approches évolutives intégrées des systèmes biologiques pour l'étude de l'évolution et de la phylogénie des eucaryotes.
... Our main objective is to analyse the phylogenetic origin of plastid-targeted genes in complex algae [1][2][3] in a fully automated fashion. To do so, we designed and developed a series of strategies and tools around a large-scale single-gene tree analysis pipeline. ...
Article
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Objectives Identifying orthology relationships among sequences is essential to understand evolution, diversity of life and ancestry among organisms. To build alignments of orthologous sequences, phylogenomic pipelines often start with all-vs-all similarity searches, followed by a clustering step. For the protein clusters (orthogroups) to be as accurate as possible, proteomes of good quality are needed. Here, our objective is to assemble a data set especially suited for the phylogenomic study of algae and formerly photosynthetic eukaryotes, which implies the proper integration of organellar data, to enable distinguishing between several copies of one gene (paralogs), taking into account their cellular compartment, if necessary. Data description We submitted 73 top-quality and taxonomically diverse proteomes to OrthoFinder. We obtained 47,266 orthogroups and identified 11,775 orthogroups with at least two algae. Whenever possible, sequences were functionally annotated with eggNOG and tagged after their genomic and target compartment(s). Then we aligned and computed phylogenetic trees for the orthogroups with IQ-TREE. Finally, these trees were further processed by identifying and pruning the subtrees exclusively composed of plastid-bearing organisms to yield a set of 31,784 clans suitable for studying photosynthetic organism genome evolution.
... Thus, mixotrophic capabilities of the euglenophyte, cryptophytes, and raphidophytes may be weaker than those of mixotrophic dinoflagellates. Euglenophyte and raphidophytes possibly have acquired photosynthetic capabilities via secondary endosymbiosis, whereas the phototrophic dinoflagellates via tertiary endosymbiosis ( Gibbs, 1978Gibbs, , 1981McFadden, 1993;Delwiche, 1999;Yoon et al., 2005;Milanowski et al., 2006;Petersen et al., 2006;Ko rený and Oborník, 2011). Endosymbiosis occurs through feeding ( Delwiche, 1999;Archibald, 2009;Keeling, 2010); thus, weaker mixotrophic ability of the euglenophyte, cryptophytes, and raphidophytes compared to mixotrophic dinoflagellates could partially be attributed to them being less evolved. ...
Article
The phototrophic euglenophyte Eutreptiella eupharyngea often causes blooms in the coastal waters of many countries, its mode of nutrition have not been assessed. This species has previously been considered as exclusively phototrophic. To explore whether E. eupharyngea is a mixotrophic species, the protoplasm of E. eupharyngea cells were examined using light, epifluorescence, and transmission electron microscopy after eubacteria, the cyanobacterium Synechococcus sp., and diverse algal species were provided as potential prey. Furthermore, the ingestion rates of E. eupharyngea on eubacteria or Synechococcus sp. as a function of prey concentration were measured. In addition, grazing by natural populations of euglenophytes on natural populations of eubacteria in Masan Bay was investigated. This study is the first to report that E. eupharyngea is a mixotrophic species. Among the prey organisms offered, E. eupharyngea fed only on eubacteria and Synechococcus sp., and the maximum ingestion rates of these two organisms measured in the laboratory were 5.7 and 0.7 cells predator-1 h-1, respectively. During the field experiments, the maximum ingestion rates and grazing impacts of euglenophytes, including E. eupharyngea, on natural populations of eubacteria were 11.8 cells predator-1 h-1 and 1.228 d-1, respectively. Therefore, euglenophytes could potentially have a considerable grazing impact on marine bacterial populations
... Moreover, whether these cryptophytes are able to feed on any other prey items such as cyanobacteria or micro-algae were not investigated. Determination of the presence or absence of a mixotrophic ability and in turn, the kind of prey that marine cryptophyte species consume are important in understanding certain evolutionary processes among photosynthetic organisms, i.e., their formation by secondary endosymbiosis and the link between red algae and dinoflagellates (Douglas and Penny, 1999;Petersen et al., 2006). The cryptophyte Teleaulax amphioxeia is one of the most wellknown marine species and has been observed in the coastal waters of many countries (Seppauml;lä and Balode, 1999;Cloern and Dufford, 2005;Novarino, 2005;Peter and Sommer, 2012;Johnson et al., 2016). ...
Article
Cryptophytes are ubiquitous and one of the major phototrophic components in marine plankton communities. They often cause red tides in the waters of many countries. Understanding the bloom dynamics of cryptophytes is, therefore, of great importance. A critical step in this understanding is unveiling their trophic modes. Prior to this study, several freshwater cryptophyte species and marine Cryptomonas sp. and Geminifera cryophila were revealed to be mixotrophic. The trophic mode of the common marine cryptophyte species, Teleaulax amphioxeia has not been investigated yet. Thus, to explore the mixotrophic ability of T. amphioxeia by assessing the types of prey species that this species is able to feed on, the protoplasms of T. amphioxeia cells were carefully examined under an epifluorescence microscope and a transmission electron microscope after adding each of the diverse prey species. Furthermore, T. amphioxeia ingestion rates were measured as a function of prey concentration, i.e., of heterotrophic bacteria and the cyanobacterium Synechococcus sp. Moreover, the feeding of natural populations of cryptophytes on natural populations of heterotrophic bacteria was assessed in Masan Bay in April, 2006. This study reported for the first time, to our knowledge, that T. amphioxeia is a mixotrophic species. Among the prey organisms offered, T. amphioxeia fed only on heterotrophic bacteria and Synechococcus sp. The ingestion rates of T. amphioxeia on heterotrophic bacteria or Synechococcus sp. rapidly increased with increasing prey concentrations up to 8.6 × 106 cells ml-1, but slowly at higher prey concentrations. The maximum prey ingestion rates of T. amphioxeia on heterotrophic bacteria and Synechococcus sp. reached 0.7 and 0.3 cells predator-1 h-1, respectively. During the field experiments, the ingestion rates and grazing impact of cryptophytes on natural populations of heterotrophic bacteria were 0.3 – 8.3 cells predator-1 h-1 and 0.012 – 0.033 d-1, respectively. Marine cryptophytes, including T. amphioxeia, are known to be excellent prey species for many mixotrophic and heterotrophic dinoflagellates and ciliates. Cryptophytes, therefore, likely play important roles in marine food webs and may exert a considerable potential grazing impact on the populations of marine bacteria.
... The plastid-targeted proteins of prokaryotic origin notably contain large numbers of proteins associated with expression of the plastid genome, such as aminoacyl-tRNA synthetases (Dorrell et al., 2017). The green algal plastid-targeted proteins identified include fundamental components of plastid metabolism, such as components of the CBB (Calvin-Benson-Bassham) cycle (Li et al., 2006;Petersen, Teich, Brinkmann, & Cerff, 2006), chlorophyll synthesis (Cihlar, Fussy, Horak, & Obornik, 2016;Hunsperger, Randhawa, & Cattolico, 2015;Li et al., 2006), and at least five components of carotenoid biosynthesis (Coesel, Obornik, Varela, Falciatore, & Bowler, 2008;Frommolt et al., 2008). The genes of green algal origin have been proposed to form the footprints of a "cryptic plastid" of green origin acquired by the stramenopile host deep in its evolutionary history (Dorrell & Smith, 2011;Moustafa et al., 2009). ...
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The stramenopiles encompass an incredible diversity of organisms, including ecologically fundamental single-celled algae such as diatoms, giant macroalgae such as kelps, as well as photo-mixotrophic and heterotrophic species. The photosynthetic species possess plastids of secondary or higher red algal origin. The diversity of stramenopile species provides an ideal system for exploring the fundamental features underpinning plastid establishment in eukaryotes, and also how plastid metabolism has diversified following endosymbiosis. In this chapter, we present an overview of stramenopile diversity and explore the chimeric origins of the stramenopile plastid, which utilises a combination of pathways derived from red algae and other sources to support its function. Next, we discuss unusual features of stramenopile plastid metabolism, some of which, responses to acute nutrient limitation and metabolic crosstalk with the mitochondria, may be specific to the diatoms and underpin their relative success in the contemporary ocean. Finally, we discuss even more dramatic transitions in the evolutionary history and life strategies of individual stramenopile groups, including evidence that stramenopiles may have given rise to some of the other major plastid lineages observed today, such as those of haptophytes and dinoflagellates, thus majorly contributing to the spread of photosynthesis through the tree of life.
... This means that the ultimate chromist ancestor was photosynthetic and that the multitude of non-autotrophic forms represents derived states that were repeatedly incurred in various divergent lineages. Despite the fact that the plastid of chromists sensu latu is generally accepted to be red algal in origin, analysis of phosphoribulokinase genes in chromalveolate representatives showed them to have green algal affiliation (Petersen et al., 2006). In addition, a complete genome analysis of certain diatoms revealed a far greater amount (470%) of green algal than red algal genes (Moustafa et al., 2009). ...
... This means that the ultimate chromist ancestor was photosynthetic and that the multitude of non-autotrophic forms represents derived states that were repeatedly incurred in various divergent lineages. Despite the fact that the plastid of chromists sensu latu is generally accepted to be red algal in origin, analysis of phosphoribulokinase genes in chromalveolate representatives showed them to have green algal affiliation (Petersen et al., 2006). In addition, a complete genome analysis of certain diatoms revealed a far greater amount (470%) of green algal than red algal genes (Moustafa et al., 2009). ...
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Microalgae have been attracting increasing attention as a renewable energy source and feedstock because of their potential for use in the production of bio-based fuels and materials. In this chapter, we provide an overview of bioproduction based on microalgae species. Specifically, we describe the taxonomic distribution of major industrially exploited microalgae species and highlight their utilities and recent advances. We also introduce recent advances in breeding and engineering techniques to improve the productivity of microalgae to enhance their biomass use.
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The phylogenetic relationships of the "golden algae", like all algae, were rarely addressed before the advent of electron microscopy because, based upon light microscopy, each group was so distinct that shared characters were not apparent. Electron microscopy has provided many new characters that have initiated phylogenetic discussions about the relationships among the "golden algae". Consequently, new taxa have been described or old ones revised, many of which now include non-algal protists and fungi. The haptophytes were first placed in the class Chrysophyceae but ultrastructural data have provided evidence to classify them separately. Molecular studies have greatly enhanced phylogenetic analyses based on morphology and have led to the description of additional new taxa. We took available nucleotide sequence data for the nuclear-encoded SSU rRNA, fucoxanthin/chlorophyll photosystem I/II, and actin genes and the plastid-encoded SSU rRNA, tufA, and rbcL genes and analysed these to evaluate phylogenetic relationships among the "golden algae", viz., the Haptophyceae (= Prymnesiophyceae) and the heterokont chromophytes (also known as chromophytes, heterokont algae, autotrophic stramenopiles). Using molecular clock calculations, we estimated the average and earliest probable time of origin of these two groups and their plastids. The origin of the haptophyte host-cell lineages appears to be more ancient than the origin of its plastid, suggesting that an endosymbiotic origin of plastids occurred late in the evolutionary history of this group. The pigmented heterokonts (heterokont chromophytes) also arose later, following an endosymbiotic event that led to the transfer of photosynthetic capacity to their heterotrophic ancestors. Photosynthetic haptophytes and heterokont chromophytes both appear to have arisen at or shortly before the Permian-Triassic boundary. Our data support the hypothesis that the haptophyte and heterokont chromophyte plastids have independent origins (i.e., two separate secondary endosymbioses) even though their plastids are similar in structure and pigmentation. Present evidence is insufficient to evaluate conclusively the possible monophyletic relationship of the haptophyte and heterokont protist host cells, even though haptophytes lack tripartite flagellar hairs. The molecular data, albeit weak, consistently fail to present the heterokont chromophytes and haptophytes as monophyletic. Phylogenetic resolution among all classes of heterokont chromophytes remains elusive even though molecular evidence has established the phylogenetic alliance of some classes (e.g., Phaeophyceae and Xanthophyceae).
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The phylogenetic relationships of the “golden algae”, like all algae, were rarely addressed before the advent of electron microscopy because, based upon light microscopy, each group was so distinct that shared characters were not apparent. Electron microscopy has provided many new characters that have initiated phylogenetic discussions about the relationships among the “golden algae”. Consequently, new taxa have been described or old ones revised, many of which now include non-algal protists and fungi. The haptophytes were first placed in the class Chrysophyceae but ultrastructural data have provided evidence to classify them separately. Molecular studies have greatly enhanced phylogenetic analyses based on morphology and have led to the description of additional new taxa. We took available nucleotide sequence data for the nuclear-encoded SSU rRNA, fucoxanthin/ chlorophyll photosystem I/II, and actin genes and the plastid-encoded SSU rRNA, tufA, and rbcL genes and analysed these to evaluate phylogenetic relationships among the “golden algae”, viz., the Haptophyceae (= Prymnesiophyceae) and the heterokont chromophytes (also known as chromophytes, heterokont algae, autotrophic stramenopiles). Using molecular clock calculations, we estimated the average and earliest probable time of origin of these two groups and their plastids. The origin of the haptophyte host-cell lineages appears to be more ancient than the origin of its plastid, suggesting that an endosymbiotic origin of plastids occurred late in the evolutionary history of this group. The pigmented heterokonts (heterokont chromophytes) also arose later, following an endosymbiotic event that led to the transfer of photosynthetic capacity to their heterotrophic ancestors. Photosynthetic haptophytes and heterokont chromophytes both appear to have arisen at or shortly before the Permian-Triassic boundary. Our data support the hypothesis that the haptophyte and heterokont chromophyte plastids have independent origins (i.e., two separate secondary endosymbioses) even though their plastids are similar in structure and pigmentation. Present evidence is insufficient to evaluate conclusively the possible monophyletic relationship of the haptophyte and heterokont protist host cells, even though haptophytes lack tripartite flagellar hairs. The molecular data, albeit weak, consistently fail to present the heterokont chromophytes and haptophytes as monophyletic. Phylogenetic resolution among all classes of heterokont chromophytes remains elusive even though molecular evidence has established the phylogenetic alliance of some classes (e.g., Phaeophyceae and Xanthophyceae).
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Plastids (photosynthetic organelles of plants and algae) are known to have spread between eukaryotic lineages by secondary endosymbiosis, that is, by the uptake of a eukaryotic alga by another eukaryote. But the number of times this has taken place is controversial. This is particularly so in the case of eukaryotes with plastids derived from red algae, which are numerous and diverse. Despite their diversity, it has been suggested that all these eukaryotes share a recent common ancestor and that their plastids originated in a single endosymbiosis, the so-called "chromalveolate hypothesis." Here we describe a novel molecular character that supports the chromalveolate hypothesis. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic and Calvin cycle enzyme that exists as two nonhomologous types, class I and class II. Red algal plastid-targeted FBA is a class I enzyme related to homologues from plants and green algae, and it would be predicted that the plastid-targeted FBA from algae with red algal secondary endosymbionts should be related to this class I enzyme. However, we show that plastid-targeted FBA of heterokonts, cryptomonads, haptophytes, and dinoflagellates (all photosynthetic chromalveolates) are class II plastid-targeted enzymes, completely unlike those of red algal plastids. The chromalveolate enzymes form a strongly supported group in FBA phylogeny, and their common possession of this unexpected plastid characteristic provides new evidence for their close relationship and a common origin for their plastids.
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The endosymbiotic, cyanobacterial nature of plastids is clearly established, but several fundamental issues concerning the origin and early evolution of plastids remain unresolved. One key question is whether plastids are monophyletic (derived from a single cyanobacterial ancestor) or polyphyletic (derived from more than one ancestor). This issue is complicated by the presence in many photosynthetic eukaryotes of secondary plastids, acquired by ingestion of a eukaryote, itself already equipped with plastids, rather than by direct ingestion of a free-living cyanobacterium. A review of the phylogenetic evidence from plastid genes indicates that the three major lineages of primary plastids (red, green, and glaucocystophyte) are probably monophyletic. Mitochondrial data further support this conclusion for red and green plastids (but are unavailable for glaucocystophytes), while nuclear data are largely unresolved. If plastids are monophyletic, then the pigment diversity of plastids must postdate their status as endosymbiotic organelles, but whether this diversity arose primarily by acquisition or loss is nuclear. Secondary endosymbiosis has greatly multiplied the variety of photosynthetic eukaryotes. A secondary origin of plastids is unequivocal for cryptomonads and chlorarachniophytes, is likely for heterokonts, haptophytes, and euglenophytes, and is suggested for the nonphotosynthetic parasites of phylum Apicomplexa. The remarkable plastid diversity of dinoflagellates appears to be the result of multiple secondary and tertiary endosymbiotic events. A consistent feature of all plastid genomes is extreme reduction relative to their cyanobacterial progenitors via outright gene loss, transfer of genes to the nuclear genome, and substitution by genes of nuclear ancestry. Most of this reduction seems to have occurred relatively soon after primary endosymbiosis, before the emergence of the major lineages of plastids, yet recent data also reveal surprising diversity of gene content among these lineages. The rubisco genes (rbcLS) of primary plastids on the red lineage are not related to those of cyanobacteria and seem to have been acquired via horizontal gene transfer.
Chapter
Chlorarachniophyte algae contain a complex, multi-membraned chloroplast derived from the endosymbiosis of a eukaryotic alga. Phylogenetic trees indicate that the host is closely related to filose amoebae and sarcomonads whereas the endosymbiont is most closely related to green algae. The endosymbiont is greatly reduced retaining only the plastid, plasmamembrane, a modicum of cytoplasm, and the nucleus. The vestigial nucleus of the endosymbiont, called the nucleomorph, contains three small linear chromosomes with a haploid genome size of 380 kb and is the smallest known eukaryotic genome. The overall gene organisation of the nucleomorph genome is extraordinarily compact making this a unique model for eukaryotic genomics.
Chapter
The endosymbiotic, cyanobacterial nature of plastids is clearly established, but several fundamental issues concerning the origin and early evolution of plastids remain unresolved. One key question is whether plastids are monophyletic (derived from a single cyanobacterial ancestor) or polyphyletic (derived from more than one ancestor). This issue is complicated by the presence in many photosynthetic eukaryotes of secondary plastids, acquired by ingestion of a eukaryote, itself already equipped with plastids, rather than by direct ingestion of a free-living cyanobacterium. A review of the phylogenetic evidence from plastid genes indicates that the three major lineages of primary plastids (red, green, and glaucocystophyte) are probably monophyletic. Mitochondrial data further support this conclusion for red and green plastids (but are unavailable for glaucocystophytes), while nuclear data are largely unresolved. If plastids are monophyletic, then the pigment diversity of plastids must postdate their status as endosymbiotic organelles, but whether this diversity arose primarily by acquisition or loss is nuclear. Secondary endosymbiosis has greatly multiplied the variety of photosynthetic eukaryotes. A secondary origin of plastids is unequivocal for cryptomonads and chlorarachniophytes, is likely for heterokonts, haptophytes, and euglenophytes, and is suggested for the nonphotosynthetic parasites of phylum Apicomplexa. The remarkable plastid diversity of dinoflagellates appears to be the result of multiple secondary and tertiary endosymbiotic events. A consistent feature of all plastid genomes is extreme reduction relative to their cyanobacterial progenitors via outright gene loss, transfer of genes to the nuclear genome, and substitution by genes of nuclear ancestry. Most of this reduction seems to have occurred relatively soon after primary endosymbiosis, before the emergence of the major lineages of plastids, yet recent data also reveal surprising diversity of gene content among these lineages. The rubisco genes (rbcLS) of primary plastids on the red lineage are not related to those of cyanobacteria and seem to have been acquired via horizontal gene transfer.
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We have analyzed DNA of Euglena gracilis for the presence of the unusual minor base β-d-glucosyl-hydroxymethyluracil or J, thus far only found in kinetoplastid flagellates and in Diplonema. Using antibodies specific for J and post-labeling of DNA digests followed by two-dimensional thin-layer chromatography of labeled nucleotides, we show that ~0.2 mole percent of Euglena DNA consists of J, an amount similar to that found in DNA of Trypanosoma brucei. By staining permeabilized Euglena cells with anti-J antibodies, we show that J is rather uniformly distributed in the Euglena nucleus, and does not co-localize to a substantial extent with (GGGTTA)n repeats, the putative telomeric repeats of Euglena. Hence, most of J in Euglena appears to be non-telomeric. Our results add to the existing evidence for a close phylogenetic relation between kinetoplastids and euglenids.