Plant Syst Evol (2015) 301:1843–1850 DOI 10.1007/s00606-015-1197-y ORIGINAL ARTICLE Use of chloroplast DNA barcodes to identify Osmunda japonica and its adulterants Si H. Zheng • Wei G. Ren • Zeng H. Wang • Lin F. Huang Received: 4 November 2013 / Accepted: 9 January 2015 / Published online: 1 February 2015 Ó Springer-Verlag Wien 2015 Abstract Osmunda japonica Thunb., a medicinal plant and its adulterants, which was helpful for further clinical newly recorded in the Chinese pharmacopoeia, has been application of the materials. used for centuries for treatment of viral influenza, dysen- tery, and bleeding. Although O. japonica and its adulter- Keywords Osmunda japonica Á Adulterants Á ants have different medicinal effects and clinical Identification Á Chloroplast Á DNA barcoding applications, it is difficult to differentiate O. japonica from its adulterants used in medicine. To distinguish O. japonica from its adulterants, two chloroplast barcodes (psbA-trnH and rbcL) were tested for the first time. Genetic distance, Introduction genetic divergence, maximum likelihood tree, barcoding gap, and identification efficiency were calculated and Osmunda japonica Thunb., a newly recorded species in the analyzed for identification of O. japonica and its adulter- Chinese Pharmacopoeia (2010 edition) (National Pharma- ants. The results showed the two barcodes could be used to copoeia Committee 2010), has been used for centuries for identify O. japonica and its adulterants, and the perfor- treatment of viral influenza, dysentery, bleeding, and for its mance of psbA-trnH was better in terms of amplification, antiviral, antitumor, and anthelminthic properties. Dryop- sequencing, genetic divergence, and variation. The psbA- teris crassirhizoma was the only fern species recorded in trnH region resulted in less overlap than rbcL, and greater the Chinese Pharmacopoeia (2005 and earlier editions); interspecific divergence. On the basis of psbA-trnH this was named ‘‘Guan Zhong’’ in traditional Chinese. In sequences, a pair of primers was designed for specific addition, another 56 fern species (belonging to 18 genera identification of O. japonica. Our findings indicated that and 11 families) were sold for medicinal use as ‘‘Guan psbA-trnH was the optimum barcode, and rbcL could be as Zhong’’ in local and commercial markets in China, and a complementary barcode for authenticating O. japonica were often confused, especially Dennstaedtia scabra, Ci- botium barometz, Cyrtomium fortunei, Matteuccia strut- Handling editor: Jochen Heinrichs. hiopteris, Pentarhizidium orientale, and Adiantum capillus-veneris, when used in medicinal slices (Lou and S. H. Zheng Á W. G. Ren Á Z. H. Wang Á L. F. Huang (&) Qin 1995). Hence, ‘‘Guan Zhong’’ was used as a homonym Institute of Medicinal Plant Development, Chinese Academy of (same name, different botanical origins) in the Chinese Medical Sciences, Peking Union Medical College, Room 402, No.151, Malianwa North Road, HaiDian District, medicinal market. However, these 58 closely related fern Beijing 100193, People’s Republic of China species might have different effects, chemical components, e-mail: [email protected] and clinical applications. For example, O. japonica, D. S. H. Zheng crassirhizoma, and C. fortunei are toxic when used clini- e-mail: [email protected] cally above a specific dose whereas M. orientalis and M. struthiopteris could be used as food (Li et al. 2012). W. G. Ren Guang’ anmen Hospital, China Academy of Chinese Medical Therefore, it was necessary to establish a method to iden- Sciences, Beijing 100053, China tify O. japonica and its adulterants. 123 1844 S. Zheng et al. DNA barcoding, a term first coined in 2003 by Hebert adulterants, to compare identification efficiency, and a pair et al. (2003a, b), uses the sequence of a short DNA segment of specific primers were designed for accurate differentia- for species discrimination. Mitochondrial gene cytochrome tion of O. japonica from its adulterants. c oxidase I (COI) was proposed as the basis of a global bio- identification system for animals. However, the perfor- mance of the COI region was not as robust in plants as in Materials and methods animals, because of low rates of substitution of mito- chondrial DNA (Hollingsworth et al. 2009). DNA barcodes Taxon sampling have been used to identify medicinal plants (Chen et al. 2010; Asahina et al. 2010; Yao et al. 2010; Huang et al. Forty-seven fresh samples comprising 17 of O. japonica 2014; Zheng et al. 2014a, b). Ferns have lost the flanking and 30 of adulterants were collected (Table 1). One-hun- trnK exons typically used to design stable priming sites. In dred and forty-seven sequences of O. japonica and its comparison, organelle regions are superior for recon- closely related species were downloaded from the Gen- structing the tree of life because they are easily amplified, Bank database for identification (Table 2). Species identi- their rates of duplication are low, and they are uniparen- fication of collected samples was performed by Professor tally inherited, unlike nuclear genomes. Therefore, previ- Lin YL (Institute of Medical Plant Development (IMP- ous study of phylogeny and identification of ferns have LAD), Chinese Academy of Medicinal Sciences) on the mainly focused on chloroplastid DNA barcodes, including basis of morphological characteristics. Samples were rbcL, rbcL-accD IGS, rbcL-atpB IGS, trnG-trnR, trnL- deposited in the IMPLAD herbarium. trnF, and psbA-trnH in the intergenic region and the trnL- F and rpL16 introns (Yatabe et al. 2001; Korall et al. 2007; DNA extraction, PCR amplification, sequencing Ma et al. 2010; Ebihara et al. 2010; Groot et al. 2011;Li et al. 2011; Dauphin et al. 2014). However, few studies Fresh leaf tissue was first dried over silica gel. The dry have focused on molecular identification of O. japonica tissue (20 mg) was powdered for 2 min at a frequency of and its adulterants, which limits further application of these 30 times/s in a FastPrep bead mill (Retsch MM400, Ger- medicinal materials and herbal drugs. many). DNA was extracted by use of a Plant Genomic In this study, two candidate chloroplast regions (psbA- DNA kit (Tiangen Biotech, China) in accordance with the trnH and rbcL) were used to identify O. japonica and its manufacturer’s instructions. We made two modifications to Table 1 Taxon sampling information for Osmunda japonica and its adulterants Collector Family Species Collection GenBank accession GenBank accession number location number (psbA-trnH) number (rbcL) WJ1–WJ2 Dennstaedtiaceae Dennstaedtia scabra ShanXi China KC795765 (WJ2) KC795808 (WJ2) JM Dicksoniaceae Cibotium barometz YunNan China KC795770 (JM) TZ1–TZ6 Dryopteridaceae Cyrtomium fortunei ShanXi China KC795774–KC795779 KC795814–KC795815 (TZ1/TZ3/TZ4/TZ5) SN1–SN6 Dryopteridaceae Cyrtomium fortunei ShanXi China KC795780–KC795785 KC795816-KC795819 (SN3/SN6) FP1 Dryopteridaceae Cyrtomium fortunei ShanXi China KC795786 KC795820 DF1–DF3 Dryopteridaceae Cyrtomium fortunei ShanXi China KC795787–KC795789 KC795821–KC795823 GZ18 Dryopteridaceae Cyrtomium fortune ShanXi China KC795790 (GZ18) MM Dryopteridaceae Dryopteris crassirhizoma JiLin China KC795769 (MM) J1–J3 Onocleaceae Matteuccia struthiopteris BeiJing China KC795771–KC795773 KC795811–KC795813 ZQ1– Osmundaceae Osmunda japonica HuNan China KC795747–KC795762 KC795791–C795805 ZQ17 (ZQ17) (ZQ2/ZQ16) Z1–Z2 Onocleaceae Pentarhizidium orientale HuNan China KC795763–KC795764 KC795806–KC795807 TX1–TX3 Pteridaceae Adiantum capillus- ShanXi China KC795766–KC795768 KC795809–KC795810 veneris (TX3) Species for which no sequences were obtained are given in parentheses 123 Use of chloroplast DNA barcodes to identify Osmunda japonica 1845 Table 2 Sequences from GenBank for identification by use of the psbA-trnH and rbcL regions Region Family Species Accession number psbA-trnH Dryopteridaceae Cyrtomium fortunei AB575729, AB575730, AB575731 Dryopteridaceae Dryopteris crassirhizoma AB575746, GU592467, GU592468, JN189435 Onocleaceae Matteuccia struthiopteris AB575690, GU592471, GU592472, HQ243691 HQ243692, HQ243693, HQ243694, HQ243695 HQ243698, HQ243699, HQ243700, HQ243701 HQ243705, HQ243706, HQ243696, HQ243697 JF912403 Osmundaceae Osmunda lancea AB575360 Osmundaceae Osmunda claytoniana AB575359 Osmundaceae Osmundastrum cinnamomeum JN575776 Osmundaceae Osmunda banksiifolia AB575358 Dennstaedtiaceae Dennstaedtia scabra AB575433 Pteridaceae Adiantum capillus-veneris AB575450 Dicksoniaceae Cibotium barometz AB575405, GQ434937, GU592448 rbcL Dryopteridaceae Cyrtomium fortune AB575105, AB575106, AB575107, AB598689 AB598690, AF537227, EF394237 Dryopteridaceae Dryopteris crassirhizoma AB575122, AY268870, EF463177, JN189543 Onocleaceae Matteuccia struthiopteris AB232415, AB575062, D43917, JF832074 MSU05930, U62032 Dennstaedtiaceae Dennstaedtia scabra AB574778, JF303966 Pteridaceae Adiantum capillus-veneris AB574796, DQ432659, JF935318, JF935320 JF935322, JF935332, JF935334, JF935345 Dicksoniaceae Cibotium barometz AB574749, AM177328, CBU05610 Osmundaceae Osmunda japonica AB024947, AB076261, AB494711, AB494712 AB494713, AB494714, AB574698, AB639162 AB639163, AB639164, AB667969, EF588701 Osmundaceae Leptopteris superba DQ646004 Osmundaceae Leptopteris hymenophylloides AB024957, EF588695, EF588696 Osmundaceae Leptopteris wilkesiana AB024958, AY612678 Osmundaceae Osmunda angustifolia FJ262723 Osmundaceae Osmunda banksiifolia EF588698, AB024955, AB024956, AB574696 Osmundaceae Osmunda claytoniana EF588700, AB024950, AB024951, AB639186 AB672747, AB574697, EF588699 Osmundaceae Osmunda hybrida