High-Throughput Sequencing of Six Bamboo Chloroplast Genomes: Phylogenetic Implications for Temperate Woody Bamboos (Poaceae: Bambusoideae)
Li D-Z (2011) High-Throughput Sequencing of Six Bamboo Chloroplast Genomes: Phylogenetic Implications for Temperate Woody
Bamboos (Poaceae: Bambusoideae). PLoS ONE 6(5): e20596. doi:10.1371/journal.pone.0020596
High-Throughput Sequencing of Six Bamboo Chloroplast Genomes: Phylogenetic Implications for Temperate Woody Bamboos (Poaceae: Bambusoideae)
Yun-Jie Zhang 0
Peng-Fei Ma 0
De-Zhu Li 0
Art F. Y. Poon, British Columbia Centre for Excellence in HIV/AIDS, Canada
0 1 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences , Kunming, Yunnan , People's Republic of China, 2 Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences , Kunming, Yunnan , People's Republic of China, 3 Graduate University of Chinese Academy of Sciences , Beijing , People's Republic of China
Background: Bambusoideae is the only subfamily that contains woody members in the grass family, Poaceae. In phylogenetic analyses, Bambusoideae, Pooideae and Ehrhartoideae formed the BEP clade, yet the internal relationships of this clade are controversial. The distinctive life history (infrequent flowering and predominance of asexual reproduction) of woody bamboos makes them an interesting but taxonomically difficult group. Phylogenetic analyses based on large DNA fragments could only provide a moderate resolution of woody bamboo relationships, although a robust phylogenetic tree is needed to elucidate their evolutionary history. Phylogenomics is an alternative choice for resolving difficult phylogenies. Methodology/Principal Findings: Here we present the complete nucleotide sequences of six woody bamboo chloroplast (cp) genomes using Illumina sequencing. These genomes are similar to those of other grasses and rather conservative in evolution. We constructed a phylogeny of Poaceae from 24 complete cp genomes including 21 grass species. Within the BEP clade, we found strong support for a sister relationship between Bambusoideae and Pooideae. In a substantial improvement over prior studies, all six nodes within Bambusoideae were supported with $0.95 posterior probability from Bayesian inference and 5/6 nodes resolved with 100% bootstrap support in maximum parsimony and maximum likelihood analyses. We found that repeats in the cp genome could provide phylogenetic information, while caution is needed when using indels in phylogenetic analyses based on few selected genes. We also identified relatively rapidly evolving cp genome regions that have the potential to be used for further phylogenetic study in Bambusoideae. Conclusions/Significance: The cp genome of Bambusoideae evolved slowly, and phylogenomics based on whole cp genome could be used to resolve major relationships within the subfamily. The difficulty in resolving the diversification among three clades of temperate woody bamboos, even with complete cp genome sequences, suggests that these lineages may have diverged very rapidly.
Funding: This project is supported by the Chinese Academy of Sciences through a project under the knowledge innovation program (KSCX-YW-N-029), National
Natural Science Foundation of China (no. 30990244) and a program of Innovation Teams of Yunnan Province, China. 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.
. These authors contributed equally to this work.
Bambusoideae is one of the largest subfamilies in the grass
family (Poaceae). Within Bambusoideae, woody bamboos are
distinguished from other species by their woody stems and by
infrequent sexual reproduction, with flowering intervals as long as
40 to 120 years . Woody bamboos are of notable economic
significance and have a long history of varied uses, ranging from
food to raw materials for furniture and housing around the world
[2,3]. They are primarily distributed in Asia, South America and
Africa, from lowlands up to about 4000 m in altitude. Many
species play important roles in their ecosystems, providing shelter
or food for many specialized and rare animal species (e.g. red
panda, mountain bongo) [4,5]. The best known may be the giant
panda of China, the country that is also home to the greatest
diversity of woody bamboos in the world, especially in the
Hengduan Mountain range of southwest China .
The grass family comprises about 11,000 species including the
most important agricultural crops, such as rice, wheat and corn,
and is one of the largest families in the angiosperms . According
to previous phylogenetic studies, Poaceae has been divided into
several basal lineages and two major lineages including the
PACMAD clade (Panicoideae, Arundinoideae, Chloridoideae,
Micrairoideae, Aristidoideae, and Danthonioideae) and the BEP
clade (Bambusoideae, Ehrhartoideae, and Pooideae) [8,9,10].
Within the BEP clade the relationships between Bambusoideae,
Ehrhartoideae, and Pooideae have long been controversial
[8,10,11]. Early work supported the sister relationship between
Bambusoideae+Ehrhartoideae and Pooideae , while others
suggested that Bambusoideae+Pooideae was sister to
Ehrhartoideae [10,11]. Bambusoideae encompasses approximately 8090
genera and 10001500 species [4,6,12] and has been resolved as a
monophyletic group, consisting of herbaceous and woody
bamboos . However, phylogenetic analyses have implied that
woody bamboos are not monophyletic and could be
geographically divided into temperate woody bamboos and tropical woody
bamboos, the latter having a sister relationship to herbaceous
bamboos [10,14]. Therefore, three tribes (Arundinarieae,
Bambuseae and Olyreae) were formulated in Bambusoideae .
Temperate woody bamboos (i.e., Arundinarieae) are highly
diverse in East Asia with varied habits and complex morphological
features [12,15]. They are notorious for being taxonomically
difficult and having a complicated taxonomy. Thus, a robust
phylogenetic tree is still needed for temperate woody bamboo
classification and elucidation of the evolutionary history of this
distinct lineage. Recent phylogenetic analyses based on multiple
chloroplast (cp) DNA regions have provided a moderate resolution
of the relationships within Arundinarieae, which was firstly
resolved to include six lineages  and then extended to ten
major lineages , i.e., Bergbamboes, the African alpine
bamboos, Chimonocalamus, the Shibataea clade, the Phyllostachys
clade, the Arundinaria clade, Thamnocalamus, Indocalamus wilsonii,
Gaoligonshania and Indocalamus sinicus. Although large DNA
sequence data (totaling 9,463 bp  and 12,943 bp ) were
used in the two studies, relationships among major lineages and
internal relationships within lineages remained unresolved.
Chloroplasts were derived from endosymbiosis between
independent living cyanobacteria and a non-photosynthetic host .
Each has its own genome that is usually nonrecombinant and
uniparentally inherited . Most higher-plant cp genomes have
conserved quadripartite structure, composed of two copies of a
large inverted repeat (IR) and two sections of unique DNA, which
are referred to as the large single copy regions and small single
copy regions (LSC and SSC, respectively) . Comparative
analysis of the cp genome architecture indicates that the gene
order and gene content are highly conserved in most cp genomes
. Cp-derived DNA sequences have been widely used for
phylogenetic studies of higher plants, and sometimes it is necessary
to use complete cp genome sequences for resolving complex
evolutionary relationships [21,22,23]. However, acquiring large
coverage of cp genomes has typically been limited by conventional
DNA sequencing. As next-generation sequencing techniques have
revolutionized DNA sequencing via high-throughput capabilities
and relatively low costs , it is now more convenient to obtain
cp genome sequences and extend gene-based phylogenetics to
While 18 complete cp genomes belonging to five Poaceae
subfamilies (Anomochlooideae, Bambusoideae, Ehrhartoideae,
Panicoideae and Pooideae) have been available in NCBI GenBank
(http://www.ncbi.nlm.nih.gov/genbank), no temperate woody
bamboo cp genome has been sequenced. To further elucidate
the phylogenetic relationships in the BEP clade and especially to
examine whether the complex evolutionary relationships of
temperate woody bamboos could be resolved by cp
phylogenomics, we completed six woody bamboo cp genomes using
nextgeneration Illumina sequencing-by-synthesis technology . The
six sequenced bamboos included one species of tropical woody
bamboos, or Bambuseae, and five species of temperate woody
bamboos, or Arundinarieae, which belong to three major lineages
according to a recent phylogenetic study . We then
investigated the evolutionary patterns of grass cp genomes with
an emphasis on Bambusoideae, and performed phylogenomic
analyses based on a data set composed of 24 complete cp genomes
in Poaceae. To date, this is the largest whole cp genome data set
used in grass phylogenetic inference and an initial attempt to
resolve the complex evolutionary relationships in Arundinarieae
using whole cp genomes.
Results and Discussion
Genome sequencing, assembly and four junctions
One tropical woody bamboo Bambusa emeiensis L. C. Chia & H.
L. Fung and five temperate woody bamboos Acidosasa purpurea
(Hsueh & T. P. Yi) P. C. Keng, Ferrocalamus rimosivaginus T. H.
Wen, Indocalamus longiauritus Handel-Mazzetti, Phyllostachys edulis
(Carrie`re) J. Houzeau, and Phyllostachys nigra var. henonis (Mitford)
Stapf ex Rendle were chosen for sequencing mainly due to their
economic significance (i.e., B. emeiensis and moso bamboo (P. edulis)
) and phylogenetic positions in recent studies [16,17].
According to Zeng et al. , I. longiauritus, P. edulis and P. nigra
var. henonis were in the Phyllostachys clade, and A. purpurea and F.
rimosivaginus belonged to the Arundinaria clade and the Shibataea
clade, respectively. Illumina paired-end (73 bp and 75 bp)
sequencing produced 225 Mb of data for each species. We aligned
1,594,119 paired-end reads of each species to the published cp
genome of Dendrocalamus latiflorus (FJ970916) , which was
chosen as a reference genome in our study with Bowtie software
. 198,446 (I. longiauritus) to 415,874 (F. rimosivaginus) paired-end
reads were mapped to the reference genome (Table 1). After de
novo and reference-guided assembly as in  with minor changes,
we obtained four complete cp genomes. The other two cp
genomes had only small gaps (two gaps per genome, averaging
372 bp according to the reference genome), which were then
finished by PCR sequencing. The summary of the assembled
contigs showing significant identity to the reference cp genome is
listed in Table 1. The N50 of contigs ranged from 607 bp to
1,560 bp and the summed length of contigs for all genomes ranged
from 101,015 bp to 118,081 bp. The mean coverage of the
genome was from 222.16 to 441.36. Successful recovery of these
genomes supposes that ,2006 sequence coverage sufficient for
assembly under this sequencing strategy and additional sequence
coverage does not improve it.
Four junction regions between IRs and SSC/LSC in each cp
genome were confirmed by PCR amplifications and Sanger
sequencing using primers (Table S1) designed on the basis of the
reference genome. The amplified sequences from six species
totaled 20,447 bp. We compared these sequences directly to the
assembled genomes, observing no nucleotide mismatches or indels.
This result also validated the accuracy of our genome sequencing
Genome features and sequence divergence
The determined nucleotide sequences of six cp genomes ranged
from 139,493 bp in B. emeiensis to 139,839 bp in P. nigra var. henonis
(Table 1). All six cp genomes showed a typical quadripartite
structure, consisting of a pair of IRs (21,79221,863 bp) separated
by the LSC (82,98883,273 bp) and SSC (12,71812,901 bp)
regions (Table 1). They encode an identical set of 131 genes with
the same gene order and gene clusters, of which 112 are unique
and 19 are duplicated in the IR regions (Figure 1). The 112 unique
genes include 4 ribosomal RNAs, 31 transfer RNAs and 77
protein-coding genes. Fifteen distinct genes (rps16, atpF, rpl16, rpl2,
ndhB, rps12, ndhA, petB, petD, trnK(UUU), trnG(UCC), trnL(UAA),
trnV(UAC), trnI(GAU), trnA(UGC)) contain one intron and only
one gene (ycf3) contains two. The cp genomes consist of 50.4% to
Aligned paired-end reads
Sum contigs length (bp)
50.7% coding regions, and the overall GC content is 38.9% for all
species except for A. purpurea (38.8%). Altogether, these six cp
genomes are highly conserved in each aspect of genome features,
such as gene content and gene order, intron and GC content.
The cp genomes of these six bamboo species are also very
similar in structure to those of other grasses. The grass cp genomes
have been under an elevated evolutionary rate in the common
grass ancestor  and are characterized by several structural
rearrangements like inversions  and gene loss [30,31,32,33].
Panicoideae, Pooideae and Ehrhartoideae have cp genomes with
average sizes of 140,766 bp, 135,686 bp and 134,509 bp,
respectively, and the cp genome of Bambusoideae averages the
second in size: 139,561 bp, with a narrow variation of 139,350 bp
to 139,839 bp. The grass cp genomes are smaller than those of
other monocots (which average 155,410 bp based on seven
representative species). The decrease in grass cp genome size
was partially due to the loss of genes such as accD, ycf1, and ycf2,
and the elimination of introns from clpP and rpoC1 [30,31,32,33].
Like other grasses, we found that these six bamboo cp genomes
also lack these genes and introns. Aside from these sequence
contractions, a unique insertion of ,400 bp in rpoC2 was observed
in Poaceae . However, the insertion in rpoC2 of F. rimosivaginus
is only 93 bp compared with tobacco , making it the shortest
documented rpoC2 gene (4,230 bp) of Poaceae (Table S2).
Therefore, we speculate that deletions may have occurred
following an initial insertion during the evolution of rpoC2 in F.
The gene order of grass cp genomes is distinct from that of
standard angiosperm cp genomes due to three typical inversions
, which also exist in these six bamboo cp genomes. The
junction positions between IRs and single copy regions often
change among various plants due to IR contraction and expansion
, and in Poaceae the termini of two genes, ndhH and ndhF,
have migrated repeatedly into and out of the adjacent IRs .
Nevertheless, the junctions are nearly identical in all six cp
genomes with ndhH extending 172195 nucleotides (data not
shown) into the IR, and ndhF confined to the SSC region, just like
most species in the BEP clade .
The genetic divergence is very low among cp genomes of
Bambusoideae. After alignment of our six cp genomes and two
other published cp genomes in Bambuseae, Bambusa oldhamii
(FJ970915) and D. latiflorus , we plotted sequence identity using
VISTA  with D. latiflorus as a reference (Figure 2). The whole
aligned sequences show high similarities with only a few regions
sequence identities falling below 90%, suggesting that bamboo cp
genomes are rather conservative. As expected, the IRs and coding
regions are more conserved than single copy and noncoding
regions, respectively. The rpoC2 gene is an exception, with lower
sequence identity due to various indels, as also found in other
grasses [31,33]. One divergent hotspot region associated with a
tRNA cluster in LSC (trnS(UGA)-trnC(GCA)) region was identified
(Figure 2), and this divergent hotspot has also been described in
other grass cp genomes [30,33]. The average genetic divergence of
the eight bamboo species, estimated by p-distance, was only 0.009.
The p-distance between Arundinarieae and Bambuseae, however,
was 0.014, a much larger divergence than that within tribes (0.002
for Arundinarieae and 0.003 for Bambuseae). These values
indicate that the majority of the extremely low sequence
divergence in Bambusoideae mainly prevails between tribes, and
that sequence divergence in Bambuseae is slightly larger than that
We divided repeats into three categories: dispersed, tandem and
palindromic repeats. For all repeat types, the minimal cut-off
identity between two copies was set to 90%. The minimal copy
size investigated were 30 bp for dispersed, 15 bp for tandem and
20 bp for palindromic repeats, respectively. In all, 228 repeats
were detected in six bamboo cp genomes (Table S3) using
REPuter . Manual verification of these identified repeats
revealed that some repeats were associated with two tRNA (e.g.
Figure 1. Gene map of the six woody bamboo chloroplast genomes. Genes shown outside the outer circle are transcribed clockwise and
those inside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. Dashed area in the inner circle
indicates the GC content of the chloroplast genome.
trnfM(CAU)) copies, or gene duplication (e.g. psaA/psaB), and these
repeats may simply be due to similarity of gene functions and thus
we classified them into another typetRNA or gene similarity
repeats (same procedure as was used in ). Tandem repeats,
accounting for 39.9% of total repeats, are the most common of the
four types (Figure 3C). The majority of repeats are located in
noncoding regions (Figure 3D), while some are found in genes
such as infA, rpoC2, rps18 and rps3. 82.7% of repeats range in size
between 15 bp and 40 bp (Figure 3A), although the defined
smallest size is 20 bp and 30 bp for palindromic and dispersed
repeats, respectively. The longest repeat is a dispersed repeat of
132 bp in B. emeiensis. Except for a 65 bp tandem repeat in P. nigra
var. henonis, all other tandem repeats are 45 bp or shorter, while
palindromic repeats occur in a narrower size range from 20 to
25 bp. Numbers of the four repeat types are similar among these
six cp genomes (Figure 3E) and their overall distribution in the cp
genome is highly conserved. Thus we investigated the repeats
shared between species using strict criteria. Repeats that had
identical lengths, and which were located in homologous regions
were defined as shared repeats. Under these criteria there were 14
repeats shared by all six bamboo species and 8 repeats shared by
the five woody temperate bamboos (excluding shared repeats with
B. emeiensis) (Figure 3B). B. emeiensis had the most unique repeats
(20), while P. edulis showed no unique repeats.
Repeat sequence may play a role in the rearrangement of cp
genomes and generating divergent regions via illegitimate
Figure 3. Repeat analyses. BE, B. emeiensis; FR, F. rimosivaginus; AP, A. purpurea; IL, I. longiauritus; PE, P. edulis; PN, P. nigra var. henonis. (A)
Histogram showing the number of repeats in the six woody bamboo chloroplast genomes. (B) Summary of shared repeats among six bamboos.
tRNA- or gene-similar repeats are excluded. (C) Composition of the 228 repeats from six bamboos. (D) Location of the 228 repeats from six bamboos.
Repeats that occurred in two regions were counted in both. (E) Histogram showing the number of four repeat types in each bamboo chloroplast
recombination and slipped-strand mispairing [31,40]. In our
sequenced genomes, divergent regions are often associated with
many repeats. For example, the rpoC2 gene contains various repeats.
However, the six cp genomes are perfectly syntenic and no significant
structural divergence was detected, hence we could not deduce
whether these repeats in bamboo cp genomes correlated with genome
rearrangement. Through the alignment of eight bamboo cp genome
sequences, we found only 12 small inversions (Table S4), which were
flanked by palindromic repeats. Five of the 12 inversions were
presumed synapomorphic inversions shared by all members from one
bamboo tribe, while all the others were probably homoplasious
inversions, judging by their random distribution in the eight bamboo
species. Therefore, we considered whether these small inversions may
provide conflicting phylogenetic information and thus should be
carefully treated when aligning sequences.
We compared the diverse repeats to determine if they could
provide phylogenetic information. Maximum parsimony (MP)
analyses of the identified repeats on the basis of their presence or
absence in the six woody bamboo cp genomes resulted in a single
most parsimonious tree with a length of 63 steps, a consistency
index (CI) of 0.968, and a retention index (RI) of 0.895 (Figure 4).
The resulting topology, with high bootstrap support (BS) values
($92%) was similar to phylogenetic trees based on DNA
sequences (see below). Thus, repeats in the cp genomes were
found to be as useful for phylogenetic reconstruction as other
genome characters such as gene content and gene order.
Four data partitions (whole cp genomes, protein coding genes,
the LSC region and the SSC region) (Table 2) from 24 grass cp
genomes were used to construct phylogenetic trees. The SSC
region had the highest percentage of parsimony informative
characters (PIs) with 13.83%. However, this data partition
contained the fewest PIs as the aligned sequence length was only
13,524 bp. Furthermore, SSC regions of bamboo cp genomes
contained a small inversion located in the rpl32-trnL (UAG)
intergenic spacer (Table S4) that had great influence on the
branching order of I. longiauritus, P. edulis and P. nigra var. henonis
(Figure S1). This inversion was considered to be of homoplasious
character because of its random distribution in Bambuseae and
Arundinarieae. However, the influence of the small inversion on a
phylogenetic tree based on complete cp genome sequences was
almost negligible. Therefore, in subsequent analysis we excluded
this inversion from the data partition of SSC region. Phylogenetic
trees with BS values and posterior probabilities (PP) based on the
four data partitions are presented in Figures 5 and 6. The
Bayesian, MP and maximum likelihood (ML) analyses yielded
similar trees in each data partition and phylogenetic trees of the
four data partitions were largely congruent with each other. The
topological differences occurred mainly within Ehrhartoideae.
Within this subfamily, three cultivated varieties of Oryza sativa
could form a monophyletic group only when using the data
partition of protein coding genes, whereas no BS or PP showed
significant support for this monophyletic relationship (Figure 6A).
The best resolution in phylogenetic relationships was achieved
using full cp genome sequences, thus we discuss the phylogenetic
relationships mainly based on Figure 5.
The BEP clade has historically been rather weakly supported
since it was first identified , and the relationships between
Bambusoideae, Pooideae and Ehrhartoideae have still not been
Figure 4. The most parsimonious tree obtained in maximum parsimony analyses of repeats in six bamboo species. Tree length is 63
steps. Consistency index is 0.968 and retention index is 0.895. Numbers at each node are bootstrap support values.
No. of nodes (BP.85%)
No. of nodes (BP.85%)
No. of nodes (PP.0.95)
Protein coding genes
Figure 5. Phylogenetic relationships of 24 Poaceae accessions as determined from whole chloroplast genomes. Support values are
shown for nodes as maximum parsimony bootstrap/maximum likelihood bootstrap/Bayesian inference posterior probability. Branch lengths were
calculated through Bayesian analysis, and scale bar denotes substitutions per site.
resolved. In this study, the BEP clade was resolved as a
monophyletic group with strong support (BS = 100%, PP = 0.98)
and within this clade Bambusoideae was revealed to be sister to
Pooideae (BS = 100%, PP = 0.99). This study was the first
successful attempt to provide well-supported relationships of the
three subfamilies in the BEP clade based on cp phylogenomic
analyses, and the results were consistent with recent phylogenetic
analyses based on selected cp DNA regions and broad sampling
[10,14]. Among these studies, Bouchenak-Khelladi et al. 
performed phylogenetic analyses based on a broad representation
of grass diversity, including 64 genera for the BEP clade. The
overall congruence between our study and Bouchenak-Khelladi et
al.  strengthens our confidence in the BEP clade relationships.
The phylogenetic tree inferred from ML and Bayesian using 43
putative orthologous cDNA sequences from nuclear genome also
had the same topology as this study, although the neighbor joining
method yielded different topology . However, further genomic
and taxon sampling, especially more taxa from Ehrhartoideae, are
deserved in further studies as phylogenomic analyses tends to
suffer from poor sampling .
Woody bamboos have long been considered to be a complex
and taxonomically difficult group because of their unique life
history, such as predominance of asexual reproduction and
infrequent flowering. Within Bambusoideae, Bambuseae and
Arundinarieae were well supported (BS = 100%, PP = 0.99) as
monophyletic. B. emeiensis was sister to B. oldhamii in Bambuseae
and this relationship was consistent with a recent phylogenetic tree
based on five cp DNA fragments , while it differs from
phylogenetic studies which included nuclear DNA sequences in
analyses [44,45]. Triplett and Clark  suggested that reticulate
evolution may be more significant in temperate woody bamboos
than previously suspected. Natural hybridization has been
reported in tropical woody bamboos as well . However,
whether the incongruence between phylogenies derived from cp
and nuclear DNA sequences was caused by hybrid events could
not be inferred from our study. Further taxon sampling and
nuclear DNA sequences will likely be necessary to explain the
Within Arundinarieae, the Phyllostachys clade was also supported
as in the previous studies [16,17], and further resolution was
achieved among the three species in the Phyllostachys clade whose
relationships had not previously been resolved . P. edulis and P.
nigra var. henonis were sister to I. longiauritus with strong support
(BS = 100%, PP = 0.98). However, the sister relationship between
P. edulis and P. nigra var. henonis was only supported by ML analyses
(BS = 95%) (Figure 6C) when using the SSC region data partition.
This data partition contained the shortest aligned sequence length
as well as the fewest PIs, which may have led to the unsatisfactory
phylogenetic resolution. From this result we concluded that
extremely low genetic divergence in Arundinarieae comprised
the main hindrance to the phylogenetic resolution of
Arundinarieae in traditional molecular phylogenetic studies. The
relationships between the Arundinaria, Shibataea and Phyllostachys clades were
unresolved in previous studies [16,17]. Despite our use of complete
cp genome sequences, the relationships between the Arundinaria
clade (A. purpurea), the Shibataea clade (F. rimosivaginus) and the
Phyllostachys clade were only supported by Bayesian analysis, which
placed the Arundinaria clade as sister to the Phyllostachys clade
(PP = 0.99). Considering the phylogenetic tree (Figure 4) based on
sequence repetition, the sister relationship between the Arundinaria
and Phyllostachys clade seems very likely. In previous cp
phylogenomic studies, it was suggested that even the entire cp
genome may be insufficient to fully resolve the rapidly radiating
lineages [22,23]. Poor resolution of the diversification among the
three clades may also be attributed to a rapid divergence early in
the evolutionary history of Arundinarieae. However, there were
only five species of Arundinarieae included in our study and
insufficient taxon sampling has been known to result in
unsatisfactory resolution as well. Therefore, more complete cp
genome sequences of Arundinarieae are necessary to confirm the
exact reason for poor resolution within the tribe. On the other
hand, the increase in phylogenetic resolution indicated that
phylogenomics based on complete cp genomes can be a useful
alternative choice to resolve the phylogeny of complex and
taxonomically difficult groups with a low rate of molecular
evolution, although its ability to completely resolve the phylogeny
of groups with complicated evolutionary history (i.e., involving
rapid radiation and reticulate evolution) can be limited.
Combining complete cp genome analysis of additional taxa with nuclear
DNA sequences may eventually elucidate the evolutionary history
of this distinct lineage.
Previous phylogenomic studies used common protein coding
genes [21,23]. In this study, the proportions of ingroup branches
with $95% support were reduced by using the protein coding
genes data partition and SSC region data partition, both of which
both contained fewer PIs than the other two data partitions
(Table 2). This result indicated that at least the LSC region was
needed to provide a good resolution of these sampled taxa. Whole
cp genomes which are perfectly collinear in Poaceae were proved
to be more effective than common protein coding genes in our
study, as evaluated by BS values and PP. Therefore, we suggest
that complete cp genomes, or even just the LSC region, could be
used for constructing the backbones of phylogenetic trees to
resolve the relationships of main clades, as well as for solving the
phylogenetic positions of some critical lineages.
Forty-five possibly informative exon indels were identified and
mapped to cp genome-based phylogenetic tree (Figure 7). Of
these, 25 indels mapped to monophyletic groups which have been
highly supported, and thus may be synapomorphies. The
remaining 20 indels may be homoplasies possibly associated with
parallel mutations or back mutations during evolutionary history.
There is not clear consensus about whether the indels should be
used for phylogenetic analyses [48,49], although most hesitancy
against them has been based on studies using one or several DNA
fragments. In our study, the 45 indels were located in 21 genes
(Table S5), and they were coded and subsequently added to the
protein coding gene matrix to perform MP analyses. Including
indel characters in the protein coding gene matrix did not change
the topology of the strict consensus tree, although it increased
three nodal support values (Figure S2). Thus, we inferred that the
influence of indels on our phylogenomic analyses on the basis of
large data sets could be neglected. Furthermore, genes in the cp
genome could contain both synapomorphic and homoplasious
indels and the proportion between synapomorphic indels and
homoplasious indels varied among different genes (Table S5). For
example, half of the indels in the ccsA gene were synapomorphic,
but three of four indels were homoplasious characters in rps18
gene. Therefore, small cp genome structural changes such as
indels should be carefully used in phylogenetic studies based only
on several DNA fragments. Mapping the indels to species whose
relationships have been well clarified could first exclude the
possible homoplasious indels and thus decrease the influence of
such homoplasious characters.
Molecular marker identification
Rates of molecular evolution are linked to life history in
flowering plants . Woody bamboos with rather long
generation times have been shown to have evolved relatively
slowly in the grass family . Since this low rate of molecular
evolution could complicate the phylogenetic study of
Bambusoideae, identifying rapidly evolving regions in bamboo cp genomes
through comparative genomics is critical. We found that
Pooideae accumulated more mutations in their cp genomes than
Bambusoideae and Panicoideae (Figure 8) as indicated by
percentage of variations (variation %). The number and
distribution pattern of variable characters in coding and
noncoding regions were rather different among Bambusoideae,
Pooideae and Panicoideae. For example, rpl32-trnL(UAG)
accumulated more variations than other noncoding regions in
Pooideae. However, it was not the most variable region (in terms
of variation percentage) in the other two subfamilies. As the
evolutionary pattern of each region is different in the three
subfamilies, it is more reasonable to select rapidly evolving
regions for phylogenetic studies specific to each subfamily.
In Bambusoideae, the proportion of variability in noncoding
regions ranged from 1.33% to 8.14% and the mean value was
3.86%, which was twice as much as in the coding regions (1.57%
on average). Correlation analysis revealed a significant positive
linear relationship between percentages of PIs and percentages of
variable sites in coding (R2 = 0.6671, P,0.001) and noncoding
(R2 = 0.7258, P,0.001) regions as expected, respectively
(Figure 9). Therefore, we choose the twenty most variable
noncoding regions as potential molecular markers for our
bamboo phylogenetic study. The variations of twenty noncoding
regions exceeded 4%, and 14 of them had a percentage of PIs
that exceeded 3% (Table S6). The 20 noncoding regions
identified in this study are listed here from high to low relative
genetic divergence: trnD(GUC)-psbM, ycf4-cemA,
trnG(UCC)trnT(GGU), ndhF-rpl32, rpl32-trnL(UAG), trnK(UUU)-rps16,
psbKpsbI, ycf3-trnS(GGA), trnT(UGU)-trnL(UAA), psbZ-trnfM(CAU),
rbcL-psaI, psaC-ndhE, trnT(GGU)-trnE(UUC), trnY(GUA)-trnD
(GUC), rps15-ndhF, trnL(UAA)-trnF(GAA), trnF(GAA)-ndhJ, rpl16
intron, psaI-ycf4, psaA-ycf3. Five of them are located in SSC region
(ndhF-rpl32, rpl32-trnL(UAG), psaC-ndhE, rpl16 intron) and
rps15ndhF is in the IRb-SSC junction. Among these regions,
rpl32trnL(UAG), trnK(UUU)-rps16, trnT(UGU)-trnL(UAA),
trnT(GGU)trnE(UUC), trnY(GUA)-trnD(GUC) and psaA-ycf3 have been used
in bamboo phylogenetic studies, and proved to be able to provide
relatively more informative characters. However, determining
whether the other 14 regions could be applied to bamboo
phylogenetic analyses requires further study.
In summary, here we completed six woody bamboo cp genomes
including one species of Bambuseae and five species of
Arundinarieae using Illumina sequencing-by-synthesis technology.
These finished cp genomes may facilitate the development of
biotechnological applications for these economically important
woody bamboos, and provide additional information about the
evolutionary history of the whole grass family. These cp genomes
are highly conserved relative to other sequenced grass cp genomes.
They possess several classes of repeat sequences, whose
distribution and types are highly similar between sequenced cp genomes,
which could provide phylogenetic information for resolving
evolutionary relationships. Phylogenomic analyses based on 24
complete cp genomes from the grass family provide strong support
for the placement of Bambusoideae in a sister relationship to
Pooideae. Furthermore, all the relationships within Bambusoideae
are well resolved with high BS and PP support, except for one
node showing support only from Bayesian inference. The
resolution decreased when using the relatively small SSC region
data set, indicating that extremely low genetic divergence is a
major hindrance for the phylogenetic resolution of Arundinarieae.
Thus, we have shown cp phylogenomics to be an efficient way for
resolving this difficult phylogeny. However, even using whole cp
genomes the relationships between the three clades of
Arundinarieae are only supported by Bayesian analysis, suggesting that
temperate woody bamboos may have diverged rapidly early in
their evolutionary history.
Materials and Methods
The six sampled species were grown in Kunming Botanical
Garden of the Kunming Institute of Botany among which F.
rimosivaginus and A. purpurea were introduced from Jinping County
of Yunnan Province, SW China in April and May 2008. The
voucher specimens for the six sampled bamboos were all deposited
at the Herbarium of Kunming Institute of Botany (KUN) and the
collectors and numbers are Zhang 08023 for A. purpurea, MPF
10170 for B. emeiensis, Zhang 08019 for F. rimosivaginus, MPF 10168
for I. longiauritus, MPF 10163 for P. edulis, and MPF 10172 for P.
nigra var. henonis. A field collection permit was obtained from the
Forestry Department of Yunnan Province, China (permit number
DNA Sequencing, genome assembly, and validation
We collected 50100 g of fresh leaves from each species for cp
DNA isolation using an improved extraction method that includes
high ionic strength buffer at low pH (3.60) buffer [20,52]. We used
5 mg of purified DNA for fragmentation by nebulization with
compressed nitrogen gas, and constructed short-insert (500 bp)
libraries following the manufacturers protocol (Illumina). DNA
Figure 8. Percentage of variable characters in homologous regions among chloroplast genomes of Panicoideae, Pooideae and
Bambusoideae. A) Coding region. B) Noncoding region. The homologous regions are oriented according to their locations in the chloroplast
from the different species was indexed by tags and pooled together
in one lane of Illuminas Genome Analyzer for sequencing at
Beijing Genomics Institute (BGI) in Shenzhen, China.
The raw sequence reads included non-cp DNA, and to
determine the proportion of cpDNA we mapped sequence reads
to the D. latiflorus cp genome using Bowtie with paired-end
alignment and a maximum of 3 mismatches (-v = 3). Subsequently,
three steps were used to assemble the cp genomes as in . First,
we assembled raw sequence reads into contigs with a minimum
length of 100 bp using SOAPdenovo  with an overlap length
of 31 bp. Second, contigs were aligned to the reference genome
using BLAST (http://blast.ncbi.nlm.nih.gov/), and aligned
contigs ($90% similarity and query coverage) were ordered according
to the reference genome. Third, gaps between the de novo contigs
were replaced with consensus sequences of raw reads mapped to
the reference genome.
Also based on the reference genome, we designed 5 and 4
primer pairs for closing gaps and verification of the four junctions
between the single-copy segments and IRs (primers in Table S1),
respectively. PCR products were sequenced using standard Sanger
protocols on ABI 3730 xl instruments. Sanger sequences and
assembled genomes were aligned using MEGA 4.0  to
determine if there were any differences.
Genome annotation and repeat analysis
Annotation of the sequenced genomes was performed using
DOGMA , coupled with manual selections for start and stop
codons and for intron/exon boundaries. We calculated the
average cp genome size of subfamilies in Poaceae on the basis of
the species listed in Table 3. We estimated the monocot mean cp
genome size based on Acorus calamus (AJ879453) , Dioscorea
elephantipes (EF380353) , Lemna minor (DQ400350) ,
Oncidium Gower Ramsey (GQ324949) , Phalaenopsis aphrodite
(AY916449) , Phoenix dactylifera (GU811709), and Typha
latifolia (GU195652) . The rpoC2 sequences from the grass
family were aligned to the rpoC2 sequences of tobacco by MEGA
4.0 to determine the insertion size in the gene. We downloaded B.
oldhamii and D. latiflorus cp genomes sequences from GenBank,
and multiple alignments of eight bamboos cp genomes were
made using MAFFT version 5 . Full alignments with
annotations were visualized using the VISTA viewer. The genetic
divergence represented by p-distance was calculated by MEGA
4.0 with species of Arundinarieae as one group and those of
Bambuseae as another.
We determined the three types of repeats, dispersed, tandem
and palindromic, by first applying the program REPuter and
then manually filtering the redundant output of REPuter. Gap
size between palindromic repeats was restricted to a maximal
length of 3 kb. Overlapping repeats were merged into one
repeat motif whenever possible. A given region in the genome
was designated as only one repeat type, and tandem repeat was
prior to dispersed repeat if one repeat motif could be identified
as both tandem and dispersed repeats. For coding, each repeat
present in a given genome was 1 and those absent were labeled
as 0. We performed MP analyses of this matrix using
PAUP*4.0b10  to implement exhaustive tree searches.
Non-parametric bootstrap analysis was conducted under 1,000
replicates with TBR branch swapping.
The six bamboo cp genome sequences, and nucleotide sequence
of the 18 publicly available grass cp genomes (Table 3) were
aligned using the program MAFFT version 5 and adjusted
manually where necessary. The unambiguously aligned DNA
sequences were used for phylogenetic tree construction. In order to
examine the phylogenetic utility of different regions, phylogenetic
analyses were performed based on the following data set: (1) the
complete cp DNA sequences; (2) a set of 74 common protein
coding genes; (3) the large single copy region; and (4) the small
single copy region. MP analyses were performed with
PAUP*4.0b10. Heuristic tree searches were conducted with
1,000 random-taxon-addition replicates and tree
bisection-reconnection (TBR) branch swapping, with multrees option in effect.
Non-parametric bootstrap analysis was conducted under 1,000
replicates with TBR branch swapping. Maximum likelihood (ML)
analyses were implemented in RAxML version 7.2.6 .
RAxML searches relied on the general time reversible (GTR)
model of nucleotide substitution with the gamma model of rate
heterogeneity. Non-parametric bootstrapping as implemented in
the fast bootstrap algorithm of RAxML used 1,000 replicates.
Bayesian analyses were performed using the program MrBayes
version 3.1.2 . The best-fitting models were determined using
the Akaike Information Criterion  as implemented in the
program Modeltest 3.7 . The Markov chain Monte Carlo
(MCMC) algorithm was run for 200,000 generations with trees
sampled every 10 generations for each data partition. The first
25% of trees from all runs were discarded as burn-in, and the
remaining trees were used to construct majority-rule consensus
tree. In all analyses, Anomochloa marantoideae was set as outgroup and
all gaps introduced by the alignment were excluded.
Exon indels were mapped onto the phylogenetic tree
determined by MP analyses of the whole cp genomes alignment
through parsimony mapping using Mesquite version 2.7
(Maddison and Maddison, http://mesquiteproject.org).
Molecular marker identification
To examine if the different cp genome regions evolved following
a unique pattern in each subfamily, both the coding and
noncoding regions longer than 200 bp were compared among
taxa from Panicoideae, Pooideae and Bambusoideae. For each
subfamily, homologous regions of cp genomes were aligned using
MAFFT version 5 and manual adjustments were made where
Saccharum officinarum cv. NCo310
Saccharum officinarum hybrid SP-80-3280
Hordeum vulgare subsp. Vulgare
Oryza sativa cv. 93-11
Oryza sativa cv. Nipponbare
Oryza sativa cv. PA64S
Phyllostachys nigra var. henonis
Morris and Duvall, 2010 
Leseberg and Duvall, 2009 
Asano et al., 2004 
Calsa et al., 2004 
Saski et al., 2007 
Maier et al., 1995 
Saski et al., 2007 
Bortiri et al., 2008 
Cahoon et al., 2010 
Saski et al., 2007 
Diekmann et al., 2008 
Ogihara et al., 2002 
Shahid et al., 2004 
Tang et al., 2004 
Hiratsuka et al., 1989 
Tang et al., 2004 
Wu et al., 2009 
Wu et al., 2009 
necessary. Subsequently, the percentage of variable characters for
each region in each subfamily was calculated. Because the aim was
to determine whether the evolution pattern of each region was
distinct in each subfamily, only numbers of nucleotide substitutions
were considered. Eight bamboo cp genomes were used to identify
rapidly evolving molecular markers which may be used for bamboo
phylogenetic studies. As the IR regions accumulate point mutations
more slowly than do the single copy regions, only fragments from
single copy regions were considered. Molecular fragments of coding
regions and noncoding regions longer than 350 bp were aligned
respectively. Because many indels in aligned sequences are
phylogenetically informative, they were scored here as well. Then
the proportion of mutational events (or variation %) for each coding
and noncoding region was calculated following the modified version
of the formula used in Gielly and Taberlet . The proportion of
mutation events = [(NS+ID)/L]6100, where NS = the number of
nucleotide substitutions, ID = the number of indels, L = the aligned
sequence length. As PIs are commonly used in phylogenetic
analyses, the proportion of PI characters was calculated as well.
Figure S1 Phylogenetic tree derived from analysis of the SSC
region (including the small inversion). Numbers at nodes indicate
bootstrap support (BP) values ($75%) from ML analyses and
posterior probability (PP) support values ($0.95) from Bayesian
inference. Branch lengths were calculated through Bayesian
analysis. The relationships in the box are different from those
resulting from the analysis based on SSC region but excluding the
Figure S2 Strict consensus tree of two parsimonious trees from
the analysis of protein coding genes (gaps were coded). Tree length
is 11,635 steps. Consistency index and retention index are 0.803
and 0.872, respectively. Numbers in nodes only show $75%
bootstrap support values, and asterisks indicate increased values
after adding gaps to the analysis.
Primers used for gap closure and junction verification.
Indels in exons of 21 genes in the grass chloroplast
We are indebted to Drs. Chun-Xia Zeng, Yu-Xiao Zhang and Zhen-Hua
Guo for various support, to Jun-Bo Yang, Prof. Song Ge and Zhi-Qiang
Wu for assistance with experiments, to Dr. Zachary Larson-Rabin for
critical reading of the manuscript.
Conceived and designed the experiments: D-ZL. Performed the
experiments: Y-JZ P-FM. Analyzed the data: Y-JZ P-FM. Contributed reagents/
materials/analysis tools: Y-JZ P-FM. Wrote the paper: Y-JZ P-FM D-ZL.
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