Medicinal pteridophytes have a long history in traditionalChinese medicine because they are an abundant source ofchemical compounds with a wide range of pharmacologicalactivities.1) Recently, Huperzia species of this group of me-dicinal plants has been used to treat patients with Alzheimer’sdisease.2) However, there is a long-standing problem of mix-ing authentic species with their adulterants in medicinalpreparations. This problem has consequences for medicalsafety as well as for the conservation of the authenticspecies.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 convenientspecies identification termed “DNA barcoding” was recentlydeveloped.8—10) DNA barcoding uses a standard DNA sequence that can be amplified by universal primers and thatpossesses sufficient variation. A segment of the mitochon-drial cytochrome c oxidase I gene is widely accepted as thestandard barcode region in animals.11) In plants, however, noequivalently accepted DNA barcode has been identified, despite the fact that many resolutions have been proposed inthe leading literature.12—16)

Previous studies has demonstrated the value of DNA bar-coding technology in authentication of medicinal plantsspecies from some taxa,17,18) but DNA-based species identifi-

cation using a large set of medicinal pteridophytes has notbeen carried out. Until now, most studies of pteridophyteDNA sequences have focused on phylogenetic place-ment.19,20) Results derived from pteridophyte samples withsmall coverage may not be readily extendable to other pteri-dophytes.12,13,21) Furthermore, some of the studies suggestingthe possibility of species identification using standard DNAsequences were carried out within a narrow pteridophytetaxon (such as the family).22) The goal of this paper was totest the feasibility of species identification in medicinal pteri-dophytes across a wider taxon range with a convenient DNAmarker. 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 Atotal of 79 medicinal pteridophyte samples were used in thisstudy (Table 1). These samples represented 51 species across24 families, and they included all 11 authentic species listedin 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 MedicinalSciences and deposited in the Herbarium IMPLAD.

In addition, 109 sequences (Table 2) representing 54species from 12 pteridophyte families were downloaded fromGenBank for species identification.

November 2010 1919Note

Species Identification of Medicinal Pteridophytes by a DNA BarcodeMarker, 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 HubeiUniversity of Chinese Medicine; Wuhan 430061, P. R. China: and c School of Bioscience and Engineering, SouthwestJiaotong 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 nosimple 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 primersand 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, andmatK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the bestcandidate 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 authenticpteridophyte 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 polymerasechain 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 analysisshowed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunctionwith the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showedthat, for 37 out of 39 separable species with at least two samples each, between-species variation was higher thanthe relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for speciesidentification in medicinal pteridophytes.

Key words medicinal pteridophyte; psbA-trnH intergenic region; species identification

Biol. Pharm. Bull. 33(11) 1919—1924 (2010)

© 2010 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: slchen@implad.ac.cn# Equal contribution with first author.

† Present address: Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine; Guangzhou510006, P. R. China.

1920 Vol. 33, No. 11

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

Voucher number Species FamilyPCR Sequencing Accession result result number

PS0085MT01 Cibotium barometz Dicksoniaceae � � GU592445—PS0085MT02 Cibotium barometz Dicksoniaceae � � GU592497PS0086MT01 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

Voucher number Species FamilyPCR Sequencing Accession result result number

PS0387MT01 Cyclosorus parasiticus Thelypteridaceae � �* NAPS0388MT01 Cyclosorus acuminatus Thelypteridaceae � �* NAPS0389MT01 Cyrtomium fortunei Dryopteridaceae � �* NAPS0392MT01 Pseudodrynaria coronans Drynariaceae � �* NAPS6005MT01 Nephrolepis exaltata Nephrolepidaceae � �* NAPS6014MT01 Adiantum reniforme var. sinense Adiantaceae � �* NAPS6018MT01 Neottopteris nidus Aspleniaceae � �* NAPS6023MT01 Selaginella labordei Selaginellaceae � � NAPS0084MT01 Woodwardia japonica Blechnaceae � NA NAPS6011MT01 Selaginella involvens Selaginellaceae � NA NAPS6020MT01 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, EU122992Adiantaceae Adiantum capillus-veneris AY178864Cyatheaceae Alsophila spinulosa FJ556581Angiopteridaceae Angiopteris evecta DQ821119Aspleniaceae Asplenium adiantum EU125551, EU125552

Asplenium antiquum EU240017Asplenium aureum EU125562Asplenium australasicum EU240008Asplenium bulbiferum EF418423Asplenium ceterach EU125557, EU125558, EU125559, EU125560, EU125561Asplenium dalhousiae EU125563, EU125564Asplenium hookerianum EF418420, EF418421, EF418422Asplenium parvifolium EU125565Asplenium trichomanes EU125553, EU125554, EU125555, EU125556

Hymenophyllaceae Callistopteris apiifolia EU122995, EU122996Crepidomanes bipunctatum EU122997, EU122998, EU338483Crepidomanes humile EU122999, EU123001Crepidomanes kurzii EU123002, EU123003Crepidomanes minutum EU123005, EU338484Didymoglossum tahitense EU123006, EU123007

Lycopodiaceae Diphasiastrum digitatum EU750650, EU750651Dryopteridaceae Dryopteris carthusiana EU750635, EU750636, EU750637

Dryopteris intermedia EU750638, EU750639, EU750640Dryopteris marginalis EU750641, EU750642, EU750643, EU750644

Equisetaceae Equisetum arvense EU750645, EU750646Equisetum hyemale EU750647, EU750649

Lycopodiaceae Huperzia appressa DQ464203Huperzia carinata DQ464213Huperzia crispate DQ464204Huperzia emeiensis DQ464205Huperzia fargesii DQ464214Huperzia fordii DQ464215Huperzia hunanensis DQ464206Huperzia lucidula AY660566Huperzia mingcheensis DQ464216Huperzia miyoshiana DQ464208Huperzia nanchuanensis DQ464209Huperzia petiolata DQ464217Huperzia phyllantha DQ464218Huperzia selago DQ464210Huperzia serrata DQ464211Huperzia serrata var. longipetiolata DQ464207Huperzia squarrosa DQ464219Huperzia sutchueniana DQ464212

Hymenophyllaceae Hymenophyllum digitatum EU123008, EU123009, EU338482Hymenophyllum flabellatum EU123010Hymenophyllum pallidum EU123011, EU123012, EU123013, EU123014, EU123015, EU338479Hymenophyllum polyanthus EU123016, EU123017, EU123018, EU338480

Identification of Universal Primers All chloroplast ge-nomic data (FJ556581 Alsophila spinulosa; DQ821119 An-giopteris evecta; AY660566 Huperzia lucidula; AY178864Adiantum capillus-veneris; AP004638 Psilotum nudum;AB197035 Selaginella uncinata) belonging to pteridophyteswas downloaded from GenBank (176.0 version). Five fre-quently recommended fragments, namely, the psbA-trnHintergenic region (hereinafter psbA-trnH), rbcL, rpoB, rpoC1,and matK, were extracted from each genomic sequence ac-cording to its annotation. Six sequences of the same kind offragment were aligned by Clustal W individually, andprimers were searched for from the alignment files by PrimerPremier 5 software (PREMIER Biosoft Int., Palo Alto, CA,U.S.A.).

DNA Extraction, Polymerase Chain Reaction (PCR)Amplification and DNA Sequencing Total DNA was extracted from silica-gel dried leaf tissues using the PlantGenomic DNA Kit (Tiangen Biotech Co., China). PCR experiments were conducted depending on the results of theanalysis, and the general PCR conditions used were the sameas described previously9,23,24) with annealing temperaturesmodified when needed. After electrophoresis, the PCR prod-ucts were purified using the Gel Band Purification Kit (Tian-gen Biotech Co., China) and then sequenced on an ABI3730XL sequencer (Applied Biosystems Inc.). Sequence editing and contig assembly were carried out using Codon-Code Aligner V 3.5.1 (CodonCode Co., U.S.A.). PCR ampli-fication efficiency and direct sequencing success was calcu-lated with species units. A species was recorded as positivewhen at least one of its individuals was amplified or sequenced successfully.

Species Discriminative Power Analysis Species identi-fication analysis employed two methods, the Basic LocalAlignment Search Tool 1 (BLAST1) method25) and the Tax-onGap method.26) Species and infraspecific taxa were treatedas conspecies. A species was recorded as positive when atleast one of its individuals was identified or separated suc-cessfully. The identification success rate was measured as thenumber of species which have the specific sequence to that ofall species suitable for dada analysis. In the BLAST1method, species determination was based on the best hit ofthe query sequence and an E-value for the match less than acutoff value. In the TaxonGap method, species discriminativepower was analyzed with the TaxonGap software (version2.4.1) by comparing the minimum distance (defined as sepa-rability) between certain species and their closest neighborand the maximum distance (defined as heterogeneity, if avail-able) within the species. The distance between a species andits closest neighbor was determined by a similarity matrixwhich was calculated using the program AlignX (Vector NTI

Suite v 9, InforMax, North Bethesda, MD, U.S.A.) with anengine based on the Clustal W algorithm. Due to display accuracy limitations of the TaxonGap method, the results required some manual calibration.

RESULTS AND DISCUSSION

DNA Marker Selection Several forward and reverseprimers were designed using Primer Premier 5 under defaultsettings in the alignment files of psbA and trnH fragments,including the pair of primers proposed by Kress et al.9) How-ever, no universal primers could be identified for the loci ofrbcL, rpoB, rpoC1, or matK. Thus, we concluded that theonly consensus primers available for wider pteridophyte taxaare those for psbA-trnH. Further tests were carried out withthe proposed psbA-trnH primers (forward primer 5�-GTTAT-GCATGAACGTAATGCTC-3�; reverse primer 5�-CGCGC-ATGGTGGATTCACAATCC-3�) to maintain consistencywith data from other taxa like flowering plants.9) The hypoth-esis was supported by a preliminary study using a singleprimer pair per locus, which showed that the PCR efficiencyof psbA-trnH was above 90%, while that of all the other lociwas below 50%. The hypothesis was also proven to a certainextent by another study that showed that universal primersdid not exist for a small amount of pteridophyte samples (7species from 3 genera) for the four loci rbcL, rpoB, rpoC1,and matK.21)

PCR Efficiency and Sequencing Success of psbA-trnHThe PCR products were subjected to electrophoresis, andbands were detected from 48 out of 51 species. This result is

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Table 3. Ambiguous Identification Cases by the BLAST1 Method

Query Best hit E value

Huperzia sutchueniana Huperzia emeiensis 1.00E-125Huperzia serrata Huperzia emeiensis 1.00E-125Huperzia emeiensis Huperzia serrata 1.00E-125Huperzia nanchuanensis Huperzia appressa 1.00E-125Huperzia miyoshiana Huperzia appressa 1.00E-125Huperzia appressa Huperzia miyoshiana 1.00E-125Huperzia mingcheensis Huperzia fordii 7.00E-124Huperzia fordii Huperzia mingcheensis 7.00E-124Selaginella tamariscina* Selaginella pulvinata 8.00E-133Selaginella pulvinata* Selaginella tamariscina 8.00E-133Asplenium parvifolium* Asplenium aureum 0Asplenium aureum* Asplenium parvifolium 0Dryopteris intermedia* Dryopteris carthusiana 0Dryopteris carthusiana* Dryopteris intermedia 0Phlegmariurus carinatus* Huperzia carinata 1.00E-131Huperzia carinata* Phlegmariurus carinatus 1.00E-131

Species followed by an asterisk were excluded from the analysis due to contro-versy in the literature and/or complex taxonomical relationships.

Table 2. Continued

Family Species Accession number

Lindsaeaceae Lindsaea digitata EU146035, EU146038, EU146039, EU146041Lindsaea divaricata EU146032, EU146033, EU146034, EU146036, EU146037, EU146040

Lycopodiaceae Lycopodium obscurum EU750652, EU750653, EU750654, EU750655Hymenophyllaceae Polyphlebium borbonicum EU123019, EU123020, EU123021, EU338477

Polyphlebium endlicherianum EU123022, EU338478Psilotaceae Psilotum nudum AP004638Selaginellaceae Selaginella uncinata AB197035

equivalent to a PCR efficiency of 94.1% (Table 1). The resultsupported our hypothesis that psbA-trnH might be amplifiedwith a single pair of primers across a wider range of pterido-phyte taxa and from a larger amount of pteridophyte samplesthan in other studies (7 and 4 species each).13,21)

We obtained sequences from 39 out of 48 species withelectrophoretic bands available, giving a success rate of81.3%. We anticipate that poly-structures may have been responsible for the sequence failure in 7 of the remaining 9species (Table 1). The high sequencing success rate sup-ported the notion that the psbA-trnH locus could becomewidely used in medicinal pteridophytes. However, the prob-lem of long mononucleotide repeats leading to poorer qualitysequences requires more attention27,28) and further testing.

Species Identification Capability of psbA-trnH The results form the BLAST1 method showed that 16 specieswas ambiguously identified (Table 3). Excluding eightspecies (please see reasons in next paragraph), we had 74identifiable 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 werespecific enough to be separated from their neighbors (forsome species, they could not be shown to be detachable butin fact had 1—3 bases different from their neighbors). Fur-thermore, for 37 out of 39 separable species having at leasttwo samples, the between-species variation was higher thanthe within-species variation. Thus, we concluded that psbA-trnH would provide sufficient variation for species identifica-tion within medicinal pteridophyte and even for a widerrange of pteridophyte taxa. This conclusion echoes the opin-ion that high sequence divergence among species makespsbA-trnH a promising barcode in flowering plants.9) Thisalso supports our notion that the psbA-trnH locus is worthfurther 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) controversialreports30) or complex relationships.31,32) These species wereexcluded from further analysis in this paper. We also notedthat eight species from GenBank in the genus Huperzia donot have a species-specific psbA-trnH sequence (Fig. 1), andthis 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 beneeded to perfect a DNA-based species identification system.

This paper proved that the chloroplast psbA-trnH inter-genic region has universal primers available and sufficientvariation to identify medicinal pteridophytes commonly usedin China and potentially even wider taxa.

Acknowledgements This work was supported by theSpecial Foundation of Ministry of Health (No. 200802043)and Beijing Municipal Science & Technology Foundation(No. D08080203640901) granted to S.L.C. This work wasalso supported by the Research Fund for the Large-scale Sci-entific Facilities of the Chinese Academy of Sciences (No.2009-LSF-GBOWS-01).

November 2010 1923

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 thewithin-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 namesof the closest relatives identified by the similarity method. Species followed by a poundsign have differences of 1—3 bases with their neighbor that could not be read due tomethodological limitations.

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