Cyanobacterial contribution to the genomes of the plastid-lacking protists.

Abstract

Eukaryotic genes with cyanobacterial ancestry in plastid-lacking protists have been regarded as important evolutionary markers implicating the presence of plastids in the early evolution of eukaryotes. Although recent genomic surveys demonstrated the presence of cyanobacterial and algal ancestry genes in the genomes of plastid-lacking protists, comparative analyses on the origin and distribution of those genes are still limited.
We identified 12 gene families with cyanobacterial ancestry in the genomes of a taxonomically wide range of plastid-lacking eukaryotes (Phytophthora [Chromalveolata], Naegleria [Excavata], Dictyostelium [Amoebozoa], Saccharomyces and Monosiga [Opisthokonta]) using a novel phylogenetic pipeline. The eukaryotic gene clades with cyanobacterial ancestry were mostly composed of genes from bikonts (Archaeplastida, Chromalveolata, Rhizaria and Excavata). We failed to find genes with cyanobacterial ancestry in Saccharomyces and Dictyostelium, except for a photorespiratory enzyme conserved among fungi. Meanwhile, we found several Monosiga genes with cyanobacterial ancestry, which were unrelated to other Opisthokonta genes.
Our data demonstrate that a considerable number of genes with cyanobacterial ancestry have contributed to the genome composition of the plastid-lacking protists, especially bikonts. The origins of those genes might be due to lateral gene transfer events, or an ancient primary or secondary endosymbiosis before the diversification of bikonts. Our data also show that all genes identified in this study constitute multi-gene families with punctate distribution among eukaryotes, suggesting that the transferred genes could have survived through rounds of gene family expansion and differential reduction.

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BioMed Central
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BMC Evolutionary Biology
Open Access
Research article
Cyanobacterial contribution to the genomes of the plastid-lacking
protists
Shinichiro Maruyama*1, Motomichi Matsuzaki1,2,3, Kazuharu Misawa1,3,3
and Hisayoshi Nozaki1
Address: 1Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan,
2Current address: Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-
0033, Japan and 3Current address: Research Program for Computational Science, Riken, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
Email: Shinichiro Maruyama* - maruyama@biol.s.u-tokyo.ac.jp; Motomichi Matsuzaki - mzaki@m.u-tokyo.ac.jp;
Kazuharu Misawa - kazumisawa@riken.jp; Hisayoshi Nozaki - nozaki@biol.s.u-tokyo.ac.jp
* Corresponding author
Abstract
Background: Eukaryotic genes with cyanobacterial ancestry in plastid-lacking protists have been
regarded as important evolutionary markers implicating the presence of plastids in the early
evolution of eukaryotes. Although recent genomic surveys demonstrated the presence of
cyanobacterial and algal ancestry genes in the genomes of plastid-lacking protists, comparative
analyses on the origin and distribution of those genes are still limited.
Results: We identified 12 gene families with cyanobacterial ancestry in the genomes of a
taxonomically wide range of plastid-lacking eukaryotes (Phytophthora [Chromalveolata], Naegleria
[Excavata], Dictyostelium [Amoebozoa], Saccharomyces and Monosiga [Opisthokonta]) using a novel
phylogenetic pipeline. The eukaryotic gene clades with cyanobacterial ancestry were mostly
composed of genes from bikonts (Archaeplastida, Chromalveolata, Rhizaria and Excavata). We
failed to find genes with cyanobacterial ancestry in Saccharomyces and Dictyostelium, except for a
photorespiratory enzyme conserved among fungi. Meanwhile, we found several Monosiga genes
with cyanobacterial ancestry, which were unrelated to other Opisthokonta genes.
Conclusion: Our data demonstrate that a considerable number of genes with cyanobacterial
ancestry have contributed to the genome composition of the plastid-lacking protists, especially
bikonts. The origins of those genes might be due to lateral gene transfer events, or an ancient
primary or secondary endosymbiosis before the diversification of bikonts. Our data also show that
all genes identified in this study constitute multi-gene families with punctate distribution among
eukaryotes, suggesting that the transferred genes could have survived through rounds of gene
family expansion and differential reduction.
Background
Cyanobacterial ancestors gave rise to plastids (chloro-
plasts) in the ancestor of a eukaryotic lineage. The birth of
the plastid had an impact on eukaryotic genome evolu-
tion, by way of endosymbiotic gene transfer (EGT), a par-
ticular form of lateral gene transfer (LGT) from
endosymbionts into the phylogenetically discontiguous
host genome [1]. Subsequently, an algal ancestor gave rise
Published: 11 August 2009
BMC Evolutionary Biology 2009, 9:197doi:10.1186/1471-2148-9-197
Received: 13 March 2009
Accepted: 11 August 2009
This article is available from: http://www.biomedcentral.com/1471-2148/9/197
© 2009 Maruyama et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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to secondary plastids in several punctate lineages of
eukaryotes. A number of these secondarily phototrophic
lineages lost their photosynthetic ability and further
diverged into secondarily heterotrophic, plastid-lacking
protists [2,3].
Although the position of the root of eukaryotes is still
uncertain, the presence of gene fusions and insertion/
deletion sequences in the marker genes have allowed us to
sort eukaryotes into at least three large groups;
Opisthokonta, Amoebozoa and bikonts (Archaeplastida,
Chromalveolata, Rhizaria and Excavata) [4-10] (Figure 1).
Most phototrophic eukaryotes harboring plastids derived
from primary endosymbiosis (primary plastids) are classi-
fied into the super-group Archaeplastida (i.e. glauco-
phytes, green plants and red algae) [10]. Although it is
widely accepted that primary plastids share a single origin
[[11-13], but see [14,15]] and the Archaeplastida are
monophyletic [[3,16], but see [17,18]], the evolutionary
history of the primary plastids is still debatable [19-21]. In
plastid-lacking protists, 'plastid imprints' can be exempli-
fied by genomic information, i.e. genes with affinity to
extant cyanobacterial or algal genes. These genes were sup-
posed to have originated from EGT events, and this
assumption should be affirmed by the resulting phyloge-
netic relationship between 'imprint' genes and the extant
relatives of the putative endosymbionts. The biggest chal-
lenge and the limitation of this 'imprint' searching process
is that the inevitable incompleteness of genome informa-
tion on lineages of interest and the ever-developing phyl-
A schematic representation of eukaryotic phylogeny
Figure 1
A schematic representation of eukaryotic phylogeny. The current consensus phylogeny and rooting of eukaryotes
based on previous studies [4,23,40]. Arrows and stars indicate plastid acquisition via endosymbiosis and alternative hypotheti-
cal time points which primary endosymbiosis occurred, respectively. The root of the eukaryotic tree on the unikonts/bikonts
boundary is hypothesized, but still controversial [5,7-9,19]. Archaeplastida are represented as a monophyletic group, but see
also [19].
Unikonts
Opisthokonta
Amoebozoa
Excavata
(Euglenida)
Rhizaria
(Chlorarachniophytes)
?
(Ciliates)
Chromalveolata
Stramenopiles
Alveolata
(Oomycetes)
Archaeplastida
(? Plantae sensu stricto)
Green plants
Red algae
Glaucophytes
Secondary endosymbiosis
Origin of
Eukaryotes
Bikonts
Primary
endosymbiosis
?
?

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ogenetic methodologies make it difficult to distinguish
EGT and ancient LGT [22]. Thus, although available
eukaryotic genome data are increasingly accumulating,
gene and genome phylogenies should be carefully inter-
preted to infer evolutionary scenarios.
Chromalveolata is a large taxonomic group of eukaryotes,
encompassing secondary phototrophs and secondarily
heterotrophic protists [10], and the 'chromalveolate
hypothesis' argues that this group originated from a com-
mon ancestor harboring the chlorophyll c-containing sec-
ondary plastid derived from a red alga (Figure 1) [23].
Among the secondarily heterotrophic chromalveolates,
several lineages have retained remnant chloroplasts for
non-photosynthetic metabolic pathways, e.g. apicoplasts
in apicomplexan parasites [24]. Recent genomic surveys
revealed the presence of plastid-derived genes, and further
suggested the presence of cryptic secondary plastids in
non-photosynthetic alveolate protists [25,26]. Further-
more, re-examination of the whole genome sequences
suggested the existence of algal genes in ciliates, another
plastid-lacking alveolate lineage, which could support the
photosynthetic ancestry of ciliates [27]. Oomycetes are
plastid-lacking stramenopiles, or chromists, classified into
Chromalveolata [10]. Although whole genome sequence
analysis showed that a number of genes with affinity to
photosynthetic organisms (cyanobacteria and algae) are
encoded in the nuclear genome, most of these 'plastid
imprints' candidates were only suggested by similarity
search and phylogenetic analyses have not yet led to fully
recovering the expected tree topology [28]. Considering
the uncertain phylogenetic affinity of the 'best hit' in sim-
ilarity search [29], reassessment of the genome informa-
tion is important to determine whether the evolutionary
history of oomycetes is comparable to ciliates [27].
One candidate of 'plastid imprints' in oomycetes has been
confirmed by studies reporting the phylogeny of gnd
genes, which encode 6-phosphogluconate dehydroge-
nase, showing that some plastid-lacking protists have
plant-like, cyanobacterium-derived gnd genes [20,21,30].
These analyses suggested that the gnd genes with cyano-
bacterial ancestry were acquired early in eukaryotic evolu-
tion, either via ancient eukaryote-to-eukaryote LGT, or
primary EGT that occurred earlier than had ever been
thought [21]. Additionally, the phylogeny of gnd genes
demonstrated that cyanobacterial genes are also present in
several Excavata protists, e.g. the heterolobosean amoebo-
flagellate Naegleria gruberi. Naegleria gruberi is a non-para-
sitic heterotrophic species related to N. fowleri, which is
the causative agent of primary amoebic meningoencepha-
litis in mammals [31]. Although the phylogenetic rela-
tionship within Excavata is still unclear, Heterolobosea,
together with Jakobida, is likely to be a sister group of
Euglenozoa [18,32].
To address how many genes have cyanobacterial ancestry
in plastid-lacking protists, and whether cyanobacterial
ancestry is limited to this gnd gene or also found in other
genes, we conducted a phylogenomic analysis using
genome sequence data of a taxonomically wide range of
plastid-lacking eukaryotes. Here we present a gene mining
study with a novel pipeline automatically producing and
summarizing one-by-one phylogenetic trees, and show
phylogenetic analyses of resultant candidate genes with
cyanobacterial ancestry, using the whole genome
sequence data from a wide range of eukaryotic lineages.
Results
To address how many genes are derived from cyanobacte-
ria in non-photosynthetic protists, we conducted cyano-
bacterial gene mining using the genome sequence data of
a wide range of the plastid-lacking eukaryotes (Additional
file 1). Using the whole genome data, we conducted
BLAST searches against all 'Bacteria' and selected queries
showing the highest similarity to genes in the available
cyanobacteria genome sequences. We then drew the
neighbor-joining (NJ) trees for genes showing homology
to cyanobacterial counterparts. After the first tree con-
struction step, we selected the gene trees where cyanobac-
teria and eukaryotes formed a monophyletic group
excluding other prokaryotes. As a result, we obtained a
shorter list of candidates, which we termed 'genes with
cyanobacterial affinity'. Subsequently we re-analyzed the
eukaryotic genes with cyanobacterial affinity by visually
checking and re-drawing the Bayesian and maximum like-
lihood (ML) trees after manually trimming operational
taxonomic units (OTUs). In total, we identified 12 plas-
tid-lacking protist genes 'with cyanobacterial ancestry' in
the genomes of the wide range of eukaryotes: two plastid-
lacking bikonts (the oomycete P. ramorum and the heter-
olobosean N. gruberi) and three unikonts (the slime mold
D. discoideum, the budding yeast S. cerevisiae and the cho-
anoflagellate M. brevicollis) (Table 1). These were the
eukaryotic genes with cyanobacterial ancestry that shared
the same origin with Archaeplastida and other eukaryotes.
They were placed within a monophyletic subclade mostly
composed of photosynthetic organisms (cyanobacteria
and plants/algae) and showed an apparent cyanobacterial
ancestry as far as was determined by tree topology (Table
1; Figures 2, 3, 4 and 5; and Additional files 2, 3, 4, 5, 6,
7, 8 and 9). We found another type of gene with cyano-
bacterial ancestry, which were the protist genes forming
monophyletic groups mostly with genes from extant
cyanobacteria (prokaryote-type genes with cyanobacterial
ancestry). Among a number of candidate genes found
through the first screening, we have presented three typi-
cal trees that were resolved with significant support values
(Additional files 10, 11 and 12). We postulate that these
prokaryote-type genes are remnants of the bacterium-to-
eukaryote LGT, which occurred 'recently' in evolution.

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Uroporphyrin III methyltransferase gene phylogeny showing the presence of genes with cyanobacterial ancestry in oomycetes
Figure 2
Uroporphyrin III methyltransferase gene phylogeny showing the presence of genes with cyanobacterial ances-
try in oomycetes. The MrBayes consensus tree with Bayesian posterior probabilities (BI) (70% or more) and maximum like-
lihood (ML) bootstrap support values (50% or more) is shown. Thick branches represent BI and ML values not lower than 100
and 95, respectively. Different phylogenetic affiliations are represented as follows: green, green plants; magenta, red algae; blue-
green, glaucophytes; orange, Chromalveolata; dark blue, Excavata; yellow, Rhizaria; gray, unikonts; sky blue, cyanobacteria.
Stars indicate plastid-lacking eukaryotes.
0.2
BI/ML
1.00
72
.82
--
.77
--
1.0
55
.97
--
.98
53
1.00
87
0.99
96
0.96
84
0.99
76
1.00
88
1.00
83
0.99
86
0.99
73
0.85
--
.72
--
0.85
--
1.00
63
0.63
65
0.70
--
0.93
70
1.00
75
0.98
71
0.84
--
1.00
98
Streptomyces avermitilis MA-4680 NP_827972
Methanocaldococcus jannaschii DSM 2661 NP_247960
Carboxydothermus hydrogenoformans Z-2901 YP_359632
Rubrobacter xylanophilus DSM 9941 YP_644736
Deinococcus geothermalis DSM 11300 YP_605594
Physcomitrella patens subsp. patens XP_001787048
Syntrophus aciditrophicus SB YP_460621
Desulfitobacterium hafniense Y51 YP_518457
Arcobacter butzleri RM4018 YP_001489309
Clostridium tetani E88 NP_781393
Methanosarcina acetivorans C2A NP_617926
Methanoculleus marisnigri JR1 YP_001046899
Synechococcus sp. BL107 ZP_01469115
Gloeobacter violaceus PCC 7421 NP_925526
Acaryochloris marina MBIC11017 YP_001518662
Cyanidium caldarium NP_045061
Cyanidioschyzon merolae CMV039C
Nitrosomonas eutropha C91 YP_747226
Alteromonas macleodii 'Deep ecotype' ZP_01110047
Gluconobacter oxydans 621H YP_192457
Emiliania huxleyi 213726
Volvox carteri 81921
Chlamydomonas reinhardtii 191432
Micromonas pusilla 22906
Micromonas sp. RCC299 87830
Ostreococcus tauri 4145
Ostreococcus lucimarinus 4668
Zea mays NP_001105724
Oryza sativa (japonica cultivar-group) NP_001043645
Vitis vinifera CAO15100
Arabidopsis thaliana NP_198901
Gloeobacter violaceus PCC 7421 NP_924180
Synechococcus elongatus PCC 6301 YP_171952
Acaryochloris marina MBIC11017 YP_001518945
Anabaena variabilis ATCC 29413 YP_323976
Cyanothece sp. CCY0110 ZP_01730125
Galdieria sulphuraria
Monosiga brevicollis MX1 XP_001742170
Pythium ultimum EL778029
Pythium oligandrum EV244605
Phytophthora ramorum 51635
Phytophthora sojae 109158
Volvox carteri 104902
Thalassiosira pseudonana
Micromonas pusilla 21172
Micromonas sp. RCC299 72207
Ostreococcus lucimarinus CCE9901 XP_001417831
Ostreococcus tauri 17331

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Cobalamin-independent methionine synthase genes in oomycetes are monophyletic with algal and cyanobacterial homologs
Figure 3
Cobalamin-independent methionine synthase genes in oomycetes are monophyletic with algal and cyanobac-
terial homologs. See legend for figure 2.
0.2
BI/ML
0.91
59
1.00
90
0.94
--
0.84
--
0.88
51
1.00
85
1.00
60
0.80
63
.99
65
1.00
79
1.00
89
0.98
72
0.92
59
1.00
100
Geobacillus sp. Y412MC10 ZP_03037227
Listeria monocytogenes str. 4b F2365 YP_014301
Bacillus halodurans C-125 NP_241304
Sulfurihydrogenibium sp. YO3AOP1 YP_001931535
Caminibacter mediatlanticus TB-2 ZP_01871179
Nitratiruptor sp. SB155-2 YP_001356355
Physcomitrella patens subsp. patens XP_001758527
Arabidopsis thaliana NP_197598
Oryza sativa Japonica Group NP_001067314
Arabidopsis thaliana NP_187028
Sclerotinia sclerotiorum 1980 XP_001588472
Coccidioides immitis RS XP_001244621
Aspergillus terreus NIH2624 XP_001214650
Neosartorya fischeri NRRL 181 XP_001267290
Parvibaculum lavamentivorans DS-1 YP_001411886
Crocosphaera watsonii WH 8501 ZP_00518984
Acaryochloris marina MBIC11017 YP_001522348
Saccharophagus degradans 2-40 YP_526112
Haemophilus somnus 2336 YP_001784615
Neisseria meningitidis 053442 YP_001599025
Methylococcus capsulatus str. Bath YP_114678
Ralstonia eutropha JMP134 YP_298442
Cyanidioschyzon merolae CMJ234C
Naegleria gruberi 59646
Bordetella bronchiseptica RB50 NP_888622
Serratia proteamaculans 568 YP_001476480
Klebsiella pneumoniae 342 YP_002241112
Escherichia coli 536 YP_671884
Lactococcus lactis subsp. lactis Il1403 NP_267411
Cyanothece sp. ATCC 51142 YP_001806235
Trichodesmium erythraeum IMS101 YP_720730
Thermosynechococcus elongatus BP-1 NP_681881
Arthrospira maxima CS-328 ZP_03272025
Synechococcus sp. JA-3-3Ab YP_473659
Phaeodactylum tricornutum 28056
Phytophthora ramorum 72019
Phytophthora sojae 108148
Galdieria sulphuraria
Chlamydomonas reinhardtii XP_001702934