全 文 ：药用植物华重楼(黑药花科)叶绿体全基因组研究
李晓娟1,2, 杨振艳1, 黄玉玲1,2, 纪运恒1*
(1. 中国科学院昆明植物研究所东亚植物多样性与生物地理学重点实验室，昆明 650201；2. 中国科学院大学，北京 100049)
摘要： 为探究华重楼(Paris polyphylla var. chinensis)的叶绿体基因组特征，利用叶绿体系统发育基因组学方法，对华重楼与其它
百合目植物的叶绿体全基因组进行了比较。结果表明，华重楼的叶绿体全基因组长158307 bp，由 4 个区组成，包括 2 个反向重
复区(IRA和IRB，27473 bp)、1 个小单拷贝区(SSC，18175 bp)和 1 个大单拷贝区(LSC，85187 bp)。其叶绿体基因组有115个
基因，包括81个编码蛋白质基因、30 个转运RNA基因和 4 个核糖体RNA基因。11 种百合目植物的叶绿体全基因组的基因组
成和基因顺序相似。华重楼的 cemA基因是假基因，其起始密码子后有多聚核苷酸 poly(A)及CA双核苷酸重复序列，编码序列
中出现多个终止密码子 , 且与北重楼(Paris verticillata)的cemA编码序列中的终止密码子位置不同。因此，华重楼叶绿体基因
组比较保守；cemA结构及假基因化现象可能具有重要的进化与系统发育信息，其编码序列中的终止密码子可以区分华重楼和
北重楼。
关键词： 叶绿体全基因组；华重楼；黑药花科；百合目；cemA 假基因化
doi: 10.11926/j.issn.1005–3395.2015.06.001
Complete Chloroplast Genome of the Medicinal Plant Paris polyphylla
var. chinensis (Melanthiaceae)
LI Xiao-juan1,2, YANG Zhen-yan1, HUANG Yu-ling1,2, JI Yun-heng1*
(1. Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming institute of Botany, Chinese Academy of Sciences, Kunming 650201,
China; 2. University of Chinese Academy of Sciences, Beijing 100049, China)
Abstract: In order to understand the characters of chloroplast genome (cp genome) in Paris polyphylla var.
chinensis, the chloroplast genome (cp genome) of P. polyphylla var. chinensis was compared with those of 10
species within Liliales by using phylogenomics methods based on complete chloroplast genomes. The results
showed that the cp genome of P. polyphylla var. chinensis was 158307 bp in length and display a typical
quadripartite structure including two inverted repeat regions (IRA and IRB, 27473 bp), one small single-
copy region (SSC, 18175 bp) and one large single-copy region (LSC, 85187 bp). It contained 115 unique
genes, including 81 protein-coding genes, 30 tRNAs and 4 rRNAs. The genome structure, gene contents and
arrangement of 10 Liliales species cp genomes were very similar. The cemA gene of P. polyphylla var. chinensis
was pseudogene with poly(A) and CA SSR patterns after the start codon, and the loci of premature stop codons
are different from those of Paris veticillata. In conclusion, the cp genome of P. polyphylla var. chinensis was
conservative. The cemA structure and pseudogenization might play an important role in the evolution and
phylogeny, and the location of the stop codons in cemA was useful for distinguishing P. polyphylla var. chinensis
from P. veticillata.
Key words: Complete chloroplast genome; Paris polyphylla var. chinensis; Melanthiaceae; Liliales; cemA
pseudogenization
热带亚热带植物学报　2015， 23(6)： 601 ~ 513
Journal of Tropical and Subtropical Botany
Received： 2015–05–12　　　　Accepted: 2015–05–27
This work was supported by the National Natural Science Foundation of China (Grant No. 30670132).
* Corresponding author. E-mail: jiyh@mail.kib.ac.cn
602 第23卷热带亚热带植物学报
The chloroplast (cp) genome in plants is about
120–160 kp in size. Because of its small genome
size and high copy numbers per cell, sequencing the
complete cpDNA genome is much more amenable
than the nuclear genome of plants[1–3]. Besides with
highly conserved gene content and order across
plant species, cp genome DNA sequences were
reported that it can provide useful information to
elucidate phylogenetic relationships among plant
taxa[4–5]. In addition, cp genome has a relatively lower
intraspecific and higher interspecific divergence than
nuclear genome, so that species identification can be
confirmed easily depending on whether a gene exist
in either of two species[6–7]. At this time, the number
of chloroplast genomes of green plants uploaded
to NCBI (http://www.ncbi.nlm.nih.gov/genomes/
GenomesGroup.cgi?taxid=2759&opt=plastid#page-
Top) has risen to 644, of which almost 496 angio-
sperms (including 139 monocots) have been available
until March 4th, 2015.
Paris polyphylla var. chinensis (Franch.) Hara.
is a perennial herb in the tribe Parideae of the family
Melanthiaceae, which is widespread in subtropical
China[8–10]. The plant (named “Chonglou” in Chinese)
is a traditional Chinese medicinal herb whose dried
rhizome is the main source of raw material for some
famous prepared Chinese medicines such as “Yunnan
Baiyao” and “Gong xue ning Capsules”, which are
used as haemostatic, antalgic and antipyretic[11–12].
More than 50 chemical compounds have been isolated
and identified from P. polyphylla var. chinensis to
date[13–15]. Modern pharmacological research has
demonstrated that steroid saponins (Diosgenin and
Pennogenin glycosides) are responsible for these
biological activities[15–17].
Although P. polyphylla var. chinensis has a great
economic and medicinal importance, no genomic
work on the plant has yet been performed. Recently,
three plastid genomes have been sequenced and
confirmed in the Melanthiaceae family, including
Veratrum patulum O. Loes[18] (tribe Melanthieae),
Chionographis japonica (Willd) Maxim[19] (tribe
Chionographideae) and its congener, Paris verticillata
M. Bieb[20]. The trnI_CAU triplication and cemA pseudo-
genization were found in the complete cp genomic
sequence of P. verticillata[20], which proposed a hypothesis
that these patterns would be useful for understanding
the phylogeny and evolution of these species by
comparing the plastid genome features among Liliales.
Here we reported the complete cp genome of P.
polyphylla var. chinensis, and contrasted it with
those of P. veticillata and other Liliales taxa. These
data will be helpful to understand the phylogenetic
relationships and evolutionary mechanism within
Parideae clade (Melanthiaceae), and to provide useful
molecular information for further research on this
important medicinal plant.
1 Material and methods
1.1 Taxon sampling, cpDNA extraction and
sequencing
In general, there are mainly three ways for
obtaining cpDNA genome sequence: the first, cpDNA
was isolated using low pH medium with high salt
method[21]; the second, amplifying cpDNA using short
range PCR primers for Sanger DNA sequencing[22–23];
the third, isolating total genomic DNA, constructing
DNA library and sequencing utilizing next-generation
sequencing[24–25]. However, these ways need large sums
of fresh leaves samples for sequencing and cpDNA
sequence obtained may be with considerable gaps. To
avoid these problems, we used the method proposed
by Yang et al[26] to amplify the whole cp genome of P.
polyphylla var. chinensis with nine universal primers.
The healthy, actively growing fresh leaves were
collected from P. polyphylla var. chinensis cultivated
in the green house of Kunming Institute of Botany,
Chinese Academy of Sciences. Total genome was
extracted from about 100 mg of these clean, fresh
leaves using CTAB method. The complete chloroplast
genome of P. polyphylla var. chinensis was amplified
by Takara PrimeSTAR GXL DNA polymerase and
nine universal pairs of primers[26]. Purified PCR
products were mixed and then broken into 200–500 bp
fragments and set paired-end libraries according to the
第6期 603李晓娟等：药用植物华重楼(黑药花科)叶绿体全基因组研究
manufacturer’s manual (Illumina). The libraries were
sequenced (2×100 bp) by Illumina Hiseq 2000.
1.2 Genome assembly
Before assembling short raw reads of P. poly-
phylla var. chinensis into contigs, sequence data were
filtered using NGS QC Tool Kit[27] for high quality
(cut-off value for percentage of read length =80, cut-
off value for PHRED quality score =30). Then the
filtered reads were conducted to contigs with the de
novo sequence assembly software, CLC Genomics
Workbench[28] V. 7 on Windows7 64bits server, and
the word size was set to 64, while the minimum contig
lengh was set to 1 kb. One cp genome which was
highly similar to that of P. polyphylla var. chinensis
was obtained after contigs were aligned using the
Basic Local Alignment Search Tool (http://blast.
ncbi.nlm.nih.gov/) with default parameters. We
arranged the aligned contigs using the highly similar
genome sequence identified in the BLAST search as
references, and joined the contigs according the orders
of contigs. At this time, contigs were assembled into
a genome sequence with some gaps. In order to get
one complete chloroplast genome, we also assembled
reads into contigs using SOAPdenovo2[29], on linux
server, set the kmers to 81. We joined the contigs
into another incomplete genome sequence following
the same steps. Last, we aligned the two incomplete
genome sequences and filled gaps. The complete
chloroplast genome sequence of P. polyphylla var.
chinensis was assembled.
1.3 Genome annotation, drawing and comparison
The P. polyphylla var. chinensis cpDNA genome
was annotated using the program DOGMA (http://
phylocluster.biosci.utexas.edu/dogma/) and start
and stop codons were adjusted using Geneious 7.0
software[30]. The tRNA genes were identified and
corrected by the tRNAscan-SE (http://selab.janelia.
org/tRNAscan-SE/). The pseudogenes were defined
in terms of terminal codons found in the middle of
protein genes coding sequence[31]. The gene introns
were showed in gene annotation tables which
Geneious[30] displayed. Then we used the annotated
chloroplast genome genome file to draw gene map
utilizing OrganellarGenomeDRAW (http://ogdraw.
mpimpgolm.mpg.de/index.shtml). We compared the
chloroplast genomic characteristic of P. polyphylla
var. chinense with 47 species chosen from some orders
of monocot plants included Liliales, Zingiberales,
Poales, Arecales, Asparagales, Dioscoreales,
Petrosaviales, Alismatales and Acorales, which data
were downloaded from NCBI (Table 1). The 48
taxa genome sequences of cemA were aligned by
MUSCLE in Geneious plugins, and the alignment
result was corrected manually.
1.4 Sequence divergence and phylogenetic analysis
The 48 complete plastid sequences representing
the nine orders of monocots (Table 1) were down-
loaded from NCBI Organelle Genome Resources
database. We extracted 79 protein-coding genes (all
protein genes except ycf15 and ycf68) from 48 species
sequences. The question marks were substituted
for missing genes of some taxa, and missing genes
sequence were aligned by MUSCEL, respectively.
These alignment results were modified manually.
Pairwise sequence divergences were calculated with
Kimura’s two parameter model using the software
MEGA 6[32].
Because the accD, ycf15, ycf68, ycf1, ycf2 are
missing in some monocot lineages, we constructed
the aligned matrix of 76 protein genes without those
five genes using Phyutility.jar[33], and used this matrix
to reconstruct phylogenetic tree with GTR+I+G
substitution model computed by Modeltest, which
is included in the PUAP GUI 2011 program. The
Markov chain Monte Carlo (MCMC) algorithm was
run for 1000000 generations with trees sampled every
100 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. We chose Acorus americanus
(Raf.) Raf. and A. calamus L. as outgroups. The
phylogenetic tree was conducted by FigTree V1.4
program.
604 第23卷热带亚热带植物学报
Table 1 Accession number of complete chloroplast genomes
Order Family Species Accession number
Acorales Acoraceae Acorus americanus NC_010093
A. calamus NC_007407
Alismatales Hydrocharitaceae Elodea canadensis NC_018541
Najas flexilis NC_021936
Araceae Colocasia esculenta NC_016753
Spirodela polyrhiza NC_015891
Lemna minor DQ400350
Wolffia australiana NC_015899
Wolffiella lingulata NC_015894
Petrosaviales Petrosaviaceae Petrosavia stellaris NC_023356
Dioscoreales Dioscoreaceae Dioscorea elephantipes EF380353
D. rotundata KJ490011
Liliales Alstroemeriaceae Luzuriaga radicans NC_025333
Bomarea edulis KM233641
Alstroemeria aurea KC968976
Smilacaeae Smilax china HM536959
Liliacceae Lilium longiflorum KC968977
Fritillaria cirrhosa NC_024728
F. hupehensis NC_024736
Melanthiaceae Veratrum patulum NC_022715
Chionographis japonica KF951065
Paris verticillata KJ433485
Zingiberales Musaceae Musa textilis NC_022926
Heliconiaceae Heliconia collinsiana NC_020362
Strelitziaceae Ravenala madagascariensis NC_022927
Marantaceae Maranta leuconeura KF601571
Zingiberaceae Zingiber spectabile NC_020363
Curcuma roscoeana NC_022928
Poales Bromeliaceae Ananas comosus NC_026220
Typhaceae Typha latifolia NC_013823
Anomochloa marantoidea NC_014062
Pharus lappulaceus NC_023245
Poaceae Leersia tisserantii NC_016677
Yushania lebigata NC_024725
Lolium perenne AM777385
Arecales Arecaceae Bismarckia nobilis NC_020366
Phoenix dactylifera NC_013991
Cocos nucifera KF285453
Elaeis guineensis NC_017602
Calamus caryotoides JX088663
Asparagales Orchidaceae Neottia nidus-avis NC_016471
Dendrobium officinale KC771275
Phalaenopsis equestris NC_017609
Oncidium NC_014056
Cymbidium aloifolium NC_021429
Amaryllidaceae Allium cepa KF728079
Asparagaceae Eustrephus latifolius KM233639
第6期 605
2 Results
2.1 Genome features
The complete cp genome of P. polyphylla var.
chinensis is 158307 base pairs (bp) in length, which
exhibits a quadripartite structure, consisting of a pair
of IRs (27473 bp) separated by the large single-copy
region (LSC, 85187 bp) and small single-copy region
(SSC, 18175 bp) (Fig. 1). The overall CG content
is 62.8%. The GC content of the IRs, LSC and SSC
regions are 41.7%, 35.5% and 31.4%, respectively.
The cp genome of P. polyphylla var. chinensis
encodes 137 predicted functional genes, of which
115 are unique, including 81 protein-coding genes,
4 rRNAs, and 30 tRNAs (Table 2). Ten protein-
coding, 8 tRNA and 4rRNA genes are duplicated
in the IR regions, however, only a part of ycf1 gene
is duplicated in the junction between IRB and SSC
regions. The LSC region includes 60 protein-coding
and 21 tRNA genes, whereas the SSC region contains
12 protein-coding (including partial ycf1 gene) and 1
tRNA genes. There are 18 intron-containing genes (12
protein-coding and 6 tRNAs) within the cp genome of
P. polyphylla var. chinensis. Among them, 9 protein-
coding genes and 6 tRNAs contain one intron, and
3 protein-coding genes (clpP, ycf3, rps12) have two
introns. The cemA, ycf15 and ycf68 are pseudogenes
judging by the presence of several terminal codons
in these coding genes regions. In the cemA gene
sequence, ploy(A) (8 bp) sequence and a small single
repeat (SSR) CA unit including 6 CA copies are found
within the coding regions (Fig. 2).
2.2 Comparison with other cp genomes in Liliales
The basic cp genomic features of P. polyphylla
var. chinensis and those of nine species from other
families within the Liliales order were compared,
including P. verticillata, C. japonica and V. patulum
(Melanthiaceae), Fritillaria hupehensis P. K. Hsiao
& K. C. Hsia.[34] and Lilium longiflorum Thunb.[35]
(Liliaceae), Smilax china L.[36] (Smilacaceae) and
Alstroemeria aurea Graham.[34] and Bomarea edulis
(Tussac) Herb.[37] (Alstroemeriaceae). The genome
size of P. polyphylla var. chinensis is the largest of the
Liliales cp genome. This variation in sequence length
is mainly attributed to the difference in the length
of the LSC region (Table 3). The AT and CG content
ratio of P. polyphylla var. chinensis (37.2% and 62.8%,
respectively) are similar to those of other species within
the Liliales. The gene content and arrangement are
similar within the Liliales lineages, except that rps16
is deleted completely in C. japonica[24] but partially in
V. patulum[18], infA lost in A. aurea[34] and S. china[36],
and ycf15 was absent from A. aurea[34], The gene
content and arrangement are similar within the Liliales
lineage. The IRB/SSC boundary is the incomplete
duplication of ycf1 in all species examined, whereas
the IRA/LSC junction expands to rps19 (P. polyphylla
var. chinensis, L. longiflorum[35] and A. aurea[34]), full
trnH_GUG (V. patulum[18], F. hupehensis[34] and B.
edulis[37]), part of rpl22 (S. china[36]) and part of rps3
(C. japonica[19] and P. verticillata[20]). The length
of intergenic spacer between rpl23 and ycf2 which
contains trnI-CAU are varied among Liliales taxa.
P. polyphylla var. chinensis cpDNA genome has the
longest IGS length (636 bp) among all Liliales species
(Table 3).
2.3 Sequence divergence of protein genes
We calculated the average pairwise sequence dis-
tance of 79 protein-coding genes among 48 monocots
taxa. The results showed that 36 genes (45.57%) have
an average sequence distance more than 0.10. The
fourteen most divergent genes (ycf1, rbl16, matK,
rbl22, clpP, rbl32, rps16, ndhA, ndhF, ccsA rps11,
ndhD, accD, infA) exhibits that the average distance
is higher than 0.15. The highest average sequence
distance is observed in ycf1 (0.23), which is located at
the IR/SSC boundary and shows a fast evolutionary
trend in monocots. The seven most conserve gene
(psbE, psbF, rps7, rps12, ndhB, rbl2 and psbL)
possess the average sequence distance less than 0.05
(Table 4).
2.4 Phylogenetic analysis
To identify the phylogenetic position of P. poly-
李晓娟等：药用植物华重楼(黑药花科)叶绿体全基因组研究
606 第23卷热带亚热带植物学报
Fig. 1 Map of Paris polyphylla var. chinensis complete chloroplast genome. Transcribed counterclockwise are shown outside of outer circle, whereas
transcribed clockwise are shown inside. Thick lines in outer circle indicate IR regions.
phylla var. chinensis, we carried out multiple sequence
alignments by using 76 protein-coding genes in the cp
genomes for 48 monocot taxa to generate a matrix of
85506 bp. Phylogenetic relationships in Melanthiaceae,
Liliales, and other orders among monocots were
reconstructed with MrBayes analysis (Fig. 3). The
results indicated that Liliales is a monophyletic group
[Bayesian posterior probabilities (BPP)=100%].
Within Liliales, Melanthiaceae is monophyletic
(BPP=100%), which is sister to Smilacaceae (S.
china) and Liliaceae (L. longiflorum, F. cirrhosa,
F. hupehensis). Also, Alstroemeriaceae (A. aurea)
is sister to Colchicaceae (Cohchicum autrumnale,
Gloriosa superba). The genus Paris (BPP=100%)
is monophyletic and sister to other taxa within
Melanthiaceae.
第6期 607
Table 2 Gene contents of complete chloroplast genome of Paris polyphylla var. chinensis
Gene type Genes
Photosystem I psaA, psaB, psaC, psaI, psaJ, ycf3**, ycf4
Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ psbK, psbL, psbM, psbN, psbT, psbZ
Cytochrome petA, petB*, petD*, petG, petL, petN
ATP synthase atpA, atpB, atpE, atpF*, atpH, atpI
Rubisco rbcL
NADH dehydrogenase ndhA*, ndhB*×2, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Ribosomal protein (large subunit) rpl2*×2, rpl14, rpl16*, rpl20, rpl22, rpl23×2, rpl32, rpl33, rpl36
Riosomal protein (small subunit) rps2, rps3, rps4, rps7×2, rps8, rps11, rps12**×2, rps14, rps15, rps16*, rps18, rps19×2
ATP-dependent protease clpP**
Cytochrome c biogenesis ccsA
Membrane protein cemA
Maturase matK
Other protein gene infA
Proteins of unknown function ycf1×2, ycf2×2, ycf15×2, ycf68×2
Ribosomal RNAs rrn23×2, rrn16×2, rrn5×2, rrn4.5×2
Transfer RNAs trnA_UGC*×2, trnH_GUG×2, trnR_ACG×2, trnI_GAU*×2, trnI_CAU×2, trnC_GCA, trnL_
CAA×2, trnV_GAC×2, trnN_GUU×2,
trnD_GUC, trnE_UUC, trnF_GAA, trnG_UCC*, trnG_UCC, trnK_UUU*, trnL_UAA*, trnV_UAC*,
trnL_UAG, trnM_CAU, trnfM_CAU, trnP_UGG, trnQ_UUG, trnR_UCU, trnS_GCU, trnS_UGA,
trnS_GGA, trnT_GGU, trnT_UGU, trnW_CCA, trnY_GUA
RNA polymerase rpoA, rpoB, rpoC1*, rpoC2
×2: Two gene copies in IR regions; *: With one intron; **: With two introns.
Fig. 2 Alignment of partial cemA sequences among 11 species of Liliales. A: Partial nucleotide sequences of cemA, gray box show SSR (CA); B:
Partial amino acid sequences of cemA, asterisks indicate the stop condon.
3 Discussions
3.1 cpDNA genome features
The gene contents and arrangement were similar
among Liliales as previously reported. However, there
were some difference in genes loss, gene structure and
pseudogenes in Liliales taxa, and some changes were
typical among Liliales species. For instance, rps16
was deleted completely in C. japonica[19], but partially
in V. patulum[18]. The infA gene lost in A. aurea[34]
608 第23卷热带亚热带植物学报
and S. china[36], while it existed as a pseudogene in
the V. patulum[18], B. edulis[37] and L. longiflorum[35]
because there were internal stop codons within the
coding regions of this gene. The accD gene was
pseudogene in S. china[36], whereas it had function
within other species of Liliales. The gene loss and
pseudogenization could be unique evolutionary events
in those species.
Because of the presence of several stop codons,
the cemA is pseudogene, and the gene is with poly (A)
sequence and SSR of CA after the start codon in the
cp genome of P. polyphylla var. chinensis as well
as in P. verticillata[25]. However, the locations of
stop codons in cemA are different between the cp
Table 3 Characteristics of chloroplast genomes among Liliales
Paris polyphylla var.
chinensis
Paris
verticillata
Chionographia
japonica
Veratrum
patulum
Fritillaria
hupehensis
Family Melanthiaceae Melanthiaceae Melanthiaceae Melanthiaceae Liliaceae
Accession number KJ433485 KF951065 KF437397 KF712486
Protein-coding genes 81 81 80 81 81
tRNAs 30 30 30 30 30
rRNAs 4 4 4 4 4
Length (bp) 158307 157379 154646 153699 152145
LSC (bp) 85187 82726 81653 83372 81898
SSC (bp) 18175 17907 18195 17607 17553
IRs (bp) 27473 28373 27399 26360 26347
AT content (%) 62.8 62.4 62.3 62.3 62.9
GC content (%) 37.2 37.6 37.7 37.7 37.1
IR/SSC junction ycf1-like ycf1-like ycf1-like ycf1-like ycf1-like
IR/LSC junction rps19 rps3-like rps3-like trnH_GUG-rps19 IGS trnH_GUG-rps19 IGS
LSC GC content (%) 35.5 36 36 35.7 34.8
SSC GC content (%) 31.4 31.1 31.4 31.4 30.5
IR GC content (%) 41.7 42 42.5 42.9 42.5
Length of IGS
(rpl23-ycf2)(bp)
636 591 303 305 307
Lilium longiflorum Smilax china Alstroemeria aurea Bomarea edulis
Family Liliaceae Smilacaceae Alstroemeriaceae Alstroemeriaceae
Accession number KC968977 HM536959 KC968976 KM233641
Protein-coding genes 81 81 79 80
tRNAs 30 30 30 30
rRNAs 4 4 4 4
Length (bp) 152793 157878 155510 154925
LSC (bp) 82230 84608 84241 84094
SSC (bp) 17523 18536 17867 17699
IRs (bp) 26520 27367 26701 26566
AT content (%) 62.98 62.75 62.74 61.8
GC content (%) 37.02 37.25 37.26 38.2
IR/SSC junction ycf1-like ycf1-like ycf1-like ycf1-like
IR/LSC junction rps19-like rpl22-like rps19-like trnH_GUG-rps19 IGS
LSC GC content (%) 34.8 35.2 36.2 36.4
SSC GC content (%) 30.8 31.4 31.8 32.2
IR GC content (%) 42.4 42.4 40.3 40.3
Length of IGS
(rpl23-ycf2)(bp)
308 308 308 307
第6期 609李晓娟等：药用植物华重楼(黑药花科)叶绿体全基因组研究
Table 4 Sequence divergence of protein genes
No. Gene
Mean sequence
distance
Standard
error Region No. Gene
Mean sequence
distance
Standard
error Region
1 ycf1 0.23203 0.01059 IR 41 rbL14 0.09637 0.01633 LSC
2 rbL16 0.19853 0.01499 LSC 42 rps18 0.09584 0.01786 LSC
3 matK 0.19165 0.01289 LSC 43 ndhk 0.09547 0.01171 LSC
4 rbL22 0.18773 0.02473 LSC 44 rbL36 0.09479 0.03008 LSC
5 clpP 0.18683 0.01317 LSC 45 rps14 0.09387 0.01794 LSC
6 rbL32 0.17464 0.03499 SSC 46 atpA 0.09227 0.00777 LSC
7 rps16 0.17059 0.01485 LSC 47 psbI 0.09194 0.03019 LSC
8 ndhA 0.16579 0.01091 SSC 48 rpoB 0.09089 0.00538 LSC
9 ndhF 0.16052 0.00926 IR 49 psaC 0.08658 0.01925 SSC
10 ccsA 0.15776 0.01411 SSC 50 rbcL 0.08635 0.00804 LSC
11 rps11 0.15721 0.02079 LSC 51 psaI 0.08381 0.02821 SSC
12 ndhD 0.15337 0.01239 SSC 52 atpB 0.08203 0.00839 LSC
13 accD 0.15177 0.01125 LSC 53 petA 0.08031 0.00945 LSC
14 infA 0.15029 0.02688 LSC 54 petL 0.08027 0.02954 LSC
15 rps15 0.14918 0.02454 SSC 55 psbJ 0.07322 0.02484 LSC
16 atpF 0.14464 0.01152 LSC 56 psbB 0.07098 0.00707 SSC
17 rbL33 0.13968 0.03189 LSC 57 petG 0.07021 0.02542 LSC
18 rbL20 0.13798 0.02018 LSC 58 psbC 0.06873 0.00708 SSC
19 rps3 0.13509 0.01494 LSC 59 atpI 0.06811 0.00964 LSC
20 rps8 0.13195 0.01904 LSC 60 psbM 0.06476 0.02546 LSC
21 rpoC2 0.13129 0.00581 LSC 61 psaA 0.06379 0.00562 LSC
22 ndhG 0.12583 0.01715 SSC 62 psaB 0.06371 0.00582 LSC
23 psbK 0.12471 0.02701 LSC 63 psbZ 0.06255 0.01791 LSC
24 cemA 0.12293 0.01419 LSC 64 psbT 0.06172 0.02421 LSC
25 rpoC1 0.11994 0.00721 LSC 65 ycf2 0.06102 0.00389 IR
26 rps19 0.11922 0.02117 IR 66 psbD 0.05992 0.00761 LSC
27 ndhC 0.11635 0.01944 LSC 67 petN 0.05889 0.02628 LSC
28 ycf3 0.11574 0.00823 LSC 68 atpH 0.05837 0.01541 LSC
29 rpoA 0.11552 0.01122 LSC 69 psbA 0.05813 0.00727 SSC
30 psaJ 0.11477 0.03141 SSC 70 psbN 0.05773 0.02098 LSC
31 atpE 0.11308 0.01693 LSC 71 psbE 0.04825 0.01389 LSC
32 ndhI 0.11087 0.01662 SSC 72 psbF 0.04687 0.01954 LSC
33 ndhH 0.10877 0.01139 SSC 73 rps7 0.04038 0.00857 IR
34 ndhE 0.10764 0.01999 SSC 74 rbL23 0.03338 0.00974 IR
35 petD 0.10743 0.01088 LSC 75 rps2 0.03274 0.01299 LSC
36 psbH 0.10627 0.02248 LSC 76 rps12 0.03245 0.00634
LSC and IR
(mainly in IR)
37 ycf4 0.09992 0.01363 LSC 77 ndhB 0.03089 0.00366 IR
38 petB 0.09797 0.01103 LSC 78 rbl12 0.03061 0.00451 IR
39 ndhJ 0.09708 0.01525 LSC 79 psbL 0.03004 0.01515 LSC
40 rps4 0.09639 0.01278 LSC
610 第23卷热带亚热带植物学报
genomes of P. polyphylla var. chinensis and P.
verticillata (Fig. 2), which can be used to distinguish
P. polyphylla var. chinensis and P. verticillata.
The cemA encoding product was found in the inner
envelope membrane of chloroplasts[38], which could
be essential to CO2 uptake in Synechocystis
[39]. The
cemA gene was found disappeared in the cp genome
of two saprophytic monocots, e.g. Neottia nidus-avi
(L.) Rich.[40], and Petrosavia stellaris Becc.[41], which
could be interpreted by the dependence on the host
plant. However, all species in the genus Paris are
autotrophic, further research is needed to clarify the
impact of cemA pseudogenization in the genus.
Gene duplication in the cp genome occurs mainly
Fig. 3 Mrbayes tree inferred from 76 protein coding genes from 48 taxa. Bayesian posterior probabilities (BPP) are shown on the right of branches.
The box displays the family Melanthiaceae taxa.
第6期 611
within the IR regions because of the IR region expan-
sion[42] and most duplication genes were tRNAs[43]. The
triplication of trnI_CAU has been reported in the
cp genome of P. verticillata[20] but was not found in
previously examined cp genomes of Melanthiaceae
and other families in the Liliales, which was proposed
to be unique to the tribe Parideae of Melanthiaceae.
However, our data revealed that the triplication
of trnI_CAU does not occur in P. polyphylla var.
chinensis. P. verticillata and P. polyphylla var.
chinensis belong two different subgenera of Paris[44].
Therefore, it is likely that the triplication of trnI_
CAU occurs only in the subgenus Paris rather than
the subgenus Daiswa. This difference may provide
information to explore the infrageneric relationships
within the genus Paris.
3.2 Phylogenetic relationships
Chloroplast genomes provide rich sources of
phylogenetic information to elucidate the evolu-
tionary relationships among angiosperms[4,45]. The
order relationships among monocots and family
relationships within Liliales defined in this study were
identical to those delineations in previous studies[10,46].
However, the generic relationships among the family
Melanthiaceae have not been satisfactory resolved
inferred from our data due to the limited taxa sam-
pling. Paris is a temperate genus with 27 species
distributed in the Eurasia[8,44]. Previous phylogenetic
studies employing one or several genes or DNA
regions placed the genus in the family Melanthiaceae
or the family Trilliaceae[9–10,47]. Our phylogeny based
on 76 chloroplast protein genes placed Paris in the
family Melanthiaceae with 100% BBP, which well
supports the treatments of APG III[10], Fuse et al[9], and
Zomlefer et al[47].
Acknowledgments　　We are very grateful to Dr. Hong-tao
Li, Zheng-shan He, Li-e Yang, Cui-xian Peng, Jian-jun Jin of
Kunming Institute of Botany for their help during processing
our data. We thank Molecular Biology Experimental Center,
Kunming Institute of Botany for experiments and materials.
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