November 2010 Note Biol. Pharm. Bull.

33(11) 1919—1924 (2010) 1919

Species Identification of Medicinal Pteridophytes by a DNA Barcode

Marker, the Chloroplast psbA-trnH Intergenic Region
Xin-Ye MA,a,† Cai-Xiang XIE,a,# Chang LIU,a Jing-Yuan SONG,a Hui YAO,a Kun LUO,b Ying-Jie ZHU,c
Ting GAO,a Xiao-Hui PANG,a Jun QIAN,a and Shi-Lin CHEN*,a
a
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences; Beijing 100193, P. R. China: b Hubei
University of Chinese Medicine; Wuhan 430061, P. R. China: and c School of Bioscience and Engineering, Southwest
Jiaotong University; Chengdu 610031, P. R. China.
Received March 14, 2010; accepted August 24, 2010; published online September 1, 2010

Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no
simple and universal way to differentiate various species of this group by morphological traits. A novel technol-
ogy termed “DNA barcoding” could discriminate species by a standard DNA sequence with universal primers
and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pterido-
phyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and
matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best
candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of
medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic
pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants.
We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase
chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Com-
bined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis
showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction
with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed
that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than
the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species
identification in medicinal pteridophytes.
Key words medicinal pteridophyte; psbA-trnH intergenic region; species identification

Medicinal pteridophytes have a long history in traditional
Chinese medicine because they are an abundant source of
chemical compounds with a wide range of pharmacological
activities.1) Recently, Huperzia species of this group of me-
dicinal plants has been used to treat patients with Alzheimer’s
disease.2) However, there is a long-standing problem of mix-
ing authentic species with their adulterants in medicinal
preparations. This problem has consequences for medical
safety as well as for the conservation of the authentic
species.3—5) The classical morphological authentication
approach was confronted with difficulties due to overly simi-
lar traits used for taxonomic characterization6,7) and an ever-
decreasing number of specialists.
A new technology for rapid, accurate and convenient
species identification termed “DNA barcoding” was recently
developed.8—10) DNA barcoding uses a standard DNA
sequence that can be amplified by universal primers and that
possesses sufficient variation. A segment of the mitochon-
drial cytochrome c oxidase I gene is widely accepted as the
standard barcode region in animals.11) In plants, however, no
equivalently accepted DNA barcode has been identified,
despite the fact that many resolutions have been proposed in
the leading literature.12—16)
Previous studies has demonstrated the value of DNA bar-
coding technology in authentication of medicinal plants
species from some taxa,17,18) but DNA-based species identifi-
†
Present address: Research Center of Chinese Herbal Resource Science
and Engineering, Guangzhou University of Chinese Medicine; Guangzhou
510006, P. R. China.
∗ To whom correspondence should be addressed. e-mail: slchen@implad.ac.cn
#
Equal contribution with first author.
cation using a large set of medicinal pteridophytes has not
been carried out. Until now, most studies of pteridophyte
DNA sequences have focused on phylogenetic place-
ment.19,20) Results derived from pteridophyte samples with
small coverage may not be readily extendable to other pteri-
dophytes.12,13,21) Furthermore, some of the studies suggesting
the possibility of species identification using standard DNA
sequences were carried out within a narrow pteridophyte
taxon (such as the family).22) The goal of this paper was to
test the feasibility of species identification in medicinal pteri-
dophytes across a wider taxon range with a convenient DNA
marker. To select the best DNA barcode, we conducted a
series of tests with criteria such as PCR amplification
efficiency, direct sequencing success rate and separability
between species.
MATERIALS AND METHODS
Samples and DNA Sequences Used in This Study A
total of 79 medicinal pteridophyte samples were used in this
study (Table 1). These samples represented 51 species across
24 families, and they included all 11 authentic species listed
in the Chinese pharmacopoeia (2005 version) and some com-
monly used adulterants. The voucher samples were authenti-
cated by Prof. Yulin Lin of IMPLAD (the Institute of Me-
dicinal Plant Development), Chinese Academy of Medicinal
Sciences and deposited in the Herbarium IMPLAD.
In addition, 109 sequences (Table 2) representing 54
species from 12 pteridophyte families were downloaded from
GenBank for species identification.
© 2010 Pharmaceutical Society of Japan
1920 Vol. 33, No. 11

Table 1. Experimental Materials Used for Species Identification with the Sequence of psbA-trnH Intergenic Region

PCR Sequencing Accession

Voucher number Species Family
result result number

PS0085MT01 Cibotium barometz Dicksoniaceae ⫹ ⫹ GU592445—

PS0085MT02 Cibotium barometz Dicksoniaceae ⫹ ⫹ GU592497
PS0086MT01 Woodwardia prolifera Blechnaceae ⫹ ⫹
PS0361MT01 Pteris multifida Pteridaceae ⫹ ⫹
PS0361MT03 Pteris multifida Pteridaceae ⫹ ⫹
PS0364MT01 Pteris vittata Pteridaceae ⫹ ⫹
PS0370MT01 Humata repens Davalliaceae ⫹ ⫹
PS0372MT01 Allantodia crenata var. crenata Athyriaceae ⫹ ⫹
PS0374MT01 Blechnum orientale Blechnaceae ⫹ ⫹
PS0374MT02 Blechnum orientale Blechnaceae ⫹ ⫹
PS0374MT03 Blechnum orientale Blechnaceae ⫹ ⫹
PS0374MT04 Blechnum orientale Blechnaceae ⫹ ⫹
PS0376MT01 Bolbitis heteroclita Bolbitidaceae ⫹ ⫹
PS0377MT01 Lemmaphyllum microphyllum Polypodiaceae ⫹ ⫹
PS0378MT01 Phymatodes cuspidata Polypodiaceae ⫹ ⫹
PS0379MT01 Colysis wrightii Polypodiaceae ⫹ ⫹
PS0382MT01 Pyrrosia lingua Polypodiaceae ⫹ ⫹
PS0382MT02 Pyrrosia lingua Polypodiaceae ⫹ ⫹
PS0384MT01 Colysis digitata Polypodiaceae ⫹ ⫹
PS0385MT01 Matteuccia struthiopteris Onocleaceae ⫹ ⫹
PS0385MT02 Matteuccia struthiopteris Onocleaceae ⫹ ⫹
PS0386MT01 Tectaria phaeocaulis Aspidiaceae ⫹ ⫹
PS0390MT03 Drynaria fortunei Drynariaceae ⫹ ⫹
PS0390MT04 Drynaria fortunei Drynariaceae ⫹ ⫹
PS0391MT01 Drynaria bonii Drynariaceae ⫹ ⫹
PS0393MT01 Drynaria sinica Drynariaceae ⫹ ⫹
PS0394MT01 Nephrolepis auriculata Nephrolepidaceae ⫹ ⫹
PS0394MT03 Nephrolepis auriculata Nephrolepidaceae ⫹ ⫹
PS0395MT01 Lygodium japonicum Lygodiaceae ⫹ ⫹
PS0395MT02 Lygodium japonicum Lygodiaceae ⫹ ⫹
PS0395MT03 Lygodium japonicum Lygodiaceae ⫹ ⫹
PS0395MT04 Lygodium japonicum Lygodiaceae ⫹ ⫹
PS0395MT05 Lygodium japonicum Lygodiaceae ⫹ ⫹
PS0395MT06 Lygodium japonicum Lygodiaceae ⫹ ⫹
PS0396MT01 Lygodium scandens Lygodiaceae ⫹ ⫹
PS0396MT02 Lygodium scandens Lygodiaceae ⫹ ⫹
PS0719MT01 Pteris semipinnata Pteridaceae ⫹ ⫹
PS0719MT02 Pteris semipinnata Pteridaceae ⫹ ⫹
PS0726MT01 Selaginella uncinata Selaginellaceae ⫹ ⫹
PS0726MT02 Selaginella uncinata Selaginellaceae ⫹ ⫹
PS0726MT03 Selaginella uncinata Selaginellaceae ⫹ ⫹
PS0727MT01 Selaginella moellendorffii Selaginellaceae ⫹ ⫹
PS0728MT01 Selaginella tamariscina Selaginellaceae ⫹ ⫹
PS0728MT02 Selaginella tamariscina Selaginellaceae ⫹ ⫹
PS0729MT01 Selaginella doederleinii Selaginellaceae ⫹ ⫹
PS0775MT01 Dicranopteris pedata Gleicheniaceae ⫹ ⫹
PS0984MT01 Equisetum pratense Equisetaceae ⫹ ⫹
PS0986MT01 Equisetum hyemale Equisetaceae ⫹ ⫹
PS1296MT01 Palhinhaea cernua Lycopodiaceae ⫹ ⫹
PS1296MT02 Palhinhaea cernua Lycopodiaceae ⫹ ⫹
PS1297MT01 Lycopodium serratum (Huperzia serrata) Lycopodiaceae ⫹ ⫹
PS6001MT01 Pyrrosia petiolosa Polypodiaceae ⫹ ⫹
PS6001MT02 Pyrrosia petiolosa Polypodiaceae ⫹ ⫹
PS6001MT03 Pyrrosia petiolosa Polypodiaceae ⫹ ⫹
PS6002MT01 Selaginella pulvinata Selaginellaceae ⫹ ⫹
PS6002MT02 Selaginella pulvinata Selaginellaceae ⫹ ⫹
PS6002MT03 Selaginella pulvinata Selaginellaceae ⫹ ⫹
PS6003MT01 Pyrrosia sheareri Polypodiaceae ⫹ ⫹
PS6003MT02 Pyrrosia sheareri Polypodiaceae ⫹ ⫹
PS6003MT03 Pyrrosia sheareri Polypodiaceae ⫹ ⫹
PS6004MT01 Coniogramme japonica Hemionitidaceae ⫹ ⫹
PS6006MT01 Parathelypteris nipponica Thelypteridaceae ⫹ ⫹
PS6007MT01 Phlegmariurus carinatus Huperziaceae ⫹ ⫹
PS6008MT01 Phlegmariurus phlegmaria Huperziaceae ⫹ ⫹
PS6017MT01 Dryopteris crassirhizoma Dryopteridaceae ⫹ ⫹
PS6017MT02 Dryopteris crassirhizoma Dryopteridaceae ⫹ ⫹
PS6017MT03 Dryopteris crassirhizoma Dryopteridaceae ⫹ ⫹
PS0371MT01 Humata tyermanni Davalliaceae ⫹ ⫺ NA
November 2010 1921

Table 1. Continued

PCR Sequencing Accession

Voucher number Species Family
result result number

PS0387MT01 Cyclosorus parasiticus Thelypteridaceae ⫹ ⫺* NA

PS0388MT01 Cyclosorus acuminatus Thelypteridaceae ⫹ ⫺* NA
PS0389MT01 Cyrtomium fortunei Dryopteridaceae ⫹ ⫺* NA
PS0392MT01 Pseudodrynaria coronans Drynariaceae ⫹ ⫺* NA
PS6005MT01 Nephrolepis exaltata Nephrolepidaceae ⫹ ⫺* NA
PS6014MT01 Adiantum reniforme var. sinense Adiantaceae ⫹ ⫺* NA
PS6018MT01 Neottopteris nidus Aspleniaceae ⫹ ⫺* NA
PS6023MT01 Selaginella labordei Selaginellaceae ⫹ ⫺ NA
PS0084MT01 Woodwardia japonica Blechnaceae ⫺ NA NA
PS6011MT01 Selaginella involvens Selaginellaceae ⫺ NA NA
PS6020MT01 Selaginella remotifolia Selaginellaceae ⫺ NA NA

Note: the “⫹” sign means a positive result; the “⫺” sign means a negative result; and “*” indicates sequencing failed due to poly-structure; NA: Not applicable.

Table 2. Sequences from GenBank as Extensions for Species Identification with the Sequence of psbA-trnH Intergenic Region

Family Species Accession number

Hymenophyllaceae Abrodictyum caudatum EU122991, EU122992

Adiantaceae Adiantum capillus-veneris AY178864
Cyatheaceae Alsophila spinulosa FJ556581
Angiopteridaceae Angiopteris evecta DQ821119
Aspleniaceae Asplenium adiantum EU125551, EU125552
Asplenium antiquum EU240017
Asplenium aureum EU125562
Asplenium australasicum EU240008
Asplenium bulbiferum EF418423
Asplenium ceterach EU125557, EU125558, EU125559, EU125560, EU125561
Asplenium dalhousiae EU125563, EU125564
Asplenium hookerianum EF418420, EF418421, EF418422
Asplenium parvifolium EU125565
Asplenium trichomanes EU125553, EU125554, EU125555, EU125556
Hymenophyllaceae Callistopteris apiifolia EU122995, EU122996
Crepidomanes bipunctatum EU122997, EU122998, EU338483
Crepidomanes humile EU122999, EU123001
Crepidomanes kurzii EU123002, EU123003
Crepidomanes minutum EU123005, EU338484
Didymoglossum tahitense EU123006, EU123007
Lycopodiaceae Diphasiastrum digitatum EU750650, EU750651
Dryopteridaceae Dryopteris carthusiana EU750635, EU750636, EU750637
Dryopteris intermedia EU750638, EU750639, EU750640
Dryopteris marginalis EU750641, EU750642, EU750643, EU750644
Equisetaceae Equisetum arvense EU750645, EU750646
Equisetum hyemale EU750647, EU750649
Lycopodiaceae Huperzia appressa DQ464203
Huperzia carinata DQ464213
Huperzia crispate DQ464204
Huperzia emeiensis DQ464205
Huperzia fargesii DQ464214
Huperzia fordii DQ464215
Huperzia hunanensis DQ464206
Huperzia lucidula AY660566
Huperzia mingcheensis DQ464216
Huperzia miyoshiana DQ464208
Huperzia nanchuanensis DQ464209
Huperzia petiolata DQ464217
Huperzia phyllantha DQ464218
Huperzia selago DQ464210
Huperzia serrata DQ464211
Huperzia serrata var. longipetiolata DQ464207
Huperzia squarrosa DQ464219
Huperzia sutchueniana DQ464212
Hymenophyllaceae Hymenophyllum digitatum EU123008, EU123009, EU338482
Hymenophyllum flabellatum EU123010
Hymenophyllum pallidum EU123011, EU123012, EU123013, EU123014, EU123015, EU338479
Hymenophyllum polyanthus EU123016, EU123017, EU123018, EU338480
1922 Vol. 33, No. 11

Table 2. Continued

Family Species Accession number

Lindsaeaceae Lindsaea digitata EU146035, EU146038, EU146039, EU146041

Lindsaea divaricata EU146032, EU146033, EU146034, EU146036, EU146037, EU146040
Lycopodiaceae Lycopodium obscurum EU750652, EU750653, EU750654, EU750655
Hymenophyllaceae Polyphlebium borbonicum EU123019, EU123020, EU123021, EU338477
Polyphlebium endlicherianum EU123022, EU338478
Psilotaceae Psilotum nudum AP004638
Selaginellaceae Selaginella uncinata AB197035

Identification of Universal Primers All chloroplast ge- Table 3. Ambiguous Identification Cases by the BLAST1 Method
nomic data (FJ556581 Alsophila spinulosa; DQ821119 An-
giopteris evecta; AY660566 Huperzia lucidula; AY178864 Query Best hit E value
Adiantum capillus-veneris; AP004638 Psilotum nudum; Huperzia sutchueniana Huperzia emeiensis 1.00E-125
AB197035 Selaginella uncinata) belonging to pteridophytes Huperzia serrata Huperzia emeiensis 1.00E-125
was downloaded from GenBank (176.0 version). Five fre- Huperzia emeiensis Huperzia serrata 1.00E-125
quently recommended fragments, namely, the psbA-trnH Huperzia nanchuanensis Huperzia appressa 1.00E-125
Huperzia miyoshiana Huperzia appressa 1.00E-125
intergenic region (hereinafter psbA-trnH), rbcL, rpoB, rpoC1,
Huperzia appressa Huperzia miyoshiana 1.00E-125
and matK, were extracted from each genomic sequence ac- Huperzia mingcheensis Huperzia fordii 7.00E-124
cording to its annotation. Six sequences of the same kind of Huperzia fordii Huperzia mingcheensis 7.00E-124
fragment were aligned by Clustal W individually, and Selaginella tamariscina* Selaginella pulvinata 8.00E-133
primers were searched for from the alignment files by Primer Selaginella pulvinata* Selaginella tamariscina 8.00E-133
Asplenium parvifolium* Asplenium aureum 0
Premier 5 software (PREMIER Biosoft Int., Palo Alto, CA, Asplenium aureum* Asplenium parvifolium 0
U.S.A.). Dryopteris intermedia* Dryopteris carthusiana 0
DNA Extraction, Polymerase Chain Reaction (PCR) Dryopteris carthusiana* Dryopteris intermedia 0
Amplification and DNA Sequencing Total DNA was Phlegmariurus carinatus* Huperzia carinata 1.00E-131
extracted from silica-gel dried leaf tissues using the Plant Huperzia carinata* Phlegmariurus carinatus 1.00E-131
Genomic DNA Kit (Tiangen Biotech Co., China). PCR Species followed by an asterisk were excluded from the analysis due to contro-
experiments were conducted depending on the results of the versy in the literature and/or complex taxonomical relationships.
analysis, and the general PCR conditions used were the same
as described previously9,23,24) with annealing temperatures Suite v 9, InforMax, North Bethesda, MD, U.S.A.) with an
modified when needed. After electrophoresis, the PCR prod- engine based on the Clustal W algorithm. Due to display
ucts were purified using the Gel Band Purification Kit (Tian- accuracy limitations of the TaxonGap method, the results
gen Biotech Co., China) and then sequenced on an ABI required some manual calibration.
3730XL sequencer (Applied Biosystems Inc.). Sequence
editing and contig assembly were carried out using Codon- RESULTS AND DISCUSSION
Code Aligner V 3.5.1 (CodonCode Co., U.S.A.). PCR ampli-
fication efficiency and direct sequencing success was calcu- DNA Marker Selection Several forward and reverse
lated with species units. A species was recorded as positive primers were designed using Primer Premier 5 under default
when at least one of its individuals was amplified or settings in the alignment files of psbA and trnH fragments,
sequenced successfully. including the pair of primers proposed by Kress et al.9) How-
Species Discriminative Power Analysis Species identi- ever, no universal primers could be identified for the loci of
fication analysis employed two methods, the Basic Local rbcL, rpoB, rpoC1, or matK. Thus, we concluded that the
Alignment Search Tool 1 (BLAST1) method25) and the Tax- only consensus primers available for wider pteridophyte taxa
onGap method.26) Species and infraspecific taxa were treated are those for psbA-trnH. Further tests were carried out with
as conspecies. A species was recorded as positive when at the proposed psbA-trnH primers (forward primer 5⬘-GTTAT-
least one of its individuals was identified or separated suc- GCATGAACGTAATGCTC-3⬘; reverse primer 5⬘-CGCGC-
cessfully. The identification success rate was measured as the ATGGTGGATTCACAATCC-3⬘) to maintain consistency
number of species which have the specific sequence to that of with data from other taxa like flowering plants.9) The hypoth-
all species suitable for dada analysis. In the BLAST1 esis was supported by a preliminary study using a single
method, species determination was based on the best hit of primer pair per locus, which showed that the PCR efficiency
the query sequence and an E-value for the match less than a of psbA-trnH was above 90%, while that of all the other loci
cutoff value. In the TaxonGap method, species discriminative was below 50%. The hypothesis was also proven to a certain
power was analyzed with the TaxonGap software (version extent by another study that showed that universal primers
2.4.1) by comparing the minimum distance (defined as sepa- did not exist for a small amount of pteridophyte samples (7
rability) between certain species and their closest neighbor species from 3 genera) for the four loci rbcL, rpoB, rpoC1,
and the maximum distance (defined as heterogeneity, if avail- and matK.21)
able) within the species. The distance between a species and PCR Efficiency and Sequencing Success of psbA-trnH
its closest neighbor was determined by a similarity matrix The PCR products were subjected to electrophoresis, and
which was calculated using the program AlignX (Vector NTI bands were detected from 48 out of 51 species. This result is
November 2010 1923

equivalent to a PCR efficiency of 94.1% (Table 1). The result

supported our hypothesis that psbA-trnH might be amplified
with a single pair of primers across a wider range of pterido-
phyte taxa and from a larger amount of pteridophyte samples
than in other studies (7 and 4 species each).13,21)
We obtained sequences from 39 out of 48 species with
electrophoretic bands available, giving a success rate of
81.3%. We anticipate that poly-structures may have been
responsible for the sequence failure in 7 of the remaining 9
species (Table 1). The high sequencing success rate sup-
ported the notion that the psbA-trnH locus could become
widely used in medicinal pteridophytes. However, the prob-
lem of long mononucleotide repeats leading to poorer quality
sequences requires more attention27,28) and further testing.
Species Identification Capability of psbA-trnH The
results form the BLAST1 method showed that 16 species
was ambiguously identified (Table 3). Excluding eight
species (please see reasons in next paragraph), we had 74
identifiable species out of 82 ones, in other words, the identi-
fication success rate was 90.2%. The result from the Taxon-
Gap method (Fig. 1, shows 82 species suitable for statistics)
also demonstrated that the sequences for 74 species were
specific enough to be separated from their neighbors (for
some species, they could not be shown to be detachable but
in fact had 1—3 bases different from their neighbors). Fur-
thermore, for 37 out of 39 separable species having at least
two samples, the between-species variation was higher than
the within-species variation. Thus, we concluded that psbA-
trnH would provide sufficient variation for species identifica-
tion within medicinal pteridophyte and even for a wider
range of pteridophyte taxa. This conclusion echoes the opin-
ion that high sequence divergence among species makes
psbA-trnH a promising barcode in flowering plants.9) This
also supports our notion that the psbA-trnH locus is worth
further testing as a candidate pteridophyte DNA barcode.
It should be noted that eight species failed to be sepa-
rated/identified for the reasons of synonyms,29) controversial
reports30) or complex relationships.31,32) These species were
excluded from further analysis in this paper. We also noted
that eight species from GenBank in the genus Huperzia do
not have a species-specific psbA-trnH sequence (Fig. 1), and
this contributed to the failed identification cases in this paper.
It suggests that the failure of psbA-trnH in species recogni-
tion may be due to insufficient variation in certain sub-
groups of pteridophytes. Therefore multiple markers will be
needed to perfect a DNA-based species identification system.
This paper proved that the chloroplast psbA-trnH inter-
genic region has universal primers available and sufficient
variation to identify medicinal pteridophytes commonly used
in China and potentially even wider taxa.

Acknowledgements This work was supported by the

Special Foundation of Ministry of Health (No. 200802043)
and Beijing Municipal Science & Technology Foundation
(No. D08080203640901) granted to S.L.C. This work was
also supported by the Research Fund for the Large-scale Sci-
entific Facilities of the Chinese Academy of Sciences (No.
2009-LSF-GBOWS-01).
Fig. 1. Species Identification Capability of the psbA-trnH Intergenic Re-
gion among Medicinal and Other Pteridophyte Species
The left panel shows the complete list of species used in this study, including se-
quences we generated and those retrieved from GenBank. The right panel depicts the
within-species heterogeneity and between-species separability for psbA-trnH as hori-
zontal light grey and dark grey bars, respectively. The right panel also shows the names
of the closest relatives identified by the similarity method. Species followed by a pound
sign have differences of 1—3 bases with their neighbor that could not be read due to
methodological limitations.
1924
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