全 文 ：Journal of Systematics and Evolution 46 (2): 142–154 (2008) doi: 10.3724/SP.J.1002.2008.07107
(formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com
Phylogenetic inference of the genus Bupleurum (Apiaceae) in Hengduan
Mountains based on chromosome counts and nuclear ribosomal DNA
ITS sequences
Qi-Zhi WANG * Xing-Jin HE ** Song-Dong ZHOU* Yun-Ke WU Yan YU Yun-Li PANG
(Laboratory of Systematic and Evolutionary Botany, College of Life Science, Sichuan University, Chengdu 610064, China)
Abstract Hengduan Mountains are the diversity center for the genus Bupleurum of Apiaceae in China. This
paper reports chromosome numbers of six species and two varieties of Bupleurum, and for four species and two
varieties their chromosome numbers are reported for the first time. The phylogeny of Bupleurum was investigated
based on the ITS region of the nuclear ribosomal DNA (nrDNA) of 14 taxa from the Hengduan Mountains, 3 taxa
from the North China (Hebei and Heilongjiang), and 16 taxa from Africa and the Mediterranean region. Varia-
tions in chromosome numbers and the ITS sequences were used to infer phylogenetic relationships between
Bupleurum species in Hengduan Mountains. The results showed that the Hengduan Mountains might represent
one of the frequency and diversity centers for Bupleurum. The ancestors of Bupleurum species in the Hengduan
Mountains may be related to the woody B. fruticosum in North Africa, or the species in the western Mediterranean
region. It is postulated that the ancestral population migrated into Hengduan Mountains through the Middle East
and the Caucasus. Furthermore, the neo-endemic B. mundtii in South Africa appeared to be a close relative of the
species in the Hengduan Mountains. In the trend of basic chromosome number evolution, x = 8 should be regarded
as the ancestral basic number, while x = 6, 7 as the derived ones. The Bupleurum species in the Hengduan Moun-
tains have been undergoing changes in the basic chromosome numbers or the ploidy level. The ITS phylogenetic
tree showed that the Chinese species were divided into two clades: one with the basic chromosome number x = 8,
and the other with x = 6, 7. The results rejected the previous infrageneric classification of Bupleurum in China.
We further suggested to raise B. marginatum var. stenophyllum to species rank based on the combined evidence
from morphology, karyology, pollen morphology, and the ITS phylogenetic tree.
Key words Bupleurum, chromosome, Hengduan Mountains, ITS, phylogeny, taxonomy.


The genus Bupleurum L., with about 180–190
species spreading in the North Temperate Zone
(mainly in Eurasia, Mediterranean and North Africa),
is one of the largest genera of the family Apiaceae
(Liu et al., 2003; Neves & Watson, 2004). Species
within Bupleurum are easily recognized by the parallel
venation of the simple leaves, conspicuous bracts and
bracteoles on the inflorescences. Pollen morphology
exhibits little variation in this genus. The fruit is
oblong to ovoid-oblong or ellipsoid, slightly laterally
compressed and the mericarp is subpentagonal (rarely
rounded) in cross section. Bupleurum is a primitive
genus in Apioideae in recent molecular studies
(Plunkett et al., 1996a, b, 1997; Downie &
Katz-Downie, 1996, 1999; Downie et al., 1998,
2000a, b; Plunkett & Downie, 1999, 2000). Despite
intensive research on Bupleurum, the infrageneric
phylogeny and classification are incomplete.
Traditionally, the woody Araliaceae is regarded
as the ancestor of the herbaceous Apiaceae (Calviňo et
al., 2006). Woody B. fruticosum L. in North Africa is
the possible ancestor of the species in genus Bupleu-
rum (Gruas-Cavagnetto & Cerceau-Larrival, 1978). A
hypothesis on the origin of Apiaceae suggests that B.
fruticosum might have migrated northward into Eura-
sia through the Middle East (Plunkett et al., 1996b).
However, Neves and Watson (2004) suggested that
the genus Bupleurum originated in the western Medi-
terranean. Although B. fruticosum was nested within
the western Mediterranean clade, it was not the most
basal taxon, thus casting doubt regarding the origin of
this genus. On the other hand, most Bupleurum spe-
cies in the Hengduan Mountains in China exhibit the
primitive pollen type of subrhomboidity to subround
to subellipse that appeared in the early Palaeocene
(Shu & Sheh, 2001). The entire region of the Heng-
duan Mountains harbors high Bupleurum diversity in
Asia. Thus, the Hengduan Mountains are another
possible center of origin for Bupleurum (Shu & Sheh,

———————————
Received: 27 August 2007 Accepted: 2 February 2008
* Co-First authors.
** Author for correspondence. E-mail: xinjinghe@126.com; Tel.: 86-28-
85415006; Fax: 86-28-85415006.
WANG et al.: Phylogenetic inference of Bupleurum in Hengduan Mountains

143
2001). By including Bupleurum species from Africa
and the western Mediterranean region, we aim to
investigate the origin and evolution of Bupleurum in
the Hengduan Mountains with a broad taxon sampling
throughout the entire genus.
Bupleurum was placed in the subtribe Carinae,
tribe Apieae, subfamily Apioideae (Drude, 1898).
Wolff (1910) and Linczevsky (1950) briefly discussed
the taxonomy of Chinese species. Su et al. (1998)
studied fourteen species, two varieties and one form of
Chinese Bupleurum in an infrageneric classification
based on morphology, anatomy and palynology. Two
subgenera of the genus were then proposed: Longifo-
lia (Wolff) Yuan and Eubupleura (Briq.) Yuan.
Subgenus Eubupleura includes two sections: Ranun-
culoidea (Wolff) Yuan and Falcata (Wolff) Yuan.
The ITS region of the nrDNA evolves at a rela-
tively rapid rate and is rich in variable and informative
sites. It has been proven to be a valuable source of
information in evaluating phylogenetic relationships at
the genus and species level (Baldwin, 1992; Baldwin
et al., 1995; Tian & Li, 2002). It has also been suc-
cessfully used to study intraspecific relationships in
Chinese Bupleurum species (Wu et al., 2005; Xie et
al., 2006). However, so far no phylogenetic study has
included species from the Hengduan Mountains.
Therefore, based on previous work, we sequenced the
ITS region of ten Bupleurum species and four varieties
from the Hengduan Mountains in South China, repre-
senting two sections in the subgenus Eubupleura
(Ranunculoidea and Falcata, sensu Su et al., 1998), as
well as B. longiradiatum Turcz. from the monotypic
subgenus Longifolia. We also sequenced B. chinense
DC. and B. scorzonerifolium Willd. from North China
in subgenus Eubupleura. Our samplings represent all
subgenera and sections in China (sensu Su et al.,
1998) (Fig. 1).
Karyotype, including the basic chromosome
numbers and ploidy level, is often used to investigate
interspecific evolution between organisms. Previous
karyotype study for Bupleurum species in the Heng-
duan Mountains was inadequate. Only two species
and one variety have had their chromosome numbers
reported (Pan & Qin, 1981; Pan et al., 1985; Qin et al.,
1989). We herein report the chromosome numbers of
six species and two varieties, and the data from four
species and two varieties were reported for the first
time (i.e., B. chaishoui R. H. Shan & M. L. Sheh, B.
gracilipes Diels, B. rockii H. Wolff, B. yunnanense
Franch., B. hamiltonii var. humile (Franch.) R. H.
Shan & M. L. Sheh and B. longicaule var. franchetii
H. Boiss.).
The questions of this study are as following: (1)
What are the relationships between the Bupleurum
species in the Hengduan Mountains and the species in
Africa and the western Mediterranean region? Did the
















Fig. 1. Infrageneric classification of the Chinese Bupleurum (sensu Su et al., 1998). All taxa are included in our sampling.
Bupleurum species in our experiment
Subgenus Eubupleura
Section Ranunculoidea
B. commelynoideum
B. longicaule var. amplexicaule
B. longicaule var. franchetii
B. petiolulatum
B. rockii
B. yunnanense
Section Falcata
B. chinense
B. chaishoui
B. gracilipes
B. hamiltonii
B. hamiltonii var. humile
B. marginatum
B. marginatum var. stenophyllum
B. microcephalum
B. scorzonerifolium
B. wenchuanense
Subgenus Longifolia
B. longiradiatum
Journal of Systematics and Evolution Vol. 46 No. 2 2008 144
genus Bupleurum originate in the Hengduan Moun-
tains? (2) Is the previous infrageneric classification of
Chinese Bupleurum (Su et al., 1998) well supported
by the molecular and karyological data? and (3) How
to delimitate of Bupleurum species in the Hengduan
Mountains (e.g., treatment of B. marginatum var.
stenophyllum (H. Wolff) R. H. Shan & Yin Li).
1 Material and methods
1.1 Material and methods of nrDNA ITS
Sample materials of Bupleurum species were ob-
tained from fresh leaves in the field, which were
collected individually in the Hengduan Mountains and
dried with silica gel. Data for the remaining 16 taxa
from African and the western Mediterranean were
obtained from GenBank. The collecting locations and
the GenBank accession numbers were listed in Table
1. Voucher specimens were deposited in SZ (Sichuan
University Herbarium), Jia Mu Si University and
Institute of Natural Medicine and Chinese Medicine
Resources.
Total genomic DNA was extracted using the
modified cetyltrimethyl-ammonium bromide (CTAB)
protocol by Doyle and Doyle (1987) or DNAsecure
plant Kit (Tiangen Biotech, Beijing, China). The
universal primers, ITS4 (5-TCCTCCGCTTATT-
GATATGC-3) and ITS5 (5-GGAAGTAAAAGTC-
GTAACAAGG-3), were used to amplify the entire
internal transcribed sequences of nrDNA sequences
(including ITS1, 5.8S and ITS2). The PCR profile was
as follows: the initial denaturation was 94 ℃ for 2.5
min and the final extension was 72 ℃ for 5 min. The
reactions were subjected to 35 cycles. Each cycle
consisted of a denaturation at 94 ℃ for 45 s, primer
annealing at 56 ℃ for 45 s, and extension at 72 ℃
for 90 s. The PCR reaction mixture contained 1 µL
DNA template, 2 µL 10× Reaction buffer (500
mmol/L KCl, 100 mmol/L Tris-HCl pH 8.4, 20
mmol/L MgCl2), 2 µL dNTP (2 mmol/L), 1 µL (10
µmol/L) for each primer, 0.5 µL DMSO, 0.2 µL (5
U/µL) Taq DNA Polymerase (SBS Genetic, Beijing,
China) and ddH2O to make up a total volume of 20
µL. If the reaction was successful, the reaction volume
was magnified to 100 µL to produce enough PCR
products, which were purified using the PCR purifica-
tion kit (SBS Genetic, Beijing, China). The purified
DNA was then sent to Invitrogen Biotech Co. Ltd.
(Shanghai, China) for sequencing. Sequencing was
done using an ABI-3730XL DNA sequencer. For each
sampled specimen, forward and reverse sequencing
reactions were performed for confirmation. Molecular
cloning was conducted on two species: B. longiradia-
tum and B. scorzonerifolium, to examine the extent of
sequence homogenization among reiterated ITS
copies. The purified ITS templates were ligated to the
pGM-T Easy vector (Tiangen Biotech, Beijing, China)
and subsequently transformed into competent cells
following the manufacturer’s protocol. The trans-
formed cells were cultured overnight at 37 ℃ on the
LB ampicillin plates. Colonies carrying the ITS insert
were identified by colors (white) and were further
verified by PCR and sequencing. For each sample, up
to 10 white colonies were checked for inserts. All ITS
sequences were aligned manually using sequential
pairwise comparisons. The boundaries of ITS1, 5.8S
and ITS2 regions were determined following the
published sequences from GenBank. All sequences
from this study were deposited in GenBank.
We chose closely related Anginon difforme (L.)
B. L. Burtt and A. paniculatum (Thunb.) B. L. Burtt
based on the Plunkett et al.’s (1996b) molecular
studying.
1.2 Molecular data Analysis
All the ITS sequences were loaded in DNASTAR
6.0 (DNASTAR Inc., 2001, Madison WI) for inspec-
tion and edition. We then used Clustal 1.81 (Thomp-
son et al., 1997) for alignment. Ambiguous sites
(19–26, 200–202) were excluded from the analyses.
The phylogenic analyses were performed in MrBayes
ver. 3.1 (Huelsenbeck & Ronquist, 2001) and
PAUP*4.0b10 (Swofford, 2002).
Maximum likelihood and Bayesian analyses were
performed using General-time-reversible + gamma
(GTR+G) model (–Ln likelihood = 2745.65) in ac-
cordance to the result of the Modeltest 3.7 (Posada &
Crandall, 1998) and MrModel test 2.2 (Posada &
Buckley, 2004). Estimated base frequencies were A =
0.2223, C = 0.2797, G = 0.2815, T = 0.2165. The
shape parameter of the Gamma distribution was
0.6760.
Bayesian tree was used by the 4 by 4 model for
base substitution and GTR+G model for optimality
criterion. Bayesian inference was estimated by run-
ning 4 simultaneous chains (Metropolis Coupling
Markov Chain Monte Carlo) for 1000000 generations.
One tree was sampled for every ten generations. The
first 2000 trees (burn in samples) were discarded, and
the remaining samples were used to construct the
majority-rule consensus tree.
In maximum parsimony (MP) analyses, heuristic
searches were carried with 1000 random addition
sequence replicates. Ten trees were saved at each
WANG et al.: Phylogenetic inference of Bupleurum in Hengduan Mountains

145
Table 1 Origin of materials and accession numbers in GenBank
Taxon Location Voucher GenBank
Acc. No.
Bupleurum album Maire Morocco Reading University/BM Expedition 532 (RNG) AF467928
B. angulosum L. Royal Botanic Garden
Edinburgh
RBGE 19861043 S. S. Neves (E)
AF469008
B. benoistii Litard. & Maire Morocco S. L. Jury et al. 18375 (E) AF477024
B. canescens Schousb. Morocco D. Bramwell et al. 265 (RNG) AF477027
B. chaishoui R. H. Shan & M. L. Sheh Mao Xian, Sichuan, China
(四川茂县)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 361536
(SZ)
EU001333
B. chinense DC. Mt. Wuling, Beijing, China
(北京雾灵山)
C. Q. Feng (冯成强) 200508 (Institute of Natural
Medicine and Chinese Medicine Resources)
EU001334
B. commelynoideum H. Boiss. Dali, Yunnan, China
(云南大理)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 374627
(SZ)
EU001335
B. dumosum Coss. & Balansa Morocco S. L. Jury et al. 14157 (RNG) AF477030
B. fruticosum L. Portugal S. S. Neves 33 (E) AF479297
B. gracilipes Diels Mao Xian, Sichuan, China
(四川茂县)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 361529
(SZ)
EU001337
B. gibraltaricum Lam. Spain S. S. Neves 51 (E) Herbarium, Royal Botanic
Garden Edinburgh, UK
AF479852
B. hamiltonii Balakr. Kunming, Yunnan, China
(云南昆明)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 348618
(SZ)
EU001338
B. hamiltonii var. Humile (Franch.) R.
H. Shan & M. L. Sheh
Lijiang, Yunnan, China
(云南丽江)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 366440
(SZ)
EU001339
B. lateriflorum Coss. Morocco S. L. Jury et al. 18323 (E) Herbarium, Royal
Botanic Garden Edinburgh, UK
AF479854
B. longicaule var. amplexicaule Wu ex
R. H. Shan & Y. Li
Kunming, Yunnan, China
(云南昆明)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 366442
(SZ)
EU001340
B. longicaule var. franchetii H. Boiss. Wenchuan, Sichuan, China
(四川汶川)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 374629
(SZ)
EU001342
B. longiradiatum Turcz. Yichun, Heilongjiang, China
(黑龙江伊春)
C. B. Wang (王长宝) 200607 (Jia Mu Si Univer-
sity)
EU001341
B. marginatum Wall. ex DC. Mao Xian, Sichuan, China
(四川茂县)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 361530
(SZ)
EU001343
B. marginatum var. stenophyllum
(H. Wolff) R. H. Shan & Y. Li
Wolong, Sichuan, China
(四川卧龙)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 374614
(SZ)
EU001344
B. microcephalum Diels Kangding, Sichuan, China
(四川康定)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 356516
(SZ)
EU001345
B. mundtii Cham. & Schltdl. South Africa RBGE Acc. No. 19972669, S. S. Neves (E)
Herbarium, Royal Botanic Garden Edinburgh, UK
AF479860
B. petiolulatum Franch. Dali, Yunnan, China
(云南大理)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 374622
(SZ)
EU001332
B. plantagineum Desf. Algeria Davis 52959 (RNG) Herbarium, Plant
Science Laboratories, University of Reading, UK
AF479865
B. rockii H. Wolff Lijiang, Yunnan, China
(云南丽江)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 361540
(SZ)
EU001346
B. rigidum L. ssp. rigidum Spain S. S. Neves 53 (E) AF481396
B. rigidum ssp. paniculatum (Brot.) H.
Wolff
Portugal F. Sales & S. S. Neves 3a (E) AF481398
B. scorzonerifolium Willd. Huanan, Heilongjiang, China
(黑龙江桦南)
C. B. Wang (王长宝) 200607 (Jia Mu Si Univer-
sity)
EU001347
B. stellatum L. Switzerland B. de Retz 88690 (MAF 145370) AF481930
B. subspinosum Maire & Weiller Morocco C. J. & A. R. Humphries 99 (BM) AF481931
B. wenchuanense R. H. Shan & Y. Li Wenchuan, Sichuan, China
(四川汶川)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 361535
(SZ)
EU001348
B. yunnanense Franch. Kunming, Yunnan, China
(云南昆明)
Q. Z. Wang & F. D. Pu (王奇志, 溥发鼎) 374574
(SZ)
EU001349
Anginon difforme (L.) B. L. Burtt South Africa All Batten AB 1018 (E) AF459742
A. paniculatum (Thunb.) B. L. Burtt South Africa H. C. Taylor 11271 (E) AF467922

Journal of Systematics and Evolution Vol. 46 No. 2 2008 146
Table 2 Length, divergence, variable sites, parsimony informative sites and mean G+C content in ITS regions of Bupleurum and outgroup
Name Length before
alignment (bp)
Divergence
(%)
Length after
alignment (bp)
Variable sites (bp) Parsimony informative
sites (bp)
Mean G+C
content (%)
ITS1 B=203–208 O=170
B=0–28.17
Both=0–52.11
B=213
O=213
B=96
Both=118
B=72
Both=97
B=60.83
Both=61.30
ITS2 B=223–231 O=231, 234
B=0–33.05
Both=0–43.93
B=239
O=239
B=106
Both=137
B=83
Both=117
B=60.96
Both=61.73
5.8S B=163, 164 O =163
B=0–6.10
Both=0–7.32
B=164
O=164
B=13
Both=16
B=10
Both=13
B=53.90
Both=54.06
ITS B=599–611 O=567–570
B=0–23.92
Both=0–37.00
B=627
O=627
B=221
Both=279
B=171
Both=235
B=58.98
Both=59.49
B=Bupleurum species; O=outgroup; Both=outgroup included.


step during stepwise addition, and tree-bisection-
reconnection (TBR) was used to swap branches.
Characters were unordered and equally weighted.
Gaps were treated as missing data and multistate data
were interpreted as uncertainty. To evaluate clade
support, bootstrap values were calculated from
1000000 replicates. Ten trees were held at each step
during stepwise addition for bootstrap.
Maximum likelihood (ML) analyses were per-
formed under GTR+G model, using gamma distribu-
tion. Other parameters were set as default. Bootstrap
values of the ML test were calculated from 1000
replicates.
1.3 Material and methods of chromosome count-
ing
Each species included at lest 2–3 populations,
and at least 10–30 individuals were observed in each
population. Root-tip cells from seed sprouts were ana-
lyzed for at least 30 cells. A minimum of 50 well
spread cells were counted for each population.
Root tips were pretreated in 0.02 mol/L
8-hydroxyquinoline solution for 2–5 h at room tem-
perature. The root tips were then fixed in ethanol:
acetic acid (3:1) solution for at least 24 h at 4 ℃.
Fixed roots were hydrolysed in 1 mol/L HCl at 60 ℃
for 2–4 min and then washed and stained in modified
Carbol fuchsin for 15–20 min. The permanent prepa-
rations were made by mounting in Canada balsam (Li,
1991). Photomicrographs of well-spread metaphase
cells were taken with an Olympus microscope for
chromosome number counting.
2 Results
2.1 Comparisons and phylogenetic analyses of
ITS sequences
Sequence characteristics of 33 Bupleurum spe-
cies were listed, excluding any ambiguous sites (Table
2). Sequences of the ITS1 region were slightly shorter
and less variable in length than those of ITS2. In the
average 227 bp ITS2 sequences, A+T was about
38.27% and G+C was only 61.73%. The overall value
of transition/transversion was 1.453, tested by the
MEGA version 3.1 program (Kumar et al., 2004). In
the average 202 bp ITS1 sequences, A+T was about
38.70% and G+C was only 61.30%. The overall value
of transition/transversion was 2.435.
In the Chinese Bupleurum group, the ITS diver-
gence was lowest (0.16%) between B. wenchuanense
and B. microcephalum from the Hengduan Mountains,
and this region also has the most divergent pair
(11.48%), B. rockii and B. microcephalum. In the MP
analysis, the highest divergence (23.92%) within
Bupleurum was found between B. rockii from the
Hengduan Mountains and B. angulosum from Medi-
terranean. The maximum parsimony analysis of the
ITS submatrix resulted in 8 most parsimonious trees
(tree length = 505, consistency index (CI, excluding
uninformative characters) = 0.80, retention index (RI)
= 0.89) (Fig. 2). The value (–Ln likelihood) was
2745.65 in the maximum likelihood tree (Fig. 3). The
Bayesian cladogram presented a 50% majority con-
sensus tree, excluding the first 2000 burn-in trees. The
posterior probabilities were mapped under the branch
in Fig. 3.
The topologies were not completely congruent in
MP, ML and Bayesian results (Figs. 2, 3). Four major
lineages (A, B, C and D) can be recognized on the
phylogeny. Clade D, representing the Mediterranean
group, was a sister group to the large clade containing
Clades A, B and C. Bupleurum mundtii was a sister
group to the clade comprising all other taxa within
Clade A. (Figs. 2, 3). Clade C included most African
taxa. The Chinese group included all species from the
Hengduan Mountains, and was divided into two major
clusters, Clade A and B. The relationships between
Clade A, B and C were still unclear (Figs. 2, 3).
In the Chinese taxa, the systematic positions of
B. commelynoideum, B. scorzonerifolium and B.
longiradiatum were clear (Clade B), though the
different positions of B. wenchuanense and B. micro-
cephalum clade confounded matters that were
WANG et al.: Phylogenetic inference of Bupleurum in Hengduan Mountains

147
B. longiradiatum (x = 6)
B. commelynoideum (x =?)



Fig. 2. Strict consensus tree of eight most parsimonious trees of 505 steps derived from maximum parsimony (MP) analysis of sequences of ITS of
Bupleurum and outgroup taxa (CI = 0.80, RI = 0.89). Numbers under branches were bootstrap estimate. Values <50% were not indicated. Chromo-
some numbers were noted in brackets. Grey boxes presented sect. Falcata. White box presented sect. Ranunculoidea.
100
B. rigidum spp. paniculatum ( x = 8)
B. rigidum spp. rigidum (x = 8)
B. dumosum (x =?)
B. rockii (x = 8)
Anginon difforme
Anginon paniculatum
B. wenchuanense ( x = 6)
B. microcephalum ( x = 6)
B. chaishou ( x = 6)
B. marginatum var. stenophyllum ( x = 7)
B. mundtii (x = 8)
B. hamiltonii var. humil ( x = 8)
B. hamiltonii (x = 8)
B. petiolulatum (x =?)
B. longicaule var. franchetii (x = 8)
B. yunnanense (x = 8)
B. longicaule var. amplexicaule (x = ?)
B. chinense ( x = 6)
B. scorzonerifolium ( x = 6)
B. marginatum ( x = 6)
B. gracilipes ( x = 6)
B. canescens (x = 8)
B. subspinosum (x = 8)
B. album (x = 8)
B. benoistii (x =?)
B. plantagineum (x = 7)
B. lateriflorum (x = 7)
B. stellatum (x =?)
B. fruticosum (x = 7)
B. angulosum (x =?)
B. gibraltaricum (x = 7)
Outgroup
Chinese group
African group
Mediterranean group
Clade A
Clade D
100
86
87
94
Clade B
Clade C
100
90
99
100
100
57
87
62
76
100
88
80
100
61
68

52
100
95
100
Journal of Systematics and Evolution Vol. 46 No. 2 2008 148
B. hamiltonii
B. rockii
1.00
1.00
0.93




































Fig. 3. I. Maximum Likelihood (ML) trees (left trees) were under GTR + G model (–Ln likelihood = 2745.65). Numbers under branches were
bootstrap estimates. Values <50% were not indicated. II. Majority rule consensus cladogram resulted from 422218 most Bayesian trees (right trees).
Numbers under branches were posterior probability values. Values <50% were not indicated. Grey boxes presented Bupleurum sect. Falcata. White
box presented sect. Ranunculoidea.

somewhat uncertain. Other groupings (e.g., B. wen-
chuanense and B. microcephalum, B. longicaule var.
amplexicaule and B. petiolulatum, B. marginatum var.
stenophyllum and B. gracilipes) were well supported
by bootstrap values (Figs. 2, 3).
2.2 Results of chromosome number examination
Somatic chromosome numbers of Bupleurum
from the Hengduan Mountains ranged from 2n = 2x =
B. mundtii
B. hamiltonii var. humil
B. petiolulatum
B. longicaule var. amplexicaule
B. longicaule var. franchetii
B. yunnanense
B. commelynoideum
B. wenchuanense
B. microcephalum
B. scorzonerifolium
B. longiradiatum
B. chinense
B. chaishoui
B. marginatum var. stenophyllum
B. marginatum
B. gracilipes
B. canescens
B. subspinosum
B. dumosum
B. benoistii
B. lateriflorum
B. stellatum
B. plantagineum
B. gibraltaricum
B. rigidum ssp. paniculatum
B. rigidum ssp. rigidum
B. fruticosum
B. angulosum
Clade A Clade A
Clade B Clade B
Clade D
Clade D
Clade C Clade C
97 100
84
65
100
99
98 94
100
94
84
96
62 97
87
61
100
1.00
1.00
1.00
0.51
0.56
1.00
1.00
1.00
0.94
1.00
1.00
1.00 1.00
0.56
0.85 0.81
1.00
100
Anginon difforme
Anginon paniculatum

1.00
0.97
0.90
100
63
0.97
B. album
WANG et al.: Phylogenetic inference of Bupleurum in Hengduan Mountains

149
12 to 2n = 2x = 16, except for the tetraploid B. margi-
natum (2n = 4x = 24) (Pan et al., 1985). Diploids
generally appear to be robust enough to grow in
various habitats. For example B. longicaule var.
franchetii shows wide distribution in forests, or grassy
places on alpine slopes from 1000 m to 4000 m.
The chromosome numbers of B. microcephalum,
B. wenchuanense, B. chaishoui and B. gracilipes were
2n = 2x = 12, and those of B. microcephalum and B.
wenchuanense were in agreement with the previous
report by Pimenov et al. (1999). The chromosome
numbers of B. yunnanense, B. rockii, B. hamiltonii
var. humile and B. longicaule var. franchetii were 2n
= 2x = 16. The chromosome numbers of B. chaishoui,
B. gracilipes, B. yunnanense and B. rockii were
reported for the first time. Two varieties, B. hamiltonii
var. humile and B. longicaule var. franchetii, were
also reported for the first time (Fig. 4).

































Fig. 4. Meiotic configurations of Bupleurum species in the Hengduan Mountains. Scale bar = 5 µm. A. B. chaishoui (2n = 12). B. B. gracilipes (2n
= 12). C. B. hamiltonii var. humile (2n = 16). D. B. yunnanense (2n = 16). E. B. microcephalum (2n = 12). F. B. wenchuanense (2n = 12). G. B. rockii
(2n = 16). H. B. longicaule var. franchetii (2n = 16).
Journal of Systematics and Evolution Vol. 46 No. 2 2008 150
3 Discussion
3.1 Origin of Bupleurum and chromosomal
evolution in Hengduan Mountains
Although Bupleurum has recently been recog-
nized as a basal group within subfamily Apioideae
(Plunkett et al., 1996a, b; Downie et al., 1996, 1998,
2000a), the origin of this genus is not clearly resolved.
Since woody growth habit has been regarded as the
ancestral characteristic in Apiaceae, it has been con-
firmed that B. fruticosum in Africa is more basal than
other species in the genus (Gruas-Cavagnetto &
Cerceau-Larrival, 1978). Plunkett et al. (1996b)
proposed that Apioideae (including Bupleurum)
originated from woody ancestors in Africa, and dis-
persed northward through the Middle East into Eura-
sia. However, Calviňo et al. (2006) believed that the
ancestor of subfamily Apioideae was herbaceous,
not woody. They suggested that, within Apiaceae, the
woody habit had evolved multiple times from their
herbaceous ancestors. Bupleurum fruticosum
was secondarily woody, and its woodiness was de-
rived from an herbaceous ancestor. Its woody
habit does not imply that it is more basal than other
Bupleurum species. Furthermore, the genus evolved
from perennial to annual. Neves and Watson (2004)
suggested that the genus Bupleurum originated
somewhere in the western Mediterranean region,
which was the early branching clade (including B.
fruticosum). In our study, although the relationships in
Clade D, including B. fruticosum, the only woody
taxon in Bupleurum, were uncertain, the Clade D was
the basal branch (Figs. 2, 3). Species from the Heng-
duan Mountains were not at the basal position, con-
trary to the hypothesis of the Hengduan Mountains as
a potential origin center (Shu & Sheh, 2001). In
accordance with previous results (Plunkett et al.,
1996b; Neves & Watson, 2004), we presume that the
woody B. fruticosum in North Africa or the species
from the western Mediterranean are the ancestors of
the species in Hengduan Mountains. It may have been
migrated northward into the Hengduan Mountains
through the Middle East and the Caucasus.
Interestingly, the native South African species, B.
mundtii, was nested in the Hengduan Mountains
group, which was a sister group to the clade compris-
ing all other taxa within Clade A in the phylogenetic
trees (Figs. 2, 3). The nucleotide composition of B.
mundtii is similar to the Chinese group, but not to the
African taxa. Furthermore, B. mundtii shares the
primitive pollen type of subrhomboidal (Cerceau-
Larrival, 1962, 1971) with plants in the Hengduan
Mountains (Shu & Sheh, 2001). Our result supports
the conclusion of Neves and Watson (2004) and
Carbonnier and Cauwet-Marc (1981) that B. mundtii
is the closest relative to the species in Eurasia. Neves
and Watson (2004) also suggested that B. mundtii was
neo-endemic to South Africa, rather than a remnant
species from the shrinking of a past larger distribution.
Our chromosomal research can increase knowl-
edge about the cytology of Bupleurum and test the
interspecific correlation between basic chromosome
numbers, which can provide evidence to the inter-
specific division of Chinese Bupleurum species. The
basic chromosome number (x = 8) is common, espe-
cially in B. mundtii (endemic to South African with
primitive pollen type) (Pan & Qin, 1981; Pan et al.,
1985; Qin et al., 1989; Jiang et al., 1994, 2002; Car-
bonnier & Cauwet-Marc, 1981). The results agree
with those of Cauwet-Marc et al.’s (1978). Eight basic
chromosomes in Bupleurum might be regarded as the
primitive state, and x = 6, 7 were derived characters.
The basic chromosome numbers of the species from
the Hengduan Mountains were x = 6, 7, 8. Most of
them were diploid, except B. marginatum, which was
tetraploid (2n = 4x = 24) (Pan et al., 1985). We pro-
pose that the genus Bupleurum in the Hengduan
Mountains may have evolved towards the changing of
the basic chromosome number and ploidy level.
With 15 species and 9 varieties (Zeng & Zeng,
1993), the Hengduan Mountains support about 40% of
the Chinese Bupleurum. The pollen morphology in the
Hengduan Mountains is generally characterized by the
primitive subrhomboidal type formed in Palaeocene
(Shu & Sheh, 2001). Both primitive and derived basic
chromosome numbers are present in this region,
indicating that the Hengduan Mountains may be one
of the modern centers of distributional frequency and
Bupleurum taxon diversity. This region is part of
Mountains of Southwest China, which is identified as
a global biodiversity hotspot (Liu et al., 2000). Its
geological history, unique ecological environment,
and micro-environmental diversity could be reasons
for the retention of primitive characteristics and for
the species abundance in the Hengduan Mountains.
3.2 Infrageneric classification of Bupleurum in
Hengduan Mountains
According to Su et al. (1998), the infrageneric
classification of the Bupleurum in China includes two
subgenera and two sections. The two subgenera
include the monotypic subgenus Longifolia (with B.
longiradiatum only) and subgenus Eubupleura (the
rest of the Chinese taxa). Eubupleura are further
divided into section Ranunculoidea and section Fal-
cata. The taxa sampled in our study represent all
WANG et al.: Phylogenetic inference of Bupleurum in Hengduan Mountains

151
subgenera and sections sensu Su et al. (1998). In our
ITS phylogenetic trees, section Falcata is indicated by
grey boxes and section Ranunculoidea by white
boxes. The two subgenera, as well the two sections,
were intermingled with each other, conflicting with
the classification by Su et al. (1998). However, our
results support two monophyletic clades within the
Chinese taxa (clade A and B), despite their uncertain
relationship with the African group. Clade A with
primitive basic chromosome number (x = 8), is mainly
distributed in grassy slopes or rocky groves habitat in
Yunnan. Clade D with derived basic chromosome
number (x = 6, 7) is mainly distributed in grassland or
the edge of forests in Sichuan, Yunnan and North
China. The only exception in clade D is B. wen-
chuanense, which also has a xerophytic distribution. A
new key to the Chinese species is compiled.

Key to the Chinese Bupleurum in our studying
1a. Bupleurum species mainly distribute in Yunnan xerophytic environment of grass slope and rock grove, rock grove and the basic
chromosome number is x = 8…………………………………………………………………………………………………………2
1b. Bupleurum species mainly distribute in grassland or the edge of forests with mesophytic environment in Sichuan, Yunnan and
North China and the basic chromosome number is x = 6, 7………………………………………………………………………..8
2a. Bracteoles large and conspicuous, ovate and subround…………………………………………………………………………….3
2b. Bracteoles small and narrow, linear-lanceolate, lanceolate and ovate lanceolate…………………………………………………..7
3a. Plant small, less than 25 cm………………………………………………………………………………B. yunnanense 云南柴胡
3b. Plant tall, more than 25 cm………………………………………………………………………………………………………..…4
4a. Cauline leaves tapering into petioles.……………………………………………………………...…..B. petiolulatum 有柄柴胡
4b. Caulines leaves no tapering into petioles…………………………………………………………………………………………….5
5a. Base leaves ovate and elliptic…………………………………..……………………………………………..…B. rockii 丽江柴胡
5b. Base leaves narrowly lanceolate……………………………………………………………………………………………………6
6a. Middle leaves lanceolate-ovate, base cordate…………………………………………B. longicaule var. amplexicaule 抱茎柴胡
6b. Middle leaves lanceolate, base narrow, not cordate……………………………………….B. longicaule var. franchetii 空心柴胡
7a. Plant small, 10–25 cm……………………………………………………………….............B. hamiltonii var. humile 矮小柴胡
7b. Plant tall, 50–80 cm…………………………………………………………………………………………B. hamiltonii 小柴胡
8a. Bracteoles large and conspicuous, ovate and subround…………………………………………………………………………….9
8b. Bracteoles small and narrow, linear-lanceolate, lanceolate and ovate lanceolate…………………………………………………10
9a. Rhizome thick; stylopodium long; petals yellow……………………………………………………..B. longiradiatum 大叶柴胡
9b. Rhizome thin; stylopodium short; petals purple……………………………………………..B. commelynoideum 紫花鸭跖柴胡
10a. Rays 1–3, leaves few and small………………………………………………………………….…..B. wenchuanense 汶川柴胡
10b. Rays more than 3, leaves numerous and large…………………………………………………………………………………….11
11a. Cauline leaves usually reflexed…………………………………………………………………………………B. chaishoui 柴首
11b. Cauline leaves no usually reflexed………………………………………………………………………………………………..12
12a. Root surfaces reddish-brown………………………………………………………………………B. Scorzonerifolium 红柴胡
12b. Root surfaces not reddish-brown………………………………………………………………………………………………..13
13a. Stem dichotomously much branched above…………………………………………………………………..B. chinense 北柴胡
13b. Stem dichotomously not much branched above………………………………………………………………………………..14
14a. Leaves linear………………………………………………………………………………………B. microcephalum 马尾柴胡
14b. Leaves long-lanceolate…………………………………………………………………………………………………………….15
15a. Leaves margin white cartilaginous………………………………………………………………………………………………16
15b. Leaves not margin white cartilaginous…………………………………………………………………..B. gracilipes 细柄柴胡
16a. Bracteoles shorter than pedicels……………………………………………………………………….B. marginatum 竹叶柴胡
16b. Bracteoles longer than pedicels………………………………………………..B. marginatum var. stenophyllum 窄竹叶柴胡

3.3 Species delimitation of Bupleurum in the
Hengduan Mountains
Bupleurum marginatum var. stenophyllum is
treated as a variety of B. marginatum in the Flora of
China. However, this variety was grouped with B.
gracilipes rather than B. marginatum, with strong
support in the ITS phylogeny (Figs. 2, 3). Bupleurum
marginatum var. stenophyllum differs from B. margi-
natum in morphology, palynology (Shu & Sheh,
2001), cytology (Qin et al., 1989; Pan et al., 1985),
and saponins compositions (Ding et al., 1986). No
intermediate individuals have been found between
them. The leaves of B. marginatum var. stenophyllum
are narrow. Its bracteoles are longer than the pedicels.
The polar/equatorial ratio is 1.3 in pollen morphology,
with a size index of 25.9. The characteristic aperture is
Journal of Systematics and Evolution Vol. 46 No. 2 2008 152
rectangular lalongate (Shu & Sheh, 2001). The basic
chromosome number is x=7 in the diploid B. margi-
natum var. stenophyllum (Qin et al., 1989). In con-
trast, the leaves of B. marginatum are wide, and the
bracteoles are shorter than the pedicels. The pollen
polar/equatorial ratio is 1.4, with the size index of
20.9. Pollen apertures are characterized by square
pores (Shu & Sheh, 2001). Bupleurum marginatum is
tetraploid with basic chromosome number x = 6 (Pan
et al., 1985). Bupleurum marginatum var. stenophyl-
lum is primarily distributed in the Hengduan Moun-
tains at high elevations of 2300–4000 m, in the alpine
forests, river banks and roadsides. Bupleurum margi-
natum has a broader distribution. It is not only found
in the Hengduan Mountains but also in southern
Gansu, Guizhou, Hubei and Qinhai provinces at
700–4000 m. This may be the result of increasing
tolerance by tetraploidy. The combined evidence
suggests that B. marginatum var. stenophyllum is not a
variety of B. marginatum. Therefore, B. marginatum
var. stenophyllum should be elevated as a species.
On the contrary, the validation of another variety,
B. hamiltonii var. humile in Flora of China, is ques-
tionable. The morphological difference between B.
hamiltonii var. humile and B. hamiltonii is that the
former is shorter (10–25 cm) and usually has red
stems throughout the plant. According to the field
observation and specimen inspection, we can not
differentiate the two plants since individuals with both
red and green stems, as well as both red and green
leaves are observed in field.
4 Conclusions
The results of our study suggest that the Heng-
duan Mountains is not a center of origin for the genus
Bupleurum, but the frequency and diversity center of
this genus. The Bupleurum species in the Hengduan
Mountains may have evolved from Mediterranean or
African ancestors. Our results disagree with the
previous classification of the Chinese taxa by Su et al.
(1998). Our ITS and cytological evidence strongly
supports the dichotomization (two clades) with
differences in basic chromosome number of the
Chinese Bupleurum. Finally, we suggest that B.
marginatum var. stenophyllum be elevated to the
species level, and that the status of B. hamiltonii var.
humile requires further investigation.
Although Bupleurum is easily recognized by its
simple leaves, conspicuous bracts and bracteoles,
interspecific identification in the Hengduan Mountains
is difficult due to wide morphological variation. More
comprehensive study is necessary to improve our
understandings of this genus.
Acknowledgements We appreciate Prof. Fa-Ding
PU’s effort to authenticate samples. We would like to
thank Prof. Jie-Mei XU from Sichuan University, Dr.
Yang WEI from Georgia University, Dr. Wen LIN
from Virginia Tech., USA, and Esther Yoon from
University of Washington-Seattle for the advices on
the manuscript. We would like to thank Prof.
Chang-Bao WANG and Prof. Cheng-Qiang FENG for
providing samples. We also thank Xiao-Ting XU,
Qiang WANG and Qi ZHENG for valuable help in the
experiment. This work was supported by the National
Natural Science Foundation of China, Grant No.
30670146, and the National Infrastructure of Natural
Resources for Science and Technology, Grant No.
2005DKA21403.
References
Baldwin BG. 1992. Phylogenetic utility of the internal
transcribed spacers of nuclear ribosomal DNA in plants:
an example from the Compositae. Molecular
Phylogenetics and Evolution 1: 3–16.
Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF,
Campbell CS, Donoghue MJ. 1995. The ITS region of
nuclear ribosomal DNA: a valuable source of evidence on
angiosperm phylogeny. Annals of the Missouri Botanical
Garden 82: 247–277.
Carbonnier J, Cauwet-Marc AM. 1981. A comparative
phytochemical investigation of the genus Bupleurum L.
(Umbelliferae). Taxon 30: 617–627.
Cauwet-Marc AM, Carbonnier J, Cerceau-Larrival MT, Dodin
R, Guyot M. 1978. Contribution à l’étude
multidisciplinaire du genre Bupleurum L. In :
Cauwet-Marc AM, Carbonnier J, eds. 1982. les
Ombellifères : contributions pluridisciplinaires à la
systématique : actes du 2ème symposium international sur
les Ombellifères. Centre Universitaire de Perpignan.
18–21 Mai 1977. St Louis. MO: Missouri Botanical
Garden. 623–651.
Calviňo CI, Tilney PM, Wyk BV, and Downie SR. 2006. A
molecular phylogenetic study of southern African
Apiaceae. American Journal of Botany 93: 1828–1847.
Cerceau-Larrival MT. 1962. plantules et pollens
d′Ombellifères. Mémoires du Muséum d’Histoire
Naturelle (Paris). Série B, Botanique 14: 1–166.
Cerceau-Larrival MT. 1971. Morphologie pollinique et
correlations phylogénétiques chez les Ombellifères. In:
Heywood VH, ed. The biology and chemistry of the
Umbelliferae. London: Academic Press. 112–113.
Ding JK, Fujino H, Kasai R, Fujimoto N, Tanaka O, Zhou J,
Matsuura H, Fuwa T. 1986. Chemical evaluation of
Bupleurum species collected in Yunnan, China. Chemical
and Pharmaceutical Bulletin 34: 1158–1167.
DNASTAR, Inc. 2001. Lasergene Sequence Analysis Software.
Madison, Wisconsin WI. 23–33.
Downie SR, Katz-Downie DS. 1996. A molecular phylogeny of
WANG et al.: Phylogenetic inference of Bupleurum in Hengduan Mountains

153
Apiaceae subfamily Apioideae: evidence from nuclear
ribosomal DNA internal transcribed spacer sequences.
American Journal of Botany 83: 234–251.
Downie SR, Ramanath S, Katz-Downie DS, Llanas E. 1998.
Molecular systematics of Apiaceae subfamily Apioideae:
phylogenetic analyses of nuclear ribosomal DNA internal
transcribed spacer and plastid rpoC1 intron sequences.
American Journal of Botany 85: 563–591.
Downie SR, Katz-Downie DS. 1999. Phylogenetic analysis of
chloroplast rps16 intron sequences reveals relationships
among woody southern African Apiaceae subfamily
Apioideae. Canadian Journal of Botany 77: 1120–1135.
Downie SR, Katz-Downie DS, Watson MF. 2000a. A
phylogeny of the flowering plant family Apiaceae based
on chloroplast DNA rpl16 and rpoC1 intron sequences:
towards a suprageneric classification of subfamily
Apioideae. American Journal of Botany 87: 273–292.
Downie SR, Watson MF, Spalik K, Katz-Downie DS. 2000b.
Molecular systematics of Old World Apioideae
(Apiaceae): relationships among some members of tribe
Peucedaneae sensu lato, the placement of several
island-endemic species, and resolution within the apioid
superclade. Canadian Journal of Botany 78: 506–528.
Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for
small quantities of fresh leaf tissue. Phytochemical
Bulletin 19: 11–15.
Drude O. 1898. Umbelliferae. In: Engler A, Prantl K, eds. Die
natürlichen. Pflanzenfamilien. Vol. 3, Part 8. Leipzig:
Wilhelm Engelman. 63–250.
Gruas-Cavagnetto C, Cerceau-Larrival MT. 1978. Presence de
pollens d’Ombellifères fossiles dans le Paleogene du
Bassin Anglo-Parisien: premiers resultats. In:
Cauwet-Marc AM, Carbonnier J, eds. 1982. Les
Ombellifères: contributions pluridisciplinaires à la
systématique: actes du 2ème Symposium International sur
les Ombellifères. Centre Universitaire de Perpignan.
18–21 Mai 1977. St Louis. MO: Missouri Botanical
Garden. 255–267.
Huelsenbeck JP, Ronquist F. 2001. MrBayes: Bayesian
inference of phylogeny. Bioinformatics 17: 754–755.
Jiang C-M (姜传明), Xu N (徐娜), Wang H-Y (王好友), Li R-J
(李瑞军), Liu M-Y (刘鸣远). 1994. Cytotaxonomical
studies on Bupleurum L. in northeast China I. karyology
analysis of six taxa and their significance of taxonomy.
Bulletin of Botanical Research (植物研究) 14: 267–274.
Jiang C-M (姜传明), Lu F (路芳), Peng Y-L (彭一良). 2002.
The number of Bupleurum L. in North of China Journal of
Heilongjiang Nongken Normal College (黑龙江农垦师专
学报) 62: 59–60.
Kumar S, Tamura K, Nei M. 2004. MEGA3: Integrated
Software for Molecular Evolutionary Genetics Analysis
and Sequence Alignment. Briefings in Bioinformatics 5:
150–163.
Liu L-H (刘伦辉), Dao Z-L (刀志灵), Guo H-Jun (郭辉军), Li
H (李恒). 2000. Flora of Gaoligong Mountains In: Li-H
(李恒), Guo H-J (郭辉军), Dao Z-L (刀志灵) eds. 2001.
Flora of Gaoligong (高黎贡山的植被). Beijing: Science
Press.
Li M-X (李懋学). 1991. Technique for plant chromosome.
Harbin: Northeast Forestry University Press. 1–31
Liu M, Shi L, Wyk BV, Tilney PM. 2003. Fruit anatomy of the
genus Bupleurum (Apiaceae) in northeastern China and
notes on systematic implications. South African Journal of
Botany 69: 151–157.
Linczevsky. 1950. Flora URSS. 16: 275–349.
Neves SS, Watson MF. 2004. Phylogenetic relationships in
Bupleurum (Apiaceae) based on nuclear ribosomal DNA
ITS sequence data. Annals of Botany 93: 379–398.
Pan Z-H (潘泽惠), Qin H-Z (秦慧贞). 1981. A report on the
chromosome numbers of Chinese umbelliferae. Acta
Phytotaxonomica Sinica (植物分类学报) 19: 447–450.
Pan Z-H (潘泽惠), Qin H-Z (秦慧贞), Wu Z-J (吴竹君), Yuan
C-Q (袁昌齐), Liu S-L (刘守炉). 1985. A report on the
chromosome numbers of Chinese umbelliferae. Acta
Phytotaxonomica Sinica (植物分类学报) 23: 97–102.
Pimenov MG, Alexeeva TV, Kljuykov EV, Bokova OM. 1999.
IOPB chromosome data 15. Newsletter Internal
Organization Poland biosystem (Pruhonice) 31: 13–16.
Plunkett GM, Soltis DE, Soltis PS. 1996a. Higher level
relationships of Apiales (Apiaceae and Araliaceae) based
on phylogenetic analysis of rbcL sequences. American
Journal of Botany 83: 499–515.
Plunkett GM, Soltis DE, Soltis PS. 1996b. Evolutionary
patterns in Apiaceae: inferences based on matK sequence
data. Systematic Botany 21: 477–495.
Plunkett GM, Soltis DE, Soltis PS. 1997. Clarification of the
relationship between Apiaceae and Araliaceae based on
matK and rbcL sequence data. American Journal of
Botany 84: 565–580.
Plunkett GM, Downie SR. 1999. Major lineages within
Apiaceae subfamily Apioideae: a comparison of
chloroplast restriction site and DNA sequence data.
American Journal of Botany 86: 1014–1026.
Plunkett GM, Downie SR. 2000. Expansion and contraction of
the chloroplast inverted repeat in Apiaceae subfamily
Apioideae. Systematic Botany 25: 648–667.
Posada D, Buckley TR. 2004. Model selection and model
averaging in phylogenetics: advantages of the AIC and
Bayesian approaches over likelihood ratio tests.
Systematic Biology 53: 793–808.
Posada D, Crandall KA. 1998. Modeltest: testing the model of
DNA substitution. Bioinformatics 14: 817–818.
Qin H-Z (秦慧贞), Pan Z-H (潘泽惠), Sheh M-L (佘孟兰), Wu
C-J (吴竹君). 1989. A report on chromosome numbers of
Chinese umbelliferae. Acta Phytotaxonomica Sinica (植物
分类学报) 27: 268–272.
Shu P (舒璞), Sheh M-L (佘孟兰). 2001. Pollen photographs
and flora of Umbelliferae in China (中国伞形科植物花粉
图志). Shanghai Scientific & Technical Publishers. 68–75.
Su P (舒璞), Yuan C-Q (袁昌齐) , Sheh M-L (佘孟兰), Liu Y-Z
(刘一真), Xiang B-R (相秉仁), An D-K (安登魁). 1998.
Numerical taxonomy of medicinal Bupleurum species in
China. Acta Botanica Boreali-Occidentalia Sinica (西北植
物学报) 18: 277– 283.
Swofford DL. 2002. PAUP*. Phylogenetic Analysis Using
Parsimony (*and other methods). Version 4.0b10.
Sunderland, Massachusetts: Sinauer Associates.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins
DG. 1997. The Clustal_X windows interface: flexible
Journal of Systematics and Evolution Vol. 46 No. 2 2008 154
strategies for multiple sequence alignment aided by
quality analysis tools. Nucleic Acids Research 24:
4876–4882.
Tian X (田欣), Li D-Z (李德铢). 2002. Application of DNA
sequences in plant phylogenetic study. Acta Botanica
Yunnanica (云南植物研究) 24: 170–184.
Wu Y (武莹), Liu C-S (刘春生), Liu Y-F (刘玉法), Yan Y-N
(阎玉凝). 2005. ITS sequence identification of Radix
Bupleuri. China Journal of Chinese Materia Medica (中国
中药杂志) 30: 732–734.
Wolff H. 1910. Bupleurum In: Engler A ed. Das Pflanzenreich
Regnis Vegetabilis Conspectus, IV. 228. Leipzig: Verlag
von Wilhelm Engelmann. 36–173.
Xie H (谢晖), Chao Z (晁志), Huo K-K (霍克克), Wu B-Y (吴
炳义 ), Pan S-L (潘胜利 ). 2006. ITS sequence of 9
Bupleurum species and its application in identification of
chaihui (Radix Bupleuri). Journal of Southern Medical
University (南方医科大学学报) 26: 1460–1463.
Zeng J-F (曾建飞), Zeng G-F (曾桂芳). 1993. Vascular plants
in Hengduan Mountains. Beijing: Science Press.
1301–1308.
基于染色体计数和 ITS序列初步探讨横断山区柴胡属植物
(伞形科)的系统发育
王奇志* 何兴金** 周颂东* 吴耘珂 余 岩 逄云莉
（四川大学生命科学学院, 植物系统进化实验室 成都 610064）

摘要 横断山区是中国柴胡属Bupleurum植物的分布中心。本文对横断山区6个种2变种进行了染色体记数报道, 其中4个种2
变种是首次报道。对横断山区的10个种4个变种、中国北方(河北和黑龙江)的3个种的nrDNA ITS进行测序, 同时从GenBank
里面下载同属的来自非洲和地中海西部的16个nrDNA ITS序列数据, 结合染色体数目变化结果, 初步探讨了横断山区柴胡属
植物的系统发育。结果表明横断山区可能是现代柴胡属植物的频度中心和多样分布中心之一。它们的祖先种可能是非洲北部
的木本柴胡属植物B. fruticosum, 或者是地中海西部的柴胡属植物, 推测是通过中东和高加索扩散而形成的, 其中与非洲南
部特有种B. mundtii的亲缘关系也较近; 染色体基数演化趋势是: 8是较原始基数, 6和7是次生基数, 其染色体异基数变异和多
倍化可能是物种形成、进化以及向外扩散的主要方式; 在ITS系统发育树中, 中国柴胡属植物染色体基数为8的种类聚为一支,
染色体基数为6和7的种类聚为了一支, 不支持舒璞等(1998)关于中国柴胡属的属下分类系统。结合已有的形态学、细胞学、
孢粉学证据和ITS系统发育树, 建议窄竹叶柴胡B. marginatum var. stenophyllum独立成种。
关键词 柴胡属; 染色体; 横断山区; ITS序列; 系统发育; 分类