Developing DNA Barcodes for Species Identification In

Journal of Systematics and Evolution 9999 (9999): 1–13 (2014) doi: 10.1111/jse.12076

Research Article Developing DNA barcodes for species identiﬁcation in Podophylloideae (Berberidaceae) 1Yun‐Rui MAO 1Yong‐Hua ZHANG 2Koh NAKAMURA 3Bi‐Cai GUAN 1Ying‐Xiong QIU* 1(Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, and Laboratory of Systematic and Evolutionary Botany and Biodiversity, College of Life Sciences, Zhejiang University, Hangzhou 310058, China) 2(Herbarium (HAST), Biodiversity Research Center, Academia Sinica, Taipei 115, Nangang, Taiwan, China) 3(College of Life Sciences and Food Engineering, Nanchang University, Nanchang 310031, China)

Abstract Species of Podophyllum, Dysosma, Sinopodophyllum, and Diphylleia, genera from Podophylloideae of Berberidaceae, have long been used in traditional herbal medicine in East Asia and/or North America. Accurate identification of the species of these four genera is crucial to their medicinal uses. In this study, we tested the utility of nine barcodes (matK, rbcL, atpH‐atpI, rpl32‐trnLUAG, rps18‐clpp, trnL‐trnF, trnL‐ndhJ, trnS‐trnfM, and internal transcribed spacer (ITS)) to discriminate different species of Podophylloideae. Thirty‐six individuals representing 12 species of Podophylloideae were collected from different locations in China, Japan, and North America. We assessed the feasibility of amplification and sequencing of all markers, examined the levels of the barcoding gap based on DNA sequence divergence between ranges of intra‐ and interspecific variation using pairwise distances, and further evaluated successful identifications using each barcode by similarity‐based and tree‐based methods. Results showed that nine barcodes, except rps18‐clpp, have a high level of primer universality and sequencing success. As a single barcode, ITS has the most variable sites, greater intra‐ and interspecific divergences, and the highest species discrimination rate (83%), followed by matK which has moderate variation and also high species discrimination rates. However, these species can also be discriminated by ITS alone, except Dysosma versipellis (Hance) M. Cheng ex T. S. Ying and D. pleiantha (Hance) Woodson. The combination of ITS þ matK did not improve species resolution over ITS alone. Thus, we propose that ITS may be used as a sole region for identification of most species in Podophylloideae. The failure of ITS to distinguish D. versipellis and D. pleiantha is likely attributed to incomplete lineage sorting due to recent divergence of the two species. Key words DNA barcoding, Podophylloideae, species identification, traditional Chinese medicine.

Podophylloideae Eaton is a small subfamily of 12 idaceae, and also conﬁrmed that Sinopodophyllum species belonging to Berberidaceae, which comprises hexandrum (Royle) T. S. Ying was sister to the ENA Sinopodophyllum (Royle) Ying (1 sp.), Dysosma Podophyllum peltatum L. (Loconte & Estes, 1989; Woodson (7 spp.), Podophyllum L. (1 sp.), and Nickol, 1995; Kim & Jansen, 1998; Liu et al., 2002; Diphylleia Michaux (3 spp.) (Loconte, 1993; Ying Wang et al., 2009), which was sister to Dysosma et al., 1993). Dysosma is restricted to China. Diphylleia versipellis (Hance) M. Cheng ex T. S. Ying; species of has an intercontinental disjunct distribution in eastern Diphylleia formed a basal clade in Podophylloideae with North America (ENA) (Diphylleia cymosa Michaux) Diphylleia cymosa from ENA, sister to Diphylleia and East Asia (Diphylleia grayi F. Schmidt and grayi–Diphylleia sinensis from East Asia (Wang Diphylleia sinensis H. L. Li). Both Sinopodophyllum et al., 2007, 2009). However, in all previous studies, and Podophyllum are monotypic, and native to the most species of Dysosma were missing except Himalaya–Hengduan Mountains and ENA, respectively D. versipellis; thus the detailed picture of species (Ying et al., 1993). Previous phylogenetic analyses relationships within the subfamily has remained unclear. using morphological and molecular data revealed that Podophylloideae species are rich sources of Podophylloideae was a monophyletic group in Berber- podophyllotoxin, an aryltetralin lignan that has impor- tant biological activities and is the precursor of semisynthetic chemotherapeutic drugs such as etopo- Received: 19 June 2013 Accepted: 19 December 2013 Author for correspondence. E‐mail: [email protected]. Tel./Fax: side and teniposide (Broomhead & Dewick, 1990; 86‐571‐86432273. Stähelin & von Wartburg, 1991). However, the

© 2014 Institute of Botany, Chinese Academy of Sciences 2 Journal of Systematics and Evolution Vol. 9999 No. 9999 2014 phytochemical compounds in quality and effect differ phyllum, Podophyllum, and Dysosma) of Podophylloi- greatly between genera and species of the subfamily deae were included in this study (Table 1). Each species (Pandey et al., 2007; Kusari et al., 2010; Jiang was represented by two to five accessions collected et al., 2012). In addition, most species in Dysosma from different populations in China, Japan, or North and Diphylleia have been listed as “endangered” or America. The species were identified based on “rare” in the China Species Red List (Wang & descriptions and keys for this subfamily in the Flora Xie, 2004) or the IUCN (Ying et al., 2011), because of China (Ying et al., 2011). Fresh leaves were dried in of their small distributional ranges, few populations, silica gel at the time of collection. Voucher specimens and small sizes of the known populations. Therefore, a were deposited in the herbarium at the Kunming rapid and accurate method for species identification of Institute of Botany, Chinese Academy of Sciences Podophylloideae is needed not only to facilitate proper (KUN) (Table 1). medicinal uses (e.g., to guide correct introduction of Eight candidate barcoding markers of chloroplast wild resources for the pharmaceutical industry), but also genome (rbcL, matK, trnL‐trnF, trnL‐ndhJ, trnS‐trnfM, to aid conservation management. atpH‐atpI, rpl32‐trnLUAG, rps18‐clpp) plus the nuclear DNA barcoding is a well‐known technique that aims ITS region were chosen to be sequenced for the study. to facilitate rapid species identification based on short, All these DNA regions have been used in previous standardized DNA sequences in cases where convention- phylogenetic studies (Taberlet et al., 1991; Demesure al taxonomic identification is not feasible (Tautz et al., 1995; Fay et al., 1997; Grivet et al., 2001; Fu et al., 2002, 2003; Hebert et al., 2003; Kress et al., et al., 2005; Kress & Erickson, 2007; Shaw et al., 2007), 2005; Savolainen et al., 2005). The use of a combination which have shown that they could be directly sequenced of DNA sequences with existing morphological charac- from polymerase chain reaction (PCR) products (also ters can facilitate fast and reliable identification of species see below) without cloning, and possessed a reasonable (Smith et al., 2005; Will et al., 2005; DeSalle, 2006; number of variable sites. Hajibabaei et al., 2007). In plants, several single‐locus or multi‐loci candidate DNA barcodes have been tested and 1.2 DNA isolation, PCR amplification, and proven to be useful for plant identification sequencing procedures (Thomas, 2009; Gao, 2010; Li et al., 2011; Sun Total genomic DNA was extracted from the dried et al., 2012). These markers include the nuclear ribosomal leaf tissue using a DNeasy plant tissue kit (Qiagen, internal transcribed spacer (ITS) and plastid gene regions Valencia, CA, USA). The PCRs were carried out in 25‐ (rpoB, rpoC1, rbcL, matK) and plastid non‐coding mL reaction mixtures containing approximately 30 ng regions (atpH‐atpI, psbK‐psbI, trnH‐psbA). However, the genomic DNA template, PCR buffer (10 mmol/L Tris, utility of each region was found to vary among taxonomic 50 mmol/L KCl buffer, pH 8.0), 2.0 mmol/L MgCl2, groups. At present, the effectiveness of these DNA 0.5 mmol/L each primer, 0.2 mmol/L each dNTP, and regions as barcodes to identify species within Podophyl- 1.0 U Taq DNA polymerase (Takara, Dalian, China). loideae remains unknown. Sequences of all the primers and annealing temper- In this study, we tested eight plastid DNA markers atures are listed in Table S1. The PCR program was as (rbcL, matK, trnL‐trnF, trnL‐ndhJ, trnS‐trnfM, atpH‐ follows: 94 °C for 3 min, followed by 34 cycles of 94 °C atpI, rpl32‐trnLUAG, rps18‐clpp) and one nuclear DNA for 30 s; 53–60 °C annealing reaction for 30 s (depend- region (ITS) for their utility as barcodes for species ing on the primers); 72 °C for 2 min; with final identification of the Podophylloideae. Our major goal extension at 72 °C for 10 min. The PCR amplifications was to develop a reliable molecular system for were carried out on a GeneAmp 9700 DNA Thermal identification of Podophylloideae species that would Cycler (Perkin‐Elmer, Foster City, CA, USA). The benefit the pharmaceutical industry and conservation. A PCR products were visualized using electrophoresis on second goal of the study was to evaluate the 1.0% agarose gels. Purification and bidirectional morphology‐based species classification using the sequencing of PCR products were completed by Beijing DNA sequence data. Genomics Institute (Shenzhen, China) using the PCR primers. All sequences have been deposited in GenBank (see Table 1 for accession numbers). 1 Material and methods 1.3 Data analysis 1.1 Plant material and loci sampling Sequences were aligned and edited in GENEIOUS A total of 36 individual samples representing all 12 version 4.5.0 (Drummond et al., 2006). For each gene species of the four genera (i.e., Diphylleia, Sinopodo- region, we examined the number of variable sites (Vs),

© 2014 Institute of Botany, Chinese Academy of Sciences 04Isiueo oay hns cdm fSciences of Academy Chinese Botany, of Institute 2014 © Table 1 Accession and GenBank data for 12 species of Podophylloideae (plus outgroup) screened for nine candidate DNA barcoding markers. The representative voucher specimens are deposited at the Herbarium of Kunming Institute of Botany, Chinese Academy of Science, Kunming, Yunnan, China (KUN) and Kyoto University Museum, Sakyo‐ku, Kyoto, Japan (KYO) Sampling locality Voucher Sample Genbank accession numbers Latitude Longitude Altitude numbers (N) (E) (m) ITS/matK/rbcL atpH‐atpI/rpl32‐trnLUAG / trnL‐trnF/trnL‐ndhJ/ rps18‐clpp trnS‐trnfM Dysosma pleiantha (Hance) Woodson Huadingshan, Zhejiang, H.‐L. Liu 0635‐0647 (KUN) 1 KC494652/KC539345/ KC539244/KC539281/ KC523318/KC523355/ 30°380 119°230 600 China KC539382 KC539318 KC523281 Deqing, Zhejiang, H.‐L. Liu 0657‐0662 (KUN) 1 KC494653/KC539346/ KC539245/KC539282/ KC523319/KC523356/ 30°420 119°480 500 China KC539383 KC539317 KC523282 Dahunshan, Taiwan, S.‐K. Yu 201005001 (KUN) 1 KC494656/KC539349/ KC539248/KC539285/ KC523320/KC523357/ 25°560 121°270 1514 China KC539386 KC539321 KC523285 Shenmuyuan, Taiwan, K. Nakamura 10393 (KUN) 1 KC494654/KC539347/ KC539246/KC539283/ KC523321/KC523358/ 24°350 121°260 1594 China KC539384 KC539319 KC523283 Yuanyanghu, Taiwan, K. Nakamura 10434 (KUN) 1 KC494655/KC539348/ KC539247/KC539284/ KC523322/KC523359/ 24°340 121°240 1699 China KC539385 KC539320 KC523284 D. versipellis (Hance) M. Cheng ex T. S. Ying Haixing, Jiangxi, China B.‐C. Guan 060401 (KUN) 1 KC494658/KC539351/ KC539250/KC539287/ KC523324/KC523361/ 29°950 116°870 700 KC539388 KC539323 KC523287 Tiantangzhai Anhui, B.‐C. Guan 060601 (KUN) 1 KC494657/KC539350/ KC539249/KC539286/ KC523323/KC523360/ 31°140 115°740 900 China KC539387 KC539322 KC523286 Dujiangyan, Sichuan, B.‐C. Guan 060801 (KUN) 1 KC494659/KC539352/ KC539251/KC539288/ KC523325/KC523362/ 31°060 107°380 800 China KC539389 KC539324 KC523288 D. majorensis (Gagnep.) Ying Emeishan, Sichuan, Y.‐R. Mao 201104005 (KUN) 1 KC494662/KC539353/ KC539254/KC539299/ KC523328/KC523365/ 29°340 103°210 1684 China KC539392 KC539327 KC523291 Jinfoshan, Chongqing, Y.‐R. Mao 201105030 (KUN) 1 KC494663/KC539354/ KC539255/KC539300/ KC523329/KC523366/ 27°500 108°450 1823 China KC539393 KC539328 KC523292 Fanjingshan, Guizhou, Y.‐R. Mao 201105035 (KUN) 1 KC494664/KC539355/ KC539256/KC539301/ KC523330/KC523367/ 29°430 94°430 522

China KC539394 KC539329 KC523293 3 Podophylloideae for barcodes DNA al.: et MAO D. difformis (Hemsl. & Wils.) T. H. Wang ex T. S. Ying Badagongshan, Hunan, Y.‐R. Mao 2011053101 (KUN) 1 KC494660/KC539359/ KC539252/KC539297/ KC523326/KC523363/ 29°450 110°030 1513 China KC539390 KC539325 KC523289 Huaping, Guangxi, Y.‐R. Mao 2011060701 (KUN) 1 KC494661/KC539360/ KC539253/KC539298/ KC523327/KC523363/ 25°380 109°540 700 China KC539391 KC539326 KC523290 D. aurantiocaulis (Handel‐Mazzetti) Hu Cangshan, Yunnan, Y.‐X. Qiu 20030701‐03 (KUN) 3 KC494665–KC494667/ KC539257–KC539259/ KC523331–KC523333/ 25°460 100°170 2646 China KC539356– KC539289– KC523368– KC539358/KC539395 KC539291/KC539330 KC523370/KC523294 –KC539397 –KC539332 –KC523296 D. tsayuensis Ying Lulang, Tibet P. Li 090978201‐03 (KUN) 3 KC494668–KC494670/ KC539260–KC539262/ KC523334–KC523336/ 29°430 94°430 3841 Autonomous Region, KC539361– KC539292– KC523371– China KC539363/KC539398 KC539294/KC539333 KC523373/KC523297 –KC539400 –KC539335 –KC523299 D. veitchii (Hemlsley & E. H. Wilson) L. K. Fu ex T. S. Ying Emeishan, Sichuan, X.‐S. Qi 07001 (KUN) 1 KC494672/KC539365/ KC539264/KC539296/ KC523337/KC523375/ 29°350 103°220 2124 China KC539402 KC539336 KC523301 Kunming, Yunnan, X.‐S. Qi 07102001 (KUN) 1 KC494671/KC539364/ KC539263/KC539295/‐ KC523338/KC523374/ 25°030 102°420 2049 China KC539401 KC523300 Continued ora fSseaisadEouinVl 99N.99 2014 9999 No. 9999 Vol. Evolution and Systematics of Journal 4 Table 1 Continued Sampling locality Voucher Sample Genbank accession numbers Latitude Longitude Altitude numbers ITS/matK/rbcL atpH‐atpI/rpl32‐trnLUAG / trnL‐trnF/trnL‐ndhJ/ (N) (E) (m) rps18‐clpp trnS‐trnfM Sinopodophyllum hexandrum (Royle) T. S. Ying Linzhi, Tibet P. Li 0909876 (KUN) 1 KC494684/KC539377/ KC539275/KC539313/ KC523350/KC523387/ 29°070 99°590 3844 Autonomous Region, KC539413 KC539337 KC523313 China Lulang, Tibet P. Li 0909873 (KUN) 1 KC494683/KC539376/ KC539276/KC539312/ KC523349/KC523386/ 29°070 99°590 3844 Autonomous Region, KC539414 KC539337 KC523312 China Kangding, Sichuan, P. Li 0909794 (KUN) 1 KC494682/KC539375/ KC539274/KC539311/‐ KC523348/KC523385/ 30°030 101°580 3154 China KC539412 KC523311 Diphylleia sinensis H. L. Li Taizishan, Gansu, Y.‐R. Mao 20110703 (KUN) 1 KC494674/KC539367/ KC539266/KC539302/ KC523343/KC523377/ 35°140 103°240 2573 China KC539403 KC539339 KC523303 Shennongjia, Hubei, Y.‐R. Mao 20110601 (KUN) 1 KC494673/KC539366/ KC539265/KC539303/‐ KC523342/KC523376/ 35°470 102°400 2501 China KC539404 KC523302 Diphylleia cymosa Michaux Linn Cove Viaduct, P. Li 100516401‐03 (KUN) 3 KC494675–KC494677/ KC539267–KC539269/ KC523339–KC523341/ 36°050 81°480 1320 North Carolina, USA KC539368– KC539304– KC523378– KC539370/KC539405 KC539306/‐ KC523380/KC523304 –KC539407 –KC523306 Diphylleia grayi F. Schmidt Mt. Daisetsu‐san, Nakamura 110701 (KYO) 1 KC494679/KC539373/ KC539271/KC539309/‐ KC523345/KC523382/ 43°420 142°490 1000 Aizankei, Japan KC539409 KC523308 Mt. Chokai‐san, Japan Nakamura 110702 (KYO) 1 KC494681/KC539374/ KC539273/KC539310/ KC523347/KC523383/ 39°070 139°080 600

04Isiueo oay hns cdm fSciences of Academy Chinese Botany, of Institute 2014 © KC539411 KC539342 KC523309 Yamagata, Tsuruoka, Nakamura 110716 (KYO) 1 KC494680/KC539371/ KC539272/KC539308/ KC523346/KC523384/ 38°310 139°530 500 Japan KC539410 KC539341 KC523310 Nagano, Hakuba, Japan Nakamura 110731 (KYO) 1 KC494678/KC539372/ KC539270/KC539307/ KC523344/KC523381/ 36°390 137°490 1400 KC539408 KC539340 KC523307 Podophyllum peltatum L. Muir Woods, WI, USA P. Li 100504301‐03 (KUN) 3 KC494685–KC494687/ KC539277–KC539279/ KC523351–KC523353/ 43°050 89°260 289 KC539378– KC539314– KC523388– KC539380/KC539415 KC539316/ KC523390/KC523314 –KC539417 KC539343 –KC523316 Epimedium simplicifolium T. S. Ying Hangzhou Botanical Y.‐R. Mao 20110801 (KUN) 1 KC494651/KC539344/ KC539243/KC539280/‐ KC523317/KC523354/ 29°150 120°070 100 Garden, Zhejiang, KC539381 KC523280 China Only one individual was sequenced. ‐, Unsuccessful sequencing. MAO et al.: DNA barcodes for Podophylloideae 5 the number of parsimony‐informative characters (PIC), the identity of a sample was determined based on that and the nucleotide diversity per site (Pi) using DnaSP the query sequence has the smallest genetic distance to version 5 (Librado & Roza, 2009). We assessed genetic all conspecific reference sequences in the database and divergences within and between species using six the distance must be less than a distance threshold (1%). metrics (Meyer & Paulay, 2005; Lahaye et al., 2008). Furthermore, the neighbor‐joining (NJ) and maxi- Three parameters were used to characterize interspecific mum parsimony (MP) methods implemented in divergence for each candidate locus: (i) average PAUP4b10 were used to evaluate whether multiple interspecific distance (Kimura 2‐parameter (K2P) samples of a species were recovered as monophyletic distances) between all species within genus; (ii) average with each barcode. Neighbor‐joining trees were theta prime (u0), where theta prime is the mean pairwise generated based on the K2P model using default distance within each genus with at least two species, PAUP4.0b10 settings. Tree nodal supports were gener- thus eliminating biases associated with different ated by 1000 bootstrap replicates. For MP analyses, full numbers of species among genera; and (iii) smallest heuristic tree searches were carried out with 100 interspecific distance, that is, the minimum interspecific replications of “random” sequence entries, tree bisec- distance within each genus with at least two species. tion–reconnection branch‐swapping, and the MulTrees Three different metrics were used to characterize option in effect. Branch support was assessed by intraspecific variation for each candidate locus: bootstrap analysis with 1000 replicates of full heuristic (i) average pairwise intraspecific difference (K2P searches under the above settings. All MP analyses distances) between all individuals sampled within were run with gaps (indels) treated as missing data. One species; (ii) average theta (u), where theta is the mean specimen of Epimedium simplicifolium T. S. Ying that pairwise distance within each species with at least two was collected in the Hangzhou Botanical Garden representatives, thereby eliminating bias associated (Zhejiang, China) was used as the outgroup in the with uneven sampling among species; and (iii) average phylogenetic analyses. coalescent depth, which is the maximum intraspecific distance within each species with at least two individuals. All analyses were carried out using 2 Results PAUP4b10 (Swofford, 2002). To evaluate intra‐ and interspecific variability for each pair of marker 2.1 Polymerase chain reaction amplification, sequences, Wilcoxon signed‐rank tests as implemented sequencing, and sequence variability in SPSS 16.0 (SPSS, Chicago, IL, USA) were used to test The PCR amplification and sequencing of all statistical significance of marker divergence differences regions was successful for all samples except rps18‐ (Kress & Erickson, 2007; Lahaye et al., 2008). Median clpp, for which the PCR success rate was 75.7% tests and Wilcoxon two‐sample tests were executed to (Table S1). The alignments of the two coding examine the extent of DNA barcoding gap/overlap chloroplast DNA (cpDNA) markers (rbcL, matK) between intra‐ and interspecific divergences (Meyer & showed no variation in sequence length among samples Paulay, 2005; Kress & Erickson, 2007). The barcoding (783 and 651 bp, respectively), whereas those of the six gaps were graphed by comparing the distributions of non‐coding cpDNA and ITS sequences showed much intra‐ and interspecific K2P distances among all variation in sequence length among samples and the samples for each candidate barcode (Meyer & alignment required many gaps (Table 2). The sequence Paulay, 2005; Lahaye et al., 2008). lengths of ITS, atpH‐atpI, rpl32‐trnLUAG, rps18‐clpp, To evaluate the success in species assignment or trnL‐trnF, trnL‐ndhJ, and trnS‐trnfM varied from 627 identification within our dataset of each gene marker, to 945, 410 to 419, 857 to 872, 1848 to 1874, 802 to BLAST1 and the nearest distance method were carried out 815, 916 to 1112, and 1080 to 1135 bp, respectively. as described previously (Ross et al., 2008; Chen Sequence variability of these regions is provided in et al., 2010). The BLAST1 searches were carried out on a Table 2. The ITS region had the highest number of local reference library constructed for each gene variable sites (14.50%) and parsimony‐informative marker. The barcode sequence of each species was sites (12.15%) and also the highest value of nucleotide queried against the corresponding reference library with diversity (3.8 102). The trnS‐trnfM region ranked the “BLASTN” command and the query sequence was second in terms of number of variable sites (12.08%), removed from the reference library. The identity of a followed by rpl32‐trnLUAG (10.20%). In terms of sample was based on the best hit and the E‐value for the parsimony‐informative sites, rpl32‐trnLUAG had the match that must be lower than the cut‐off value (102). second highest (6.90%), followed by matK (5.10%). In comparison, for the nearest genetic distance method, Sequences of rbcL were the most highly conserved with

Table 2 Variability of nine candidate DNA barcodes for Podophylloideae evaluated in this study DNA barcode No. of Aligned No. information No. variable Nucleotide Indels Length samples length (bp) sites (%) sites (%) diversity range (bp) matK 37 783 40 (5.10) 40 (5.10) 0.00980 0 783 rbcL 37 651 8 (1.23) 9 (1.38) 0.00284 0 651 atpH‐atpI 37 427 9 (2.11) 26 (6.09) 0.00436 5 410–419 rpl32‐trnLUAG 37 884 61 (6.90) 90 (10.2) 0.02065 8 857–872 rps18‐clpp 37 1914 23 (1.20) 97 (3.81) 0.00346 14 1848–1874 trnL‐trnF 37 831 26 (3.13) 55 (6.62) 0.00593 7 802–815 trnL‐ndhJ 37 1136 35 (3.08) 74 (6.51) 0.00720 8 916–1112 trnS‐trnfM 37 1192 31 (2.60) 144 (12.08) 0.00536 17 1080–1135 ITS 37 634 77 (12.15) 92 (14.5) 0.03828 7 627–945 Number in bracket indicates percentage of parsimony informative sites; Number in bracket indicates percentage of variable sites. ITS, internal transcribed spacer. the lowest number of variable sites (1.38%) and found that the maximum intraspecific distance within parsimony‐informative sites (1.23%). Dysosma versipellis was close to the minimum interspecific distance between D. versipellis and 2.2 Intra‐ and interspecific DNA sequence D. pleiantha (Hance) Woodson, leading to the narrow divergence overlapping distance (Fig. 1). As expected, when Six metrics estimated in PAUP (see 1.3 Data D. versipellis was excluded from the “barcoding gap” analysis) were used to characterize inter‐ versus test, ITS showed a clear barcoding gap (Fig. 2). For all intraspecific variation. The results showed that the nine barcodes examined, intraspecific differences were mean interspecific K2P distances of 0.0079 with a range significantly lower than those of interspecific diver- between 0.0000 and 0.0463 across all species pairs for gences based on results from both the median and the nine candidate loci were approximately 40‐fold Wilcoxon two‐sample tests. The difference between higher than the mean intraspecific K2P distance of intra‐ and interspecific distances was the greatest in ITS 0.0002 with a range between 0.0000 and 0.0032. The (e.g., Wilcoxon two‐sample test, P 7.82 1015), highest interspecific divergence was observed in ITS followed by trnL‐ndhJ (P 3.90 1013) (Table S4). with all three metrics (K2P distance, theta prime, minimum interspecific distance), followed by rpl32‐ 2.4 Identification efficiency of the DNA barcodes UAG trnL , whereas the lowest interspecific divergence Two methods (BLAST1 and the nearest genetic was observed in rbcL (Table 3). Based on these distance) were used to test the applicability of the nine estimates of three metrics for intraspecific divergence barcodes for species identification. Our results revealed (K2P distance, theta, coalescent depth), the highest that ITS possessed the highest species identification intraspecific divergence was also recorded for ITS, efficiency at 100% using both methods. In contrast, the followed by trnL‐trnF, whereas the lowest was rbcL, rate of successful species identification using rbcL was atpH‐atpI, and trnS‐trnfM (Table 3). Similar results the lowest (BLAST1 method, 37.84%; distance method, were obtained using Wilcoxon signed‐rank tests 35.14%) (Table 4). (Tables S2, S3). 2.5 Monophyletic test based on phylogenetic trees 2.3 DNA barcoding gap To evaluate whether species were recovered as To evaluate whether a “barcode gap” is present in monophyletic under each barcode, we constructed each of the nine barcodes, we examined their phylogenetic trees with single candidate loci. Among distributions of intra‐ versus interspecific sequence single‐locus analyses, ITS and matK attained the divergences at a scale of 0.002 distance units (Fig. 1). highest score of monophyletic species, with a success As can be seen from the graph (Fig. 1), barcoding gaps rates of 83.3% (i.e., 10 correctly identified out of 12 were not found in any of the nine candidate loci. Each species) under both MP and NJ, followed by rpl32‐ distribution graph showed an overlap between intra‐ trnLUAG (75%), trnL‐ndhJ (66.7% under NJ), and trnL‐ and interspecific distances. However, ITS showed the trnF (66.7% under MP), whereas rbcL had the lowest least overlap and a relatively large range distribution of discrimination level (33.3%) under both MP and NJ inter‐ and intraspecific distances compared with other (Table 4). Due to the similarity in tree topologies based loci (Fig. 1). The interspecific distances ranged mainly on NJ and MP, tree‐based DNA identification was from 0.004 to 0.048, whereas intraspecific distances assessed using the NJ tree. The NJ analysis of the were less than 0.002. We examined the source data, and ITS þ matK dataset resulted in very similar topology to

that of ITS, thus providing the same score of 0.0009 0.0014 0.0111 0.0144 0.0133 0.0010 monophyletic species (83.3%). We note, however, ITS that ITS, matK, and ITS þ matK failed to distinguish D. versipellis and D. pleiantha. The NJ analysis of 37 individuals for ITS sequences (Fig. 3) recovered three major clades (clade 1, Dysosma; clade 2, Sinopodo- 0.0000 0.0005 0.0000 0.0008 0.0026 0.0297 0.0028 0.0197 0.0026 0.0147 0.0000 0.0006 phyllum and Podophyllum; clade 3, Diphylleia), trnfM ‐ corresponding to the previously recognized genera (Wang et al., 2007). In clade 1, a monophyletic D. pleiantha fell among samples of D. versipellis (Fig. 3). Based on the present ITS data, all Podophyl- 0.0010 0.0000 0.0030 0.0042 0.0039 0.0030 0.0031 0.0020 0.0008 0.0000 0.0005 0.0000

ndhJ trnS loideae species with multiple individuals, except for ‐ D. versipellis, were recovered as monophyletic.

3 Discussion 0.0011 0.0005 0.0024 0.0042 0.0032 0.0035 0.0029 0.0023 0.0009 0.0005 0.0006 0.0002 trnF trnL ‐ 3.1 Evaluation of potential barcodes for Podophylloideae An ideal DNA barcode sequence should have adequately conserved flanking regions for the design of 0.0010 0.0007 0.0011 0.0034 0.0013 0.0029 0.0014 0.0018 0.0008 0.0006 0.0007 0.0004 clpp trnL ‐ universal primers, high PCR amplification efficiency, and sufficient variability that can be used for species rps18 identification (CBOL Plant Working Group, 2009). In the present study, ITS showed suitable aligned

UAG sequence lengths (approximately 634 bp), the most 0.0004 0.0006 0.0071 0.0029 0.0083 0.0027 0.0084 0.0017 0.0003 0.0003 0.0002 0.0005 ‐ fi trnL variation, greater intra and interspeci c divergences, ‐ the highest species discrimination rate (83.3%), and a relatively well‐defined gap between intra‐ and interspecific divergences (Tables 2–4; Fig. 1) among the markers examined. These features make it the best 0.0000 0.0001 0.0030 0.0131 0.0038 0.0104 0.0038 0.0068 0.0000 0.0001 0.0000 0.0001 atpI rpl32 ‐ choice as a DNA barcode in Podophylloideae. Nuclear ITS was initially proposed as a DNA barcode for plants due to its high sequence divergence (Kress et al., 2005; Cowan et al., 2006). However, it was rejected from incorporation into the core plant barcode due to inherent 0.0000 0.0000 0.0017 0.0029 0.0015 0.0027 0.0014 0.0020 0.0000 0.0000 0.0000 0.0000 problems such as the possible presence of paralogous ITS copies or incongruence between ITS1 and ITS2 (e.g., Álvarez & Wendel, 2003; Chase et al., 2007; Starr et al., 2009; Hollingsworth et al., 2011). These potential

c divergences for nine DNA barcodes for Podophylloideae drawbacks of ITS were not found in this study. Due to ﬁ 0.0004 0.0000 0.0054 0.0023 0.0052 0.0015 0.0047 0.0008 0.0003 0.0000 0.0003 0.0000 ﬁ dif culties in amplifying both ITS1 and ITS2 regions as matK rbcL atpH a whole by PCR and high variability of the ITS1 region in some lineages of plants, the ITS2 region alone has

and intraspeci been proposed as the plant DNA barcode marker ‐ (Hollingsworth et al., 2009; Chen et al., 2010). In the present study, we found that the ITS1 region should also be included in the ITS barcode in Podophylloideae, because ITS2 alone only provided a 66.7% species

Estimates of inter discrimination rate (data not shown). The chloroplast rbcL has been selected as one of the core plant barcodes due to its high universality, sequence quality, and high Coalescent depth 0.0001 Average interspecific distance 0.0084 Markers Theta prime 0.0054 Table 3 Minimum interspecific distance 0.0040 Average intraspecific distance 0.0001 Theta 0.0001 discrimination power at family and genus level (Kress

Fig. 1. Relative distribution of inter‐ and intraspeciﬁc Kimura 2‐parameter (K2P) distances for nine DNA barcodes for Podophylloideae. A, matK. B, rbcL. C, atpH‐atpI. D, rpl32‐trnLUAG. E, rps18‐clpp. F, trnL‐trnF. G, trnL‐ndhJ. H, trnS‐trnfM. I, Internal transcribed spacer (ITS). Barcoding gaps were assessed with median tests and Wilcoxon two‐sample tests, and all were highly signiﬁcant (P < 0.0001).

& Erickson, 2007; Hollingsworth et al., 2009; Liu chloroplast coding region matK was also recommended et al., 2011), although it possesses very low interspecific as one of the core plant barcodes by CBOL, yet its variation (Kress et al., 2005), especially between universality was questioned in some groups (Chase closely related species (Newmaster et al., 2008). In et al., 2007). In our case, all samples surveyed were our study, we similarly found that rbcL contained the successfully amplified and sequenced using universal lowest number of parsimony‐informative sites and the matK primer pairs (Table S1), indicating a very high lowest genetic variability (Table 2). Moreover, rbcL universality for Podophylloideae species. The matK also performed poorest in species discrimination with gene also attained the highest score of monophyletic less than half the species (33.3%) successfully identi- species in our tree‐based species identification tests. fied. Consequently, we proposed that rbcL is not a good However, the overlap of intraspecific and interspecific choice for barcoding Podophylloideae species. The divergence was too substantial to be of use for species

tion (Kress et al., 2005). Wilcoxon signed‐rank tests showed that rpl32‐trnLUAG was outstanding in its interspeciﬁc variation compared to atpH‐atpI, rps18‐ clpp, trnL‐trnF, trnL‐ndhJ, trnS‐trnfM. The rpl32‐ trnLUAG region also provided a relatively high rate of success of species identiﬁcation (75%), but all of these species can also be discriminated by ITS sequences alone. Taken together, we suggest that ITS alone can serve as the only DNA barcode in Podophylloideae.

3.2 Taxonomy and species identification Multiple individuals (2–5) per species were collected in this study. Of all 12 morphologically defined species in Podophylloideae, the NJ tree based on ITS sequence data identified 10 monophyletic species, and each was well supported with high bootstrap values (>95%) (Fig. 3). The genetic differentiation between species is generally larger Fig. 2. Relative distribution of inter‐ and intraspecific Kimura 2‐ parameter (K2P) distances for internal transcribed spacer (ITS) sequences than within species, as indicated by smaller intraspecific among all samples of 11 species of Podophylloideae (excluding Dysosma than interspecific distances (Table 3). Furthermore, we versipellis). observed no incongruence between the plastid matK and nuclear ITS trees, suggesting the present dataset discrimination (Fig. 1). To further explore the potential involved no natural hybridization. In the monograph of of matK as candidate barcode in Podophylloideae, we Epimedium, Stearn (2002) included two genera in the constructed trees of ITS þ matK, which provided subfamily Podophylloideae, Diphylleia and Podophyl- 83.3% species resolution (data not shown). Considering lum. Shaw (2009) divided Podophyllum into three that ITS alone provided up to 83.3% species resolution, genera: Podophyllum with a single species P. peltatum it appears impractical and unnecessary to combine ITS from ENA; Dysosma for the Eastern Asiatic taxa; and with matK. Of the seven plastid intergenic spacer Sinopodophyllum for the Himalayan species often regions, rps18‐clpp had the lowest amplification known as P. hexandrum Royle. The decision to efficiency (75.7%; Table S1), which thus disqualifies recognize Sinopodophyllum is based on several this marker as a DNA barcode in Podophylloideae. As characters that are often used at generic level, such as expected, indels are common in plastid intergenic pollen exine sculpture, pollen grains released in tetrads, spacer regions of Podophylloideae species (Table 2), and the unique shape of the first leaf (Ying, 1979). In which resulted in greater length variation and increased this study, the samples from three genera clustered in a difficulty in alignment. In contrast with the problems of monophyletic clade in both MP and NJ trees based on indels for sequence alignment, indels will ultimately ITS nucleotide sequences, even though they were provide the information needed for species discrimina- collected from a very wide distribution range,

Table 4 Identiﬁcation efﬁciency of nine DNA barcodes for 12 species in Podophylloideae using similarity‐based (BLAST1 and nearest distance) and tree‐ based (neighbor‐joining (NJ) and maximum parsimony (MP)) methods DNA barcodes BLAST1 method Distance method NJ method MP method Successfully Ambiguous Successfully Ambiguous Percentage Percentage identified (%) (%) identified (%) (%) of species of species monophyly (%) monophyly (%) matK 83.78 16.22 83.78 16.22 83.3 83.3 rbcL 37.84 62.16 35.14 64.86 33.3 33.3 atpH‐atpI 67.57 32.43 40.54 59.46 41.7 41.7 rpl32‐trnLUAG 72.97 27.03 72.97 27.03 75.0 75.0 rps18‐clpp 92.59 7.41 92.59 7.41 41.7 41.7 trnL‐trnF 78.38 21.62 78.38 21.62 58.3 66.7 trnL‐ndhJ 100 0 89.19 10.81 66.7 50.0 trnS‐trnfM 89.19 10.81 67.57 32.43 58.3 58.3 ITS 100 0 100 0 83.3 83.3 ITS, internal transcribed spacer.

Fig. 3. Neighbor‐joining tree based on the internal transcribed spacer sequences with the Kimura 2‐parameter distance model. Bootstrap values (>50%) are shown above the relevant branches.

© 2014 Institute of Botany, Chinese Academy of Sciences MAO et al.: DNA barcodes for Podophylloideae 11 supporting a closer relationship of three genera. outcome of such prospective studies, with the compre- Monophyletic Diphylleia is recognized by ITS and hensive species sampling used in the present study, our combined ITS þ matK data, which is congruent with results indicated that ITS sequencing provided a reliable the previous molecular phylogenetic studies (Liu and highly efficient and effective means for the et al., 2002; Wang et al., 2007) and also supported by discrimination of most Podophylloideae species, morphological characters such as rhizomes with leaf although not all. scars, flowers in erect, many‐flowed cymes, anthers opening by up‐rolling flaps, and a small, blue berry Acknowledgements We thank Dr. Shota SAKAGU- (Ying et al., 2011). Fifteen individuals collected from CHI (Kyoto University, Kyoto, Japan), Pan LI (Zhejiang seven morphologically defined species in Dysosma University, Hangzhou, China) for help during fieldwork, formed a monophyletic clade, providing convincing Hans Peter COMES (Salzburg University, Salzburg, evidence for the monophyly of Dysosma. Of the 12 Austria) for very insightful comments that improved Podophylloideae species, D. versipellis did not form earlier drafts of the paper, and Hui YAO (Peking Union species‐specific monophyletic clades. Rather, three Medical College, Beijing, China) for computational samples of D. versipellis clustered with monophyletic assistance. The authors also thank the editor for D. pleiantha. However, D. pleiantha and D. versipellis providing valuable comments on the manuscript for are easily distinguishable by morphological characters improvement. This research was supported by the (Ying et al., 1993, 2011). Thus, the seeming para- National Natural Science Foundation of China (Grant phyletic structure of D. versipellis might be explained Nos. 31170200, 30900082), the Zhejiang Provincial by incomplete lineage sorting between two species due Funds for Distinguished Young Scientists (Grant No. to recent divergence. This situation prevents using ITS LR12C02001), the Fundamental Research Funds for the as the barcode to identify D. versipellis. To fully Central Universities (Grant No. 2011QNA6013), the understand this situation, however, more sampling and Qianjiang talent project from the Bureau of Science and detailed morphological and molecular studies on both Technology of Zhejiang Province, China (Grant No. species are needed. 2010R10090); and the Main Direction Program of DNA barcoding can be used for species identifica- Knowledge Innovation of the Chinese Academy of tion. It can provide a deeper understanding of Sciences (Grant No. KSCX2‐EWZ‐1). biodiversity at large and species boundaries. DNA barcoding is only complementary, not to replace morphological taxonomic approaches (Yang et al., References 2012). Identification of all species of a taxonomic group Álvarez I, Wendel JF. 2003. Ribosomal ITS sequences and plant using DNA barcoding requires comprehensive species phylogenetic inference. Molecular Phylogenetics and sampling to facilitate a high rate of success of species Evolution 29: 417–434. identification (Hollingsworth et al., 2011). However, Broomhead AJ, Dewick PM. 1990. Tumor inhibitory aryltralin lignans in Podophyllum versipelle, Diphylleia cymosa and how many specimens are needed to construct a reliable – fi Diphylleia grayi. Phytochemistry 29: 3831 3837. reference for a given species for reliable identi cation is CBOL Plant Working Group. 2009. A DNA barcode for land still an open question. Some authors suggested plants. 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