The Bryopsis hypnoides Plastid Genome: Multimeric
Forms and Complete Nucleotide Sequence
Fang Lu ¨1,3., Wei Xu ¨2,3., Chao Tian1, Guangce Wang1*, Jiangfeng Niu1, Guanghua Pan4, Songnian Hu3
1Institute of Oceanology, The Chinese Academy of Sciences (IOCAS), Qingdao, China, 2Beijing Genomics Institute, The Chinese Academy of Sciences (BGICAS), Beijing,
China, 3Graduate University of the Chinese Academy of Sciences, Beijing, China, 4College of Marine Science and Engineering, Tianjin University of Science and
Technology, Tianjin, China
Background: Bryopsis hypnoides Lamouroux is a siphonous green alga, and its extruded protoplasm can aggregate
spontaneously in seawater and develop into mature individuals. The chloroplast of B. hypnoides is the biggest organelle in
the cell and shows strong autonomy. To better understand this organelle, we sequenced and analyzed the chloroplast
genome of this green alga.
Principal Findings: A total of 111 functional genes, including 69 potential protein-coding genes, 5 ribosomal RNA genes,
and 37 tRNA genes were identified. The genome size (153,429 bp), arrangement, and inverted-repeat (IR)-lacking structure
of the B. hypnoides chloroplast DNA (cpDNA) closely resembles that of Chlorella vulgaris. Furthermore, our cytogenomic
investigations using pulsed-field gel electrophoresis (PFGE) and southern blotting methods showed that the B. hypnoides
cpDNA had multimeric forms, including monomer, dimer, trimer, tetramer, and even higher multimers, which is similar to
the higher order organization observed previously for higher plant cpDNA. The relative amounts of the four multimeric
cpDNA forms were estimated to be about 1, 1/2, 1/4, and 1/8 based on molecular hybridization analysis. Phylogenetic
analyses based on a concatenated alignment of chloroplast protein sequences suggested that B. hypnoides is sister to all
Chlorophyceae and this placement received moderate support.
Conclusion: All of the results suggest that the autonomy of the chloroplasts of B. hypnoides has little to do with the size and
gene content of the cpDNA, and the IR-lacking structure of the chloroplasts indirectly demonstrated that the multimeric
molecules might result from the random cleavage and fusion of replication intermediates instead of recombinational
Citation: Lu ¨ F, Xu ¨ W, Tian C, Wang G, Niu J, et al. (2011) The Bryopsis hypnoides Plastid Genome: Multimeric Forms and Complete Nucleotide Sequence. PLoS
ONE 6(2): e14663. doi:10.1371/journal.pone.0014663
Editor: Shin-Han Shiu, Michigan State University, United States of America
Received May 18, 2010; Accepted January 11, 2011; Published February 14, 2011
Copyright: ? 2011 Lu ¨ et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by the National Natural Science Foundation of China (30970302, 40806063, and 30830015). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
The chloroplast of plants is thought to be descended from an
originally free-living cyanobacterium; most of the genes in the
genome of the cyanobacterium were transferred to the nucleus of
the host cell during the evolutionary transformation of the
endosymbiont into the chloroplast [1,2]. Nevertheless, the
chloroplast retained its own genome, which performs some
essential metabolic and biosynthetic pathways, such as photosyn-
thesis and amino acid biosynthesis .
Chloroplast genomes (cpDNA) were the first plant genomes to
be characterized because of their small size, limited number of
repeated elements, and abundance of foliar tissues. At present,
complete chloroplast genome sequences have been obtained from
virtually all of the major higher plant and algal lineages.
Comparative analyses of those complete cpDNA sequences not
only highlight considerable differences at the organizational level,
but also offer clarification of the evolutionary relationships among
the main groups of algae and higher plants [3–6].
Compared with higher plants, algal chloroplast genomes,
especially those of the green algae, exhibit numerous extreme
features . The cpDNA of Helicosporidium sp. , a parasitic, non-
photosynthetic green alga, is only 37.5 kb in size, which is the
smallest among the cpDNAs characterized, and it lacks all genes
for proteins that function in photosynthesis. The cpDNA size of
the siphonous alga Acetabularia sp. is more than 2000 kb, which is
the largest known cpDNA of photosynthetic organisms [1,9].
The antecedents of higher plants are thought to lie within the
green algal lineage [10,11], so algal plastid genomics offer useful
experimental guides for the higher plants. In the past few years,
studies of the algal chloroplast genome have been increasing. To
date, 14 complete chloroplast genomes have been sequenced for
representatives of the chlorophyte lineage: the prasinophytes
Nephroselmis olivacea , Ostreococcus tauri , Pyramimonas parkeae
, Pycnococcus provasolii , and Monomastix sp. OKE-1 ; the
trebouxiophytes Chlorella vulgaris  and Leptosira terrestris ; the
ulvophytes Pseudendoclonium akinetum , Oltmannsiellopsis viridis ,
and Helicosporidium sp. ; and the chlorophytes Chlamydomonas
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reinhardtii  Scenedesmus obliquus , Stigeoclonium helveticum ,
and Oedogonium cardiacum . Because the divergence order of
these lineages has remained contentious, more sequence data and
data from additional taxa are necessary.
In addition to cpDNA sequencing, many studies now are
focused on the organization of cpDNA with other elements, such
as subgenomic minicircular, plasmid-like molecules  and the
cpDNA conformation observation [23–27]. Using electron
microscopy, Kolodner and Tewari  found that instead of
only monomers, some dimers of cpDNA existed in plant cells.
Deng et al.  reported that cpDNA can exist in trimer and
tetramer form. Using in-gel procedures, including pulsed-field gel
electrophoresis (PFGE), restriction fragment mapping, and
fluorescence microscopy, Oldenburg and Bendich  found that
most of the cpDNA from maize seedlings was in linear or complex
branched forms rather than in circles. Multimeric forms of cpDNA
also have been found in brassicas using field-inversion gel
electrophoresis (FIGE)  and in tobacco using fluorescence
hybridization in situ (FISH) . In contrast, algal cpDNAs, like
their higher plant counterparts, have circular restriction maps, and
higher order organization (such as multimeric or anomalous forms)
has not been identified in algal cpDNAs to date .
Bryopsis sp., which is a siphonous green alga, is a unicellular
coenocytic giant cell. The total contents of the multinucleate cell
can be squeezed out, leaving only the cell membranes and walls
[29,30]. The extruded protoplasm without a cell membrane
maintains sufficient viability to regenerate into a mature individual
. The regenerated individual can grow up to 58 cm, which is
three times longer than the wild thalli , suggesting that
regenerated alga have an advantage over wild individuals in terms
of growth. Furthermore, the Bryopsis chloroplast is the largest
organelle in the cell and is thought to play an important role in
the protoplast regeneration process , thus we inferred that its
cpDNA may be special regarding the genome size and coding
gene compared with other algae. However, previous studies of
Bryopsis almost always have been focused on morphology and
mechanisms of regeneration, and the chloroplasts, especially the
cpDNAs, have received little attention.
Here we describe the complete sequence of the B. hypnoides
chloroplast genome and present chloroplast phylogenies based on
the genomic data currently available for higher plants and algae.
Additionally, we report for the first time the presence of
multimeric forms of cpDNA from B. hypnoides, a phenomenon
that previously was known only for higher plant cpDNAs.
Growth of the germinated aggregation of protoplasts of
The protoplasm extruded from wild B. hypnoides immediately
aggregated into numerous balls and fine strands when they were
mixed with natural seawater (Figure 1A). The aggregations were
covered with gelatinous envelopes within 3 min (Figure 1B). Some
of the aggregations germinated after 24 hours (Figure 1C–E), and
the germinated aggregations then developed into mature individ-
uals (Figure 1F). As in wild B. hypnoides, the mature regenerated
alga could also develop into the rhizoid, which was used as an
anchor, and the thallus.
Dimensions of chloroplasts in the B. hypnoides thallus
The size of chloroplasts in the B. hypnoides thallus varied from 3 to
16 mm,but most were 8–12 mm in length (Figure2A), suggesting that
the size diversity of the chloroplasts existed in the B. hypnoides thallus.
This result was confirmed by the results of sucrose density gradient
centrifugation (Figure 2B), in which five clear discrete green bands
appeared in the centrifugation tubes. Then all the bands were
recovered separately and observed under the light microscope. We
found that all the five bands were intact chloroplasts except for a few
cell debris; the chloroplasts in the five bands were of different sizes,
demonstrating the presence of five kinds of chloroplasts. When
Figure 1. Formation and germination of protoplasts from B. hypnoides. A: formation of aggregation of protoplasts within 1 min; B: formation
of a gelatinous envelope within 3 min; C–E: germination of the aggregation of B. hypnoides protoplasts; F: the germinated aggregation was
developed into a mature individual. Bars, 10 mm (A–E), 1cm (F).
The B. hypnoides plastid genome
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cultured under the same conditions, both the wild and the
regenerated algal thallus displayed the same results.
Structure and gene organization of the B. hypnoides
The B. hypnoides cpDNA sequence assembles into a circle of
153,429 bp; Figure 3 illustrates its gene map. Overall, the GC
content of the cpDNA is 33.1%, which is comparable with that of
Chlamydomonas (34.5%), Chlorella (31.6%), Pseudendoclonium (31.5%),
and Pyramimonas (34.7%). Like its C. sertularoides and C. fragile
homologs, the chloroplast genome of B. hypnoides does not contain
the inverted repeat that is commonly found in many chloroplast
genomes. A total of 111 functional genes, including 69 potential
protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes,
were identified (Table 1).In addition, 29 open reading frames(ORFs)
were identified with a threshold of 300 bp. All genes are present in a
single copy, and this gene content is typical for chlorophyte cpDNAs.
The sequence of B. hypnoides was most similar to that of C. vulgaris
when compared with other completely sequenced chlorophyte
cpDNAs. Table 2 compares the gene content of B. hypnoides cpDNA
with that of other Ulvophyceae, Trebouxiophyceae, and Chloro-
phyceae (UTC) algal cpDNAs that have been completely sequenced
to date. A common set of 84 genes is shared by these genomes.
Relative to ulvophytes (O. viridis and P. akinetum), seven protein genes
(chlI, minD, psaI, psaM, rpl19, ycf1, and ycf20) are absent from B.
hypnoides cpDNA. Two genes, cysA and cysT, that encode sulfate
transport proteins are absent in the ulvophytes O. viridis and P.
akinetum but are present in the trebouxiophytes C. vulgaris and L.
terrestris and in our B. hypnoides cpDNA.
In terms of gene organization, many derived gene clusters are
shared specifically between B. hypnoides, C. vulgaris, O. viridis, and P.
akinetum cpDNAs (i.e., rpl16-rpl14-rpl5-rps8-infA-rpl36-rps11-rpoA,
rps2-atpI-atpH-atpF-atpA, psbE-psbF-psbL-psbJ, and ccsA-chlL-chlN
(not in P. akinetum)), as are gene pairs (i.e., atpB-atpE, petB-petD,
rpl2-rps19, rps12-rps7, psbD-psbC, rpoC1-rpoC2, rps19-rps3, rps9-rpl12,
rps2-atpI, rpl20-rps18, psbK-ycf12, and psaA-psaB (not in P. akinetum))
as well as two ancestral gene pairs (psbB-psbT and rpl23-rpl2). The
gene pairs rps3-rpl16, rpoB-rpoC1, and tufA-rpl19 and the petA-petL-
petG cluster are missing from our B. hypnoides cpDNA.
Eleven introns in the B. hypnoides chloroplast are distributed
among ten genes, among which rrs exhibits two introns and atpA,
psaA, psbB, rbcL, rpl2, rpl5, rpl23, trnL-UAA and ycf3 each contain
one intron. These introns vary from 348 to 2466 bp in size.
According to their secondary structures, five introns belong to the
group I family , and three of these carry an internal ORF
encoding a putative LAGLIDADG homing endonuclease. The
intron of rpl2 is commonly present in the chloroplast genomes of
land plants, however it not found in the completely sequenced
chlorophyte cpDNAs. The introns of rpl5 and rpl23 in Bryopsis are
the first found in the known chloroplast genomes of Viridiplantae,
and blast searches of these two intron sequences against the
GenBank database failed to detect any homologous introns in
PFGE analysis of cpDNA
Figure 4A shows the results of PFGE analysis of cpDNAs of the
wild B. hypnoides. In the cpDNAs from the different types of
chloroplasts purified by sucrose density gradient centrifugation, at
least four clear bands can be seen in every lane, each
corresponding to a type of B. hypnoides chloroplast. The southern
hybridization with labeled probes of the rbcL gene showed all the
four bands were positive (Figure 5), suggesting that the four bands
represented all of the cpDNAs from B. hypnoides. The four bands
were located at 150 kb, 300 kb, 450 kb, and 600 kb, all of which
were in multiple relations. The relative amounts of the four bands
in PFGE, which corresponded to monomers, dimers, trimers, and
tetramers of the B. hypnoides cpDNAs, were estimated to be about
1, 1/2, 1/4, and 1/8 based on molecular hybridization.
Figure 4B shows the results of PFGE analysis of cpDNAs from
the regenerated B. hypnoides. These results are almost the same as
those shown in Figure 4A, which confirms that the cpDNAs from
the regenerated individual are similar to those of the wild alga.
Phylogenetic position of B. hypnoides chloroplasts
To elucidate the overall position of B. hypnoides in the plastid
phylogeny of algae/land plants, a global analysis was performed
using a subset of 31 taxa (see supplemental data) and 14,160
aligned characters. Thirty-one organisms were included as
representatives of algae and higher plants and included the green
lineage (streptophyte and chlorophyte lineage) and the non-green
lineage (red and chromist lineage). The best tree identified two
distinct lineages: the green lineage and the non-green lineage.
Figure 2. Dimensions of chloroplasts in the B. hypnoides thallus. (A) The size distribution of chloroplasts in Bryopsis hypnoides thallus as
inferred by optical microscope (ZEISS HBO 50, Germany). Ten fields of vision (6200) were selected randomly and all of the chloroplasts with different
sizes were determined with an eyepiece micrometer. (B) The chloroplasts separated by sucrose density gradient centrifugation from the wild B.
hypnoides. Numbers indicate the five discrete bands.
The B. hypnoides plastid genome
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Moreover, the chlorophytes and streptophytes formed two distinct
green lineages (Figure 5). Unexpectedly, the trees inferred with
ML and MP methods both identified a clade uniting B. hypnoides
and four complete sequenced members of the Chlorophyceae, this
clade received 90% and 62% bootstrap value in MP and ML
analyses, respectively. For other chlorophytes, N. olivacea represents
the first branch of the Chlorophyta, which is also reported in
recent phylogenetic analyses [6,12], but the branching order of the
other four Prasinophyceae (O. tauri, P. parkeae, P. provasolii and
Monomastix sp.) showed the different topology in the ML and MP
analyses. In ML tree, P. provasolii is sister to all other UTC algae
(bootstrap 51%); in the MP topology, it forms a moderately
supported clade with O. tauri, Monomastix sp. and P. parkeae. For the
UTC algae, the topological differences are also seen in the C.
vulgaris clade, which was clustered with two Ulvophyceae (O. viridis
and P. akinetum) in the MP tree, whereas in the ML tree, it located
at the basal position within the UTC algae.
The relationships observed for the non-green algae and strepto-
phytes taxa in the phylogeny are congruent with recently published
phylogenies based on whole chloroplast genome sequences [6,33].
Within the Streptophyta, the clade uniting Chlorokybus and Mesostigma
was placed basally and received strong bootstrap support. The other
Figure 3. Gene map of the B. hypnoides chloroplast genome. Position 0 is in the 12 o’clock position. The CDs of the genes are shown in blue,
tRNA genes are indicated as red, and genes of rRNA are shown in violet.
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four Charophyceae were clustered with two higher plants (N. tabacum
study, P. purpurea and G. tenuistipitate var. liui formed a strongly
supported lineage that is sister to the clade uniting the G. theta, R.
salina, O. sinensis and E. huxleyi. Two other red algae, C. merolae and C.
caldarium, robustly clustered in a separate clade.
The autonomy of the B. hypnoides chloroplast
Our previous studies showed that the chloroplasts of B. hypnoides
had strong autonomy for the following reasons: (1) The chloroplasts
in B. hypnoides had greater vitality than other organelles under
unfavorable conditions ; (2) chloroplasts purified by sucrose
density gradient centrifugation can aggregate into spheres, although
they cannot develop into mature individuals ; and (3) The
chloroplasts from siphonous algae possess great vitality and can
symbiotic association exists between siphonaceous green algae
(Acetabularia, Bryopsis, Caulerpa, and Codium) plastids and some marine
sea slug species that are highly specialized herbivores that feed on
siphonalean algae by puncturing the cells and sucking out the
contents. The chloroplasts of siphonous algae are not always
digested and can lodge in the body of the animal and conduct
photosynthesis for at least 3 months [36–41]. Moreover, genes
supporting photosynthesis have been acquired by the host animal
via horizontal gene transfer, and the encoded proteins are
retargeted to the chloroplast . In summary, compared with
chloroplasts of other algae and higher plants, B. hypnoides
chloroplasts have great vitality and independence. Because of these
extraordinary traits, we conducted research both on the genome
sequence and the conformation of the cpDNA.
Distinctive features of the B. hypnoides chloroplast
The genome size, arrangement, and IR-lacking structure of the
chloroplast genome of B. hypnoides more closely resemble that of C.
vulgaris cpDNA  than its O. viridis  and P. akinetum 
homologs. In addition to the 84 conserved genes that exist in all of
the completely sequenced UTC algal cpDNAs, B. hypnoides shares
an additional 14 genes with C. vulgaris cpDNA, compared to 12
and 10 genes with O. viridis and P. akinetum, respectively (Table 2).
Among the chlorophycean algal plastids investigated to date, the
inverted repeat is lost in the prasinophytes (Monomastix and Pycnococcus
); trebouxiophytes (Chlorella , Helicosporidium , and Leptosira
); ulvophytes (Caulerpa  and Codium ); and the chlorophyte
Stigeoclonium , suggesting that this ancestral character has been
independently lost in those lineages. Furthermore, considering the
cpDNAs identified previously, the chloroplast genome of the
Ulvophyceae likely has evolved under relaxed constraints .
The most notable feature of the B. hypnoides chloroplast genome
is that the rRNA locus consists of five genes: rrn23, rrn16, rrn7, rrn5,
and rrn3. The same situation can be found only in C. reinhardtii
cpDNA , as genes rrn7 and rrn3 are absent from all other
completely sequenced chlorophyte cpDNAs. Similar to C.
reinhardti, the rRNA gene cluster of B. hypnoides is arranged in the
order rrn16- rrn5- rrn23- rrn3-rrn7; however, the typical rRNA
operon in B. hypnoides has been broken in half and the SSU and
LSU genes are distributed at opposite ends of the gene map circle,
as is found in the ulvophytes C. sertularoides  and C. fragile ;
this might be an outcome of the loss of the inverted repeat.
Another surprising feature of the B. hypnoides chloroplast gene
repertoire is the presence of 10 unusual tRNA genes that have not
been found in other completely sequenced chlorophyte cpDNAs.
Five of them (trnA-AGC, trnE-CUC, trnI-AAU, trnV-CAC, and trnV-
AAC) correspond to those identified in some embryophyte cpDNAs,
whereas the other five (trnA-CGC, trnK-CUU, trnP-AGG, trnQ-CUG,
and trnT-AGU) have been found previously only in some bacterial
genomes. These unusual tRNA genes may not be essential for plastid
function in green algae or may not be functional genes; they also
might be involved in the special physiological functions of B. hypnoides.
Wolfe et al.  reported that the cp genome of Epifagus
virginiana, a plant with non-photosynthetic chloroplasts, encodes
Table 1. Genes contained in B. hypnoides chloroplast DNA.
Gene products Genes
Photosystem IpsaAb, B, C, J
Photosystem IIpsbA, Ba, C, D, E, F, H, I, J, K, L, M, N, T, Z
Cytochrome b6/f petA, B, D, G, L
ATP synthase atpAa, B, E, F, H, I
Chlorophyll biosynthesis chlB, L, N
Large subunit ribosomal proteinsrpl2b, 5b, 12, 14, 16, 20, 23b, 32, 36
Small subunit ribosomal proteinsrps2, 3, 4, 7, 8, 9, 11, 12, 14, 18, 19
RNA polymerase rpoA, B, C1, C2
Other proteinsaccD, ccsA, cemA, clpP, cysA, cysT
Proteins of unknown functionycf3b, 4, 12
Ribosomal RNAsrrn23, 16aa, 7, 5, 3
Transfer RNAstrnA(UGC), A(CGC), A(AGC), C(GCA), D(GUC), E(UUC), E(CUC), F(GAA), G(UCC), G(GCC), H(GUG), I(AAU), I(GAU),
K(CUU), K(UUU), L(CAA), L(UAA)a, L(UAG), M(CAU), N(GUU), P(UGG), P(AGG), Q(CUG), Q(UUG), R(ACG), R(CCU),
R(UCG), R(UCU), S(GCU), S(UGA), T(AGU), T(UGU), V(AAC), V(CAC), V(UAC), W(CCA), Y(GUA)
aGenes containing one- and two- group I introns.
bGenes containing group II introns.
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only 25 proteins because the photosynthetic machinery and the
corresponding genes are not needed. Glo ¨ckner et al.  identified
several genes unique to the cp genome of C. caldarium that correlate
to its special environmental conditions. Thus, it seems that loss or
gain of function is accompanied by changes of genes in the cp
genome. Herein we originally presumed that the size and gene
Table 2. Gene Content in B. hypnoides and Other UTC Algal cpDNAs.
GeneCv LtOv Pa By Cr So Sh Oc
Cv: Chlorella vulgaris, Lt: Leptosira terrestris, Ov: Oltmannsiellopsis viridis, Pa: Pseudendoclonium akinetum, By: Bryopsis hypnoide, Cr: Chlamydomonas reinhardtii,
So: Scenedesmus obliquus, Sh: Stigeoclonium helveticum, Oc: Oedogonium cardiacum. A filled/open circle denotes the presence/absence of a gene. Only the genes that
are missing in one or more genomes are indicated. A total of 84 genes are shared by all compared cpDNAs: atpA, B, E, F, H, I, cemA, clpP, petB, D, G, L, psaA, B, C, J, psbA, B,
C, D, E, F, H, I, J, K, L, M, N, T, Z, rbcL, rpl2, 5, 14, 16, 20, 23, 36, rpoA, B, C2, rps2, 3, 4, 7, 8, 9, 11, 12, 14, 18, 19, rrf, rrl, rrs, tufA, ycf3, 4, trnA(ugc), C(gca), D(guc), E(uuc), F(gaa),
G(gcc), G(ucc), H(gug), I(gau), K(uuu), L(uaa), L(uag), Me(cau), Mf(cau), N(guu), P(ugg), Q(uug), R(acg), R(ucu), S(gcu), S(uga), T(ugu), V(uac), W(cca), Y(gua).
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content of the chloroplast genome of B. hypnoides should be
extraordinary in order to support its autonomy. However, we
found that B. hypnoides possesses the usual chloroplast genome of
153 kbp and that the gene repertoire is typical for chlorophyte
cpDNAs. Thus, the autonomy of chloroplasts of B. hypnoides seems
to have little to do with the size and gene content of the cpDNA.
The multimeric forms conformation of B. hypnoides
Figure 4 shows that the cpDNA of B. hypnoides has multimeric
forms, including at least monomers, dimers, trimers, and
tetramers, and these are similar to the multimeric cpDNA forms
of higher plants, such as Arabidopsis, tobacco, and peas [24,26,47].
The five different sizes of B. hypnoides chloroplasts showed the same
cpDNA characteristics in multimeric forms, suggesting that the
relative amounts of the different forms of B. hypnoides cpDNA had
no relationship to chloroplast dimension.
Green alga is thought to have been the progenitor of higher plants
[10–12]; B. hypnoides is a green alga thus, its cpDNA conformation
may be similar to that of higher plants. However, a PFGE study of
the C. reinhardtii chloroplast genome did not reveal the higher order
organization that was observed previously for higher plant cpDNAs
. Our phylogenetic tree revealed that B. hypnoides is located in the
same clade as C. reinhardtii (Figure 5), whereas their cpDNA
conformations were quite different. This finding indicates that the
cpDNA of green algae is structurally plastic.
Lilly et al.  proposed that the formation of higher order
multimeric molecules may result from recombinational events or
the random cleavage and fusion of replication intermediates.
Recombinational events correlate to the presence of IRs, as
revealed in both Arabidopsis and tobacco [26,48]. Recombination
between IRs that occurs on two separate monomers results in a
dimer ; subsequent recombination events between molecules
presumably produce multimers. However, our study demonstrated
that multimeric forms of cpDNA also exist in the IR-lacking
chloroplast genome, which suggests that the higher order
organization of the chloroplast genome maybe less related to the
presence of IRs than was previously thought. Our results indirectly
support the alternative explanation that multimeric molecules
were produced by the random cleavage and fusion of replication
intermediates. However, this explanation requires confirmation
Similar conformation of cpDNA between wild and
regenerated B. hypnoides
Our previous studies showed that the regenerated alga had an
advantage over the wild individual in terms of growth and that
biochemical compositions differed between the wild and the
regenerated alga [30,34]. The regenerated alga can grow up to
58 cm in length, which is three times longer than the wild form
. Wang and Tseng  reported that when the regenerated
alga was on the decline, the organelles aggregated in the thallus
and then moved to the outside; next, one organelle aggregation
located outside of the thallus germinated and developed into a
mature alga. From this, we inferred that the DNA in the organelles
of the regenerated alga, especially the cpDNAs, underwent
changes during organelle aggregation and later development into
a mature individual. The cpDNAs from the regenerated B.
hypnoides were similar in size and conformation to the cpDNA of
the wild alga (Figure 4), and both exhibited monomeric, dimeric,
trimeric, and tetrameric forms of cpDNAs. Thus, the differences
between the wild alga and the regenerated individual probably
result from gene expression rather than alteration of the genome,
especially the cpDNAs.
Evolution of the chlorophycean chloroplast genome
The basal position of the Prasinophyceae in the Chlorophyta is
well established, but the branching order of the Ulvophyceae,
[5,51]. There are two hypotheses concerning the divergence order
of the UTC lineages: 1) phylogenetic inferences from cpDNA-
encoded proteins and genes favor the hypothesis that the
Ulvophyceae is a sister to the Trebouxiophyceae ; and 2)
chloroplast phylogenies inferred from gene order  and
mitochondrial phylogenies inferred from proteins or genes 
suggest that the Ulvophyceae share a sister relationship with the
Chlorophyceae. Our phylogenetic analyses of 42 chloroplast
proteins revealed that the Ulvophyceae clade I (O. viridis and P.
akinetum) is a sister to the Trebouxiophyceae I (C. vulgaris).
However, the B. hypnoides (Ulvophyceae II) chloroplast genome is
closely related to the Chlorophyceae. Although the B. hypnoides
chloroplast genome shares similarities with its O. viridis and P.
akinetum counterparts in terms of gene order, its IR-lacking
structure and gene content are quite different; we therefore
inferred that Ulvophyceae was non-monophyletic group, B.
hypnoides is located in the different phylogenetic lineage with O.
viridis and P. akinetum. Moreover, our phylogenetic analyses favor
the premise that the Trebouxiophyceae and Prasinophyceae were
also non-monophyletic groups, thus it was difficult to state a
precise taxonomic relationship among the UTC lineages in this
study. These different phylogenetic results may be caused by the
insufficient taxon sampling. So studies on additional chloroplast
genome data, especially from the UTC algae, will be very useful
for determining the phylogenetic relationships among the major
lineages of Chlorophyta.
Figure 4. PFGE showing multimeric forms of chloroplasts from
the wild (A) and the regenerated (B) B. hypnoides. (A) lane 1:
molecular standard marker; lane 2: cpDNAs from crude chloroplasts of
B. hypnoides without purification by sucrose density gradient centrifu-
gation; lanes 3–7: cpDNAs from different chloroplasts separated by
sucrose density gradient centrifugation; lane 8: the cpDNA hybridized
with the labeled probe of rbcL gene. (B) lane 1: molecular standard
marker; lanes 2–6: cpDNAs from different chloroplasts separated by
sucrose density gradient centrifugation (corresponding to the bands
1–5 in Fig. 3); lane 7: the cpDNA hybridized with the labeled probe of
The B. hypnoides plastid genome
PLoS ONE | www.plosone.org7February 2011 | Volume 6 | Issue 2 | e14663
Materials and Methods
Bryopsis hypnoides Lamouroux was collected from the intertidal
zone of Zhanqiao Pier, Qingdao, China (36u39N, 120u199E). The
fresh algae, which were rinsed with plenty of autoclaved seawater
and brushed with a soft brush to remove the surface microbial and
epiphytic organisms, were cultured in autoclaved sea water under
irradiance of 25 mmol m22? s21with a 16 h light: 8 h dark regime
at room temperature .
The aggregation of organelles in the protoplasm
Thalli of B. hypnoides were cut into small pieces and then placed
in eight layers of sterilized gauze to squeeze out the protoplasm.
The extruded protoplasm was mixed with an equal volume of
sterilized seawater (pH 8.3) and gently rocked. The organelles in
the protoplasm aggregated into spheres of different sizes, and the
aggregated spheres were cultured into mature B. hypnoides (the
regenerated B. hypnoides) under the culture conditions described
Chloroplast and cpDNA purification
The protoplasts squeezed from the wild B. hypnoides were added
to double volume ice-cold extraction buffer (400 mM sucrose,
50 mM Tris, 20 mM EDTA, 0.2% BSA, 0.2% b-mercaptoetha-
nol, pH 7.8) and then filtered through four layers of cheese cloth.
The filtrate was centrifuged at 800 g at 4uC for 10 min, and the
pellet was suspended in the extracted buffer. Most of the pellets
Figure 5. Phylogenetic position of B. hypnoides as inferred by ML analyses of 42 cpDNA-encoded proteins. Family-level affinities are
shown on the right of the diagram. Cyanophora paradoxa were used as outgroup. Numbers in each branch indicated maximum-likelihood (ML) /
maximum parsimony (MP) bootstrap values; and the dashed lines indicated the MP topologies which were different from the ML tree.
The B. hypnoides plastid genome
PLoS ONE | www.plosone.org8 February 2011 | Volume 6 | Issue 2 | e14663
were found to be chloroplasts under microscopic examination, so
they were layered onto the sucrose density gradient from 10% to
60%. The gradient was centrifuged at 150,000g for 90 min, and
five clear green bands appeared in the tube after centrifugation.
The bands were removed from the tube separately, and dialyzed
against the rinse buffer (400 mM sucrose, 50 mM Tris, 0.5%, BSA
pH 7.8) for 5 hours to remove sucrose, then were observed under
The purified chloroplasts were washed twice with the buffer
(50 mM Tris-HCl, pH 8.0, 25 mM EDTA), suspended in the lysis
buffer (50 mM Tris, pH 8.0, 25 mM EDTA, 2% SDS, 50 mg/ml
proteinase K), and incubated at 40uC for 3 hours with gently
shaking, following by being centrifuged at 10 000 g at 4uC for
15 min. The supernatant was extracted several times with phenol/
chloroform and then precipitated with cold ethanol. CsCl density
gradient ultracentrifugation was used for further purification of the
cpDNA. CsCl and Hoechst dye No. 33258 were added to the
crude cpDNA, and the mixture was centrifuged at 240,000g at
20uC for 38 hours with a Beckman Ti 80 rotor. cpDNA bands
were visualized under UV illumination and then recovered. The
cpDNA was precipitated and dissolved in TE buffer after both the
Hoechst dye 33258 and CsCl were removed. The cpDNA from
the regenerated B. hypnoides then was purified as described above.
The purified cpDNA was sheared by nebulization, and
1,50063,000 bp fragments were recovered by electroelution after
agarose gel electrophoresis. These fragments were treated with T4
DNA polymerase and cloned into the SmaI site of PUC18. After
transformation of electrocompetent E. coli TOP10 cells (Invitro-
gen, Carlsbad, CA), recombinant plasmids were isolated and
nucleotide sequences were determined with the PRISM dye
terminator cycle sequencing kit (Applied Biosystems, Foster City,
CA) on a DNA sequencer (model 373; Applied Biosystems) using
T3 and T7 primers. Sequencing data were accumulated to 106
coverage for all PCR fragments; remaining gaps were cloned by
PCR. The determined sequences were accumulated, trimmed,
aligned, and assembled using the Phred-Phrap (Phil Green,
University of Washington, Seattle, WA, USA) and Consed
programs . The fully annotated B. hypnoides chloroplast
genome sequence has been deposited in GenBank with accession
PFGE and southern blot analysis
The PFGE assay was performed by the method described by
Sambrook and Russell . Brifely, the purified chloroplasts were
mixed with an equal volume of low melting point agarose, which
was dissolved in rinse buffer containing 20 mM EDTA, and 90 ml
of the mixture was added into every pole of the mould (Bio-Rad,
Richmond, Calif), then solidified at 4uC. The solid gels were put
into the lysis buffer (0.01 M Tris, 0.45 M EDTA, pH 7.8, 2%
SLS, 10 mg/ml proteinase K) and incubated at 50uC for 40 h,
during which the buffer was changed twice. The gels then were
rinsed six times with TE (10 mM Tris, 1 mM EDTA, pH 8.0) and
then stored at 4uC. Pulsed-field gel electrophoresis (PFGE) was
performed on a Bio-Rad CHEF Mapper TM according to the
manufacturer’s instructions (CHEF Mapper TM and CHEF
Mapper XA Pulsed Field Electrophoresis Systems). After PFGE,
the gel was dyed with 0.5 mg/ml EB for 30 min, and the result was
observed by Pharmacia Biotech ImagemasterH VDS.
A DNA fragment encoding the B. hypnoides rbcL gene (1145 bp;
GenBank accession no. AY566304) was prepared from genome
DNA as described previously . The rbcL gene then was labeled
according to the directions provided in the Dig High Prime DNA
Labeling and Detection Starter Kit I (Roche, Germany) and used
as a probe for southern blot analysis. DNA in the gel was
denatured and transferred to the positively charged nylon
membrane (Osmonics, Westborough, MA, USA)  and cross-
linked under UV for 90 seconds, then hybridized with the probe
following the instructions for the Roche Kit.
Sequence and phylogenetic analyses of cpDNA
Genes were identified using Blast homology searches provided
by the National Center for Biotechnology Information (NCBI)
server (http://www.ncbi.nlm.nih.gov/BLAST/). Protein-coding
genes and positions of ORFs were determined using ORFFIN-
DER at NCBI. tRNA genes were annotated using the tRNAscan-
SE program . Intron boundaries were located by modeling
intron secondary structures [32,56] and by comparing the
sequences of intron-containing genes with those of intron-less
homologues using FRAMEALIGN of the Wisconsin package.
Circle graphs were generated using the CGView program .
Phylogenetic analysis was conducted using forty-two cp protein
sequences (atpA, atpB, atpE, atpF, atpH, petB, petD, petG, psaA, psaB,
psaC, psaJ, psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK,
psbN, psbT, psbZ, rpl2, rpl14, rpl16, rpl20, rpl36, rps2, rps3, rps4, rps7,
rps8, rps11, rps12, rps14, rps18, rps19, ycf3, and ycf4) from 31 algal/
land plant organisms (see Data S1). The concatenated protein
sequences were aligned using the multiple sequence alignment
tools in CLUSTAL X version 1.81 with the default settings .
The adjusted alignment after manual correction was used for
phylogenetic analyses by maximum likelihood (ML) and maxi-
mum parsimony (MP) methods. ML trees were computed with
PHYML 3.0  under the cpREV45+C+I model of amino acid
substitutions  and bootstrap support for each node was
calculated using 100 replicates. MP trees were calculated with
MEGA 4.0  by 1,000 bootstrap replications, which were
obtained usingthe Close-Neighbor-Interchange
MEGA 4.0 was used for visualization and printing of the trees.
the phylogenetic analyses
Found at: doi:10.1371/journal.pone.0014663.s001 (0.02 MB
Algal and land plant chloroplast genomes examined in
Conceived and designed the experiments: FL GW. Performed the
experiments: FL WX CT. Analyzed the data: FL WX GW. Contributed
reagents/materials/analysis tools: FL JN GP SH. Wrote the paper: FL.
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