全 文 ：Journal of Systematics and Evolution 46 (2): 213–219 (2008) doi: 10.3724/SP.J.1002.2008.07036
(formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com
A molecular phylogenetic study of Huperziaceae based on chloroplast
rbcL and psbA-trnH sequences
1, 3Sheng-Guo JI 2Ke-Ke HUO 1Jun WANG 1Sheng-Li PAN*
1(School of Pharmacy, Fudan University, Shanghai 200032, China)
2(School of Life Sciences, Fudan University, Shanghai 200433, China)
3(Guangdong Pharmaceutical University, Guangzhou 510006, China)
Abstract Phylogenetic relationships within Huperziaceae were studied using chloroplast rbcL gene and
psbA-trnH intergenic spacer sequences. In the rbcL sequence analysis, 25 species were analyzed and they were
resolved into two major clades. One clade corresponded with sections Huperzia and Serratae of the genus Hu-
perzia, while the other clade contained Phlegmariurus and the tropical species of Huperzia. Therefore, Phleg-
mariurus is derived from Huperzia. In the psbA-trnH sequence analysis, 17 species were sampled and they were
resolved into two major clades with high bootstrap values. Both Chinese species of Huperzia and Phlegmariurus
formed sister groups, and within Chinese Huperzia, sect. Huperzia and sect. Serratae formed sister groups.
Key words Huperziaceae, phylogenetic, psbA-trnH, rbcL.
Huperziaceae are an ancient group of plants dis-
tributed mainly in China, the Americas and Europe,
members of which are often commonly known as
firmosses or fir clubmosses. In China, the Hu-
perziaceae have received considerable interest from
the scientific community as members of this family
have been used extensively in the treatment of human
ailments. Indeed, recent reports have revealed that
approximately 33 species are used for medicinal
purposes, the medicinal properties of which maybe
partly explained by the presence of biologically active
alkaloids. Recent evidence has demonstrated that
these alkaloids, particularly huperzine A, have a wide
range of biological activities particularly towards
neurodegenerative diseases (Ma et al., 2006). Given
the potential pharmacological importance of this
family, it is imperative that we gain further insights
into the taxonomic relationships of species in this
family, particularly those found in China, that have
yet been subjected to any major phylogenetic analysis.
In 1944 the family Huperziaceae was comprised
of a single genus, Huperzia Bernh. (Rothmaler, 1944).
However, since the proposed inclusion of the genus
Phlegmariurus (Herter) Holub by Holub (1964), the
taxonomy of this family has become complicated and
hotly debated among taxonomists. Ching (1981, 1982)
proposed a taxonomic treatment for Huperziaceae in
China, dividing Huperziaceae into two separate genera
Huperzia Bernh. and Phlegmariurus (Herter) Holub.
Øllgaard (1987) recognized 22 informal groups in
Huperziaceae and Lycopodiaceae. Zhang and Kung
(1998, 2000a) supported Ching’s viewpoint and
further divided the genus Huperzia into two sections
namely, sect. Huperzia and sect. Serratae (Rothm.)
Holub. Each of these sections has unique morpho-
logical characters; species of sect. Huperzia have
entire leaves, whereas those of sect. Serratae possess
serrate or denticulate leaves. The genus Phlegmariu-
rus in China has been divided into three sections: sect.
Huperzioides H. S. Kung & L. B. Zhang, sect. Cari-
naturus (Herter) H. S. Kung & L. B. Zhang, and sect.
Phlegmariurus (Zhang & Kung, 1999, 2000b). Wik-
ström et al. (1999) and Wikström and Kenrik (2000a,
b) examined phylogenetic relationships of Hu-
perziaceae and Lycopodiaceae based on nucleotide
sequences of the chloroplast rbcL gene and trnL-trnF
intergenic spacer. Their results supported the species
groups recognized by Øllgaard (1987), and recognized
a clade containing the neotropical and paleotropical
species of Huperzia. However, the rbcL sequence
divergence among neotropical species of the ancient
genus Huperzia is extremely low and additional data
are necessary to resolve the relationships among
epiphytes and ground-living species. Yatesentyk et al.
(2001) used nucleotide sequences of a chloroplast
rDNA to examine relationships of Lycopodiaceae, and
suggested that Lycopodium and Lycopodiella were
sister taxa, and both genera had higher variability
than the species of Huperzia. Their study also sug-
gested that the H. selago group was separated into a
genus.
In this study a phylogenetic analysis of Hu-
perziaceae was conducted using sequence data from

———————————
Received: 5 March 2007 Accepted: 14 July 2007
* Author for correspondence. E-mail: .
Journal of Systematics and Evolution Vol. 46 No. 2 2008 214
the chloroplast gene rbcL and psbA-trnH intergenic
spacer to develop a more detailed phylogenetic
framework for this family.
1 Material and methods
1.1 Plant materials
Fifteen species were obtained from China and
one species from Borneo of Malaysia (Table 1). Total
genomic DNA was extracted using a plant DNA
Extraction Mini Kit (Watson Biotechnologies, Inc.)
from 2 g of fresh or 1 g of silica gel dried plant mate-
rial.
1.2 PCR amplification and direct sequencing
1.2.1 Primer design The forward primer for
amplifying the psbA-trnH intergenic spacer was
designed to correspond to the region of the conserved
psbA gene in Nicotiana tabacum L. of Solanaceae
(Shinozaki et al., 1986) and Brassica napus L. of
Brassicaceae (GenBank No. M36720). The reverse
primer is located in a region of the trnH gene that is
conserved among Nicotiana tabacum, Helianthus
annuus L. (Asteraceae) (GenBank No. X60428), and
Arabidopsis thaliana (L.) Heynh. (Brassicaceae)
(GenBank No. X79898). The rbcL gene primers
(Wikström & Kenrick, 1997) are shown in Table 2.
1.2.2 PCR amplification The polymerase chain
reaction (PCR) amplification was carried out in a
PTC-100TM Peltier Thermal Cycle (MJ Research,
INC), in a reaction volume of 50 µL. To amplify rbcL,
each reaction contained 5.0 µL of 10×reaction buffer
and 0.5 µL (1 U) of Taq DNA polymerase (Takara
Biotechnology (Dalian) Co. Ltd), 3.0 µL of dNTPs
(dATP dCTP dGTP dTTP each at 2.5 mmol/L), 2.0
µL each of primers rbcL 1F and rbcL 2R at 10
mmol/L, and 6.0 µL (20 ng) of genomic DNA. The
reaction was adjusted to the final volume of 50 µL
with ddH2O. PCR amplification reactions were con-
ducted using the following settings: pre-denaturing at
95 ℃ for 7 min; followed by 30 cycles of 45 s at 95
℃ denaturing, 70 s at 55 ℃ annealing, and 50 s at
72 ℃ extension; and a final extension for 5 min at 72
℃. To amplify psbA-trnH spacer, each PCR reaction
contained 5.0 µL of 10×reaction buffer, 0.5 µL (1U)
of Taq DNA polymerase, 2.5 µL of dNTPs, 2.0 µL
each of primers psbA-trnH F and psbA-trnH R at 10
mmol/L, and 5.0 µL (20 ng) genomic DNA. The
reaction was adjusted with ddH2O to a final volume of
50 µL. PCR amplification reaction conditions are as
follows: pre-denaturing at 94 ℃ for 5 min; 25 cycles
of 45 s at 94 ℃ denaturing, 45 s at 50 ℃ annealing,
and 50 s at 72 ℃ extension; and a final extension for
5 min at 72 ℃.
1.2.3 Purification and sequencing PCR products
were subsequently purified using a Gel Extraction
Mini Kit (Watson Biotechnologies, Inc.). The ABI
Prism™, BigDye™ Terminator and Cycle Sequencing
Ready Reaction Kit was used for sequencing with
primers rbcL 1F, rbcL 2F, rbcL 3F, and rbcL 1R for
the rbcL gene, and primers psbA-trnH F and
psbA-trnH R for psbA-trnH intergenic spacer. The
sequences were analyzed by an ABI prism 377 DNA
Analyzer (Applied Biosystems, Foster City, CA).
Several clones of the same PCR product were
sequenced to check whether there were errors intro-
duced by Taq polymerase. In addition, the DNA
fragments were sequenced from ten samples and the
consensus sequences were obtained from all sampled
species.
1.3 DNA sequence analysis and phylogenetic
analysis
Sequence alignment was performed using Clustal
X version 1.8 (Thompson et al., 1997). Phylogenetic
analyses were done by the maximum parsimony (MP)
and neighbor-joining (NJ) methods using PHYLIP
(Felsenstein, 1993) and Mega 3.1 (Kumar et al., 2004)
software packages. For all analyses, gaps were treated
as missing data, and no sites containing insertion/
deletions were excluded. NJ analysis was conducted
employing an HKY85 model (Hasegawa et al., 1985)
to estimate the distances between sequences. Parsi-
mony search options included 100 random addition
sequences, tree bisection-reconnection branch-swap-
ping, and retention of multiple parsimonious trees.
The internal support was evaluated by bootstrap
analysis (Felsenstein, 1985) and decay indices
(Bremer, 1988, 1994). In the parsimony analysis, each
of the 1000 bootstrap replicates was analyzed with the
heuristic search option using one random additional
replicate, and without the retention of multiple parsi-
monious trees.
2 Results
2.1 rbcL gene
The obtained sequences have been submitted to
GenBank (Table 1). The entire rbcL gene was
1366–1429 bp long, with 136 variable sites, and 58
parsimony informative sites. Sixteen equally most
parsimonious trees have been found with a length of
322 steps, a consistency index (CI) of 0.848 and a
retention index (RI) of 0.859. The topologies of the
JI et al.: A molecular phylogenetic study of Huperziaceae

215
Table 1 Source of materials
GenBank accession No. Taxon Voucher Locality Species group or section
rbcL psbA-trnH
Huperzia appressa (Desv.) Á. Löve & D.
Löve (伏贴石杉)
S. L. Pan (潘胜利)
WJ0409048 (SHMU)
Mt. Changbai, Jilin,
China (吉林长白山)
sect. Huperzia Bernh. DQ464220 DQ464203
H. crispata (Ching ex H. S. Kung) Ching
(皱边石杉)
S. L. Pan (潘胜利)
WJ0503019 (SHMU)
Mt. Xuefeng, Hunan,
China (湖南雪峰山)
sect. Serratae (Rothm.)
Holub.
DQ464221 DQ464204
H. emeiensis (Ching & H. S. Kung)
Ching & H. S. Kung (峨眉石杉)
S. L. Pan (潘胜利)
WJ0409023 (SHMU)
Xishui, Guizhou,
China (贵州习水)
sect. Huperzia DQ464222 DQ464205
H. serrata var. longipetiolata (Spring)
H.-M. Chang (长柄石杉)
S. L. Pan (潘胜利)
JSG0504181 (SHMU)
Mt. Xuefeng, Hunan,
China (湖南雪峰山)
sect. Serratae DQ464224 DQ464207
H. miyoshiana (Makino) Ching (东北石
杉)
S. L. Pan (潘胜利)
WJ0409101 (SHMU)
Mt. Changbai, Jilin,
China (吉林长白山)
sect. Huperzia DQ464225 DQ464208
H. nanchuanensis (Ching & H. S. Kung)
Ching & H. S. Kung (南川石杉)
S. L. Pan (潘胜利)
WJ0408011 (SHMU)
Nanchuan, Chongqing,
China（重庆南川）
sect. Huperzia DQ464226 DQ464209
H. selago (L.) Bernh. ex Schrank &
Mart. (小杉兰)
S. L. Pan (潘胜利)
WJ0409041 (SHMU)
Mt. Changbai, Jilin,
China (吉林长白山)
sect. Huperzia DQ464227 DQ464210
H. serrata (Thunb. ex Murray ) Trevis.
(蛇足石杉)
S. L. Pan (潘胜利)
WJ0408261 (SHMU)
Mt. Changbai, Jilin,
China (吉林长白山)
sect. Serratae DQ464228 DQ464211
H. sutchueniana (Herter ) Ching (四川石
杉)
S. L. Pan (潘胜利)
JSG0504192 (SHMU)
Mt. Xuefeng, Hunan,
China (湖南雪峰山)
sect. Serratae DQ464212
Phlegmariurus carinatus (Desv.) Ching
(龙骨马尾杉)
S. L. Pan (潘胜利)
WJ0207129 (SHMU)
Xishuangbanna,
Yunnan, China (云南
西双版纳)
sect. Carinaturus
(Herter) H. S. Kung &
L. B. Zhang
DQ464229 DQ464213
Ph. fargesii (Herter) Ching (金丝条马尾
杉)
S. L. Pan (潘胜利)
WJ0404031 (SHMU)
Mt. Dayao, Guangxi,
China (广西大瑶山)
sect. Carinaturus DQ464230 DQ464214
Ph. fordii (Baker) Ching (福氏马尾杉) S. L. Pan (潘胜利)
WJ0207151 (SHMU)
Changting, Fujian,
China (福建长汀)
sect. Huperzioides H.
S. Kung & L. B. Zhang
DQ464231 DQ464215
Ph. minchegensis (Ching) L. B. Zhang
(闽浙马尾杉)
S. L. Pan (潘胜利)
WJ0112127 (SHMU)
Liancheng, Fujian,
China (福建连城)
sect. Huperzioides DQ464232 DQ464216
Ph. petiolatus (C. B. Clarke) H. S. Kung
& L. B. Zhang (有柄马尾杉)
S. L. Pan (潘胜利)
WJ0107181 (SHMU)
Sanming, Fujian,
China (福建三明)
sect. Huperzioides DQ464233 DQ464217
H. phyllantha (Hook. & Arn.) Holub S. L. Pan (潘胜利)
MLB- 03001 (SHMU)
Malaysia DQ464234 DQ464218
Ph. squarrosus (Forst.) L. Löve & D.
Löve (粗糙马尾杉)
S. L. Pan (潘胜利)
WJ0207111 (SHMU)
Xishuangbanna,
Yunnan, China (云南
西双版纳)
sect. Huperzioides DQ464235 DQ464219
Huperzia campiana B. Øllg. H. phlegmaria group X98282
H. cumingii (Nessel) Holub H. saururus and H.
brevifolia group
Y07930
H. hippuridea (Christ) Holub H. brongniartii group Y07931
H. linifolia (L.) Trevis. H. linifolia group Y07932
H. selago (L.) Bernh. ex Schrank &
Mart. (小杉兰)
H. selago group Y07934
H. wilsonii (Underw. & F. E. Lloyd) B.
Øllg.
H. mandiocana group Y07933
H. billardieri (Spring) Trevis. H. myrtifolia group AJ133894
H. hippuris (Poiret) Trevis. H. hippuris group AJ133895
H. phlegmarioides (Gaudich.) Rothm. H. phlegmaria group AJ133896
H. verticillata (L. f.) Rothm. H. verticillata group AJ133897
H. lucidula (Michx.) Trevis. H. selago group NC006861 NC006861
Lycopodium scariosum G. Forst. Lycopodium group AJ133255
Lycopodium jussiaei Desv. Lycopodium group AJ133256
Lycopodiella cernua (L.) Pichi-Serm. Lycopodiella group AJ133258
Lycopodiella glaucescens (C. Presl) B.
Øllg.
Lycopodiella group AJ133260
Phylloglossum drummondii Kunze Y07939
Selaginella selaginoides (L.) Link Out group Y07940
Selaginella uncinata (Desv.) Spring Out group NC007625




Journal of Systematics and Evolution Vol. 46 No. 2 2008 216
Table 2 Primers used to amplify and sequence rbcL gene and psbA-trnH intergenic spacers in Huperziaceae
Direction Primers name Primer sequences (5′→3′) 5′ position
Forward rbcL 1F ATGTCACCACAAACGGA 1
Forward rbcL 2F CGTCCTTTATTAGGATGTAC 500
Forward rbcL 3F GTCTACTTCTTCACATTCAC 863
Reverse rbcL 1R GCCAAAGCATTACGTATGTC 944
Reverse rbcL 2R TCAAATTCAAACTTGATTTCTTTCCA 1409
Forward psbA-trnH F GTTATGCATGAACGTAATGCTC 1
Reverse psbA-trnH R CGCGCATGGTGGATTCACAATC 416


MP tree and NJ tree were similar. However, different
bootstrap values were obtained for some branches
(Fig. 1).
Phylloglossum was sister to Huperziaceae (99%
and 100%). There were two clades in the family: the
neotropical-paleotropical clade (designated as A)
included some Huperzia species and those of Phleg-
mariurus (bootstrap = 100%), and the other contained
the remaining species of Huperzia (B, bootstrap =
98%, 100%). Within clade B there were two subclades
corresponding to sections Huperzia (C) and Serratae
(D), respectively. Neither section (Carinaturus and
Huperzioides) within Chinese Phlegmariurus formed
clades.
2.2 psbA-trnH intergenic spacer
The entire psbA-trnH intergenic spacer is 325 bp
long, with 36 variable sites, and 28 parsimony infor-
mative sites. Parsimony analyses produced fourteen
equally most parsimonious trees of 131 steps, with a
CI of 0.9465 and a RI of 0.9271. The MP and NJ trees
showed similar relationships, but different bootstrap
values were observed in some branches (Fig. 2).
Huperzia and Phlegmariurus each formed robust
clades; however, no tropical species of Huperzia was
included in the analyses due to the lack of material. As
in the rbcL tree, Huperzia included two major groups,
sect. Huperzia (bootstrap 96% and 98%) and sect.
Serratae (bootstrap 95%). As in the rbcL analyses,
monophyly of neither section (Carinaturus and Hu-
perzioides) was recovered in Phlegmariurus.
3 Discussion
3.1 Huperziaceae and Lycopodiaceae
Some botanists recognized only two genera of
isosporous Lycopsida (Lycopodium and Phylloglos-
sum, e.g., Filin, 1978) in Lycopodiaceae, while others
divided Lycopodium into seven genera (Wagner &
Beitel, 1992). Øllgaard (1987) considered Lycopodi-
aceae as having four genera (Huperzia, Lycopodium,
Lycopodiella, and Phylloglossum). In the rbcL tree
(Fig. 1), Phlegmariurus is embedded within Huperzia,
suggesting that the genus should be merged within
Huperzia. Therefore, Huperziaceae can be considered
monotypic, and Huperziaceae should be combined
with Lycopodiaceae (Holub, 1985; Øllgaard, 1987;
Crane, 1990; Wagner & Beitel, 1992).
3.2 Interspecific relationships within Huperzi-
aceae
Huperzia species are small, ground-growing
plants or epiphytes with upright stem. The sporophyll
is smaller than the trophophyll. Leaves are serrate or
entire. Phlegmariurus plants are larger than Huperzia
plants. They are epiphytes with bent or drooping
caudex. The sporophyll is similar to or different from
trophophyll. Leaves are entire. Therefore, Ching
(1981, 1982) recognized the two genera based on the
morphological differences. In the rbcL tree, however,
some tropical species of Huperzia are more closely
related to those of Phlegmariurus than to other species
of Huperzia. Therefore, our rbcL data do not support
the separation of the two genera.
Within the clade B of Huperzia (Figs. 1, 2) there
are two major groups: sect. Huperzia and sect. Hu-
perzioides. Thus our results agree with Zhang and
Kung’s treatment (1998, 2000a). Ching (1981, 1982)
divided Chinese Phlegmariurus into sections Carina-
turus and Huperzioides. In both rbcL and psbA-trnH
trees (Figs. 1, 2), neither section formed clades.
Therefore, detailed morphological analyses of the
species are needed to identify potential synapomor-
phies for subgroups of Chinese Phlegmariurus.
3.3 Systematic positions of some Chinese species
Leaves of H. nanchuanensis are entire, incurvate
and narrowly lanceolate, suggesting that it is closely
related to sect. Huperzia (Zhang & Kung, 1998). In
the rbcL tree, H. nanchuanensis was placed in the
sect. Serratae clade with weak support. However, in
the psbA-trnH tree, it fell in the sect. Huperzia clade.
It may be appropriate to include H. nanchuanensis in
sect. Huperzia. Nevertheless, more data are needed to
further test the systematic position of this species.
JI et al.: A molecular phylogenetic study of Huperziaceae

217


Fig. 1. The strict consensus tree constructed based on rbcL gene sequences of 25 species of Huperziaceae using MP and NJ
analysis in Mega 3.1 package. Bootstrap values (1000 replicates) are shown above branches (MP) and under branch (NJ). Taxa
underlined were sequenced in this study.


Huperzia serrata var. longipetiolata is similar to
H. serrata in morphology. Zhang and Kung (1998)
treated this species as a variant of H. serrata. How-
ever, Yang (1989) has treated it as a species distinct
from H. serrata. In the rbcL tree, H. serrata var.
longipetiolata is grouped with H. serrata with moder-
ate bootstrap support (81%, 84%). In the psbA-trnH
tree, H. serrata is clustered with H. emeiensis and H.
sutchueniana in one clade with poor bootstrap support
(57%, 61%), and then together they are grouped with
H. serrata var. longipetiolata with 78% and 82%
bootstrap support. Therefore, as suggested by Zhang
and Kung (1998), the four species are closely related
and they may represent a species complex.
Journal of Systematics and Evolution Vol. 46 No. 2 2008 218



Fig. 2. The strict consensus tree constructed based on psbA-trnH intergenic spacer sequences of 17 species of Huperziaceae using
MP and NJ analysis in Mega 3.1 package. Bootstrap values (1000 replicates) are shown above branches (MP analysis) and under
branch (NJ analysis).


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基于叶绿体基因 rbcL和 psbA-trnH间区探讨石杉科
植物系统关系
1,3姬生国 2霍克克 1王 峻 1潘胜利*
1(复旦大学药学院 上海 200032)
2(复旦大学生命科学学院 上海 200433)
3(广东药学院中药学院 广州 510006)
摘要 关于石杉科Huperziaceae植物的分类, 一直存在一些争议。在旧的分类体系中石杉科植物被包含在一个混合的石松科
Lycopodiaceae和多谱系的石松属Lycopodium中。本文利用叶绿体rbcL基因和psbA-trnH基因间区序列探讨石杉科植物的系统
位置及石杉科内部的分类关系, 用最大简约法和邻接法对自测序列结合由GenBank下载的rbcL及psbA-trnH基因间区序列进
行系统发育分析。结果显示, 石杉科与Phylloglossum属关系较近, 与石松科关系较疏远。在石杉科中热带石杉属Huperzia植
物和马尾杉属Phlegmariurus植物的关系要比它们与其他石杉属植物更近。所以, 我们的rbcL基因数据不支持秦仁昌关于石杉
科分为石杉属和马尾杉属的分类处理。但是, 因为我们的psbA-trnH序列没有包括热带种类, 对石杉属植物和马尾杉属植物的
关系无验证。因此需要更多的样品和序列数据进一步探讨石杉科的演化关系。
关键词 石杉科; 系统发育; psbA-trnH; rbcL基因