Table of Contents

01 June 2011, Volume 49 Issue 3
Cover illustration: Conceptual sketch for plant DNA barcoding. A coordinated effort has been made across research groups to enhance the creation of a reference database for plant barcoding in China. See Editorial by LI et al., pp. 165–168 in this issue. Designed and illustrated by Ling WANG & Hong WANG.
  • De-Zhu LI, Jian-Quan LIU,Zhi-Duan CHEN,Hong WANG,Xue-Jun GE,Shi-Liang ZHOU,Lian-Ming GAO,Cheng-Xin FU,Shi-Lin CHEN
    J Syst Evol. 2011, 49(3): 165-168.
  • Research Articles
  • Yan LI,Lian-Ming GAO, Ram C. POUDEL, De-Zhu Li, Alan FORREST
    J Syst Evol. 2011, 49(3): 169-175.
    DNA barcoding is a tool to provide rapid and accurate taxonomic identification using a standard DNA region. A two-marker combination of matK+rbcL was formally proposed as the core barcode for land plants by the Consortium for the Barcode of Life Plant Working Group. However, there are currently no barcoding primers for matK showing high universality in gymnosperms. We used 57 gymnosperm species representing 40 genera, 11 families and four subclasses to evaluate the universality of nine candidate matK primers and one rbcL primer in this study. Primer (1F/724R) of rbcL is proposed here as a universal primer for gymnosperms due to high universality. One of the nine candidate matK primers (Gym_F1A/Gym_R1A) is proposed as the best “universal”matK primer for gymnosperms because of high polymerase chain reaction success and routine generation of high quality bidirectional sequences. A specific matK primer for Ephedra was newly designed in this study, which performed well on the sampled species. The primers proposed here for rbcL and matK can be easily and successfully amplified for most gymnosperms.
  • Jing YU, Jian-Hua XUE, Shi-Liang ZHOU
    J Syst Evol. 2011, 49(3): 176-181.
    The chloroplast maturase K gene (matK) is one of the most variable coding genes of angiosperms and has been suggested to be a “barcode” for land plants. However, matK exhibits low amplification and sequencing rates due to low universality of currently available primers and mononucleotide repeats. To resolve these technical problems, we evaluated the entire matK region to find a region of 600–800 bp that is highly variable, represents the best of all matK regions with priming sites conservative enough to design universal primers, and avoids the mononucleotide repeats. After careful evaluation, a region in the middle was chosen and a pair of primers named matK472F and matK1248R was designed to amplify and sequence the matK fragment of approximately 776 bp. This region encompasses the most variable sites, represents the entire matK region best, and also exhibits high amplification rates and quality of sequences. The universality of this primer pair was tested using 58 species from 47 families of angiosperm plants. The primers showed a strong amplification (93.1%) and sequencing (92.6%) successes in the species tested. We propose that the new primers will solve, in part, the problems encountered when using matK and promote the adoption of matK as a DNA barcode for angiosperms.
  • Qian WANG,Qiu-Shi YU,Jian-Quan LIU
    J Syst Evol. 2011, 49(3): 182-188.
    Considerable debate remains as to which DNA region should be used to barcode plants. Several different chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA) and nuclear ribosomal internal transcribed regions (ITS) have been suggested as suitable barcodes in plants. Recently, low-copy nuclear loci were also suggested to be potentially ideal barcode regions. The aim of the present study was to test the effectiveness of these proposed DNA fragments and five additional low-copy loci (CHS, DET1, COP1, PGIC1, and RPS2; comprising both coding and non-coding regions) in barcoding closely related species. We examined the divergences within and between two species of Pugionium (Brassicaceae). We failed to find any interspecific variation from three cpDNA fragments with which to discriminate the two species. However, a single base mutation in the internal transcribed spacer (ITS) could discriminate between the two species consistently. We found more variations among all individuals of the two species using each of the other five low-copy nuclear loci. However, only alleles from one locus (DET1) of the five low-copy loci related to flowering regulations was able to distinguish the sampled individuals into two species. We failed to amplify the corresponding fragments out of Brassicaceae using the designed DET1 primers. We further discussed the discrimination power of different loci due to incomplete lineage sorting, gene flow, and species-specific evolution. Our results highlight the possibility of using the nuclear ITS as a core or complementary fragment to barcode recent diverged species.
  • Li-Na DONG,Alexandra H. Wortley,Hong WANG, De-Zhu LI,Lu LU
    J Syst Evol. 2011, 49(3): 189-202.
    DNA barcoding is becoming an increasingly popular means to identify species. The obscure discrimination in the genus Pterygiella calls into question the re-assessment of the criterion for species delimitation. We collected 20 individuals, representing all five described species of this genus in its distributional range. The aim was to use three proposed barcode DNA regions (rbcL, matK, and ITS) to diagnose Pterygiella species, and examine which barcode is more suitable for discerning the congeneric and related species. The results showed that the core barcodes matK and rbcL were comparatively less effective. However, the ITS region, especially ITS-1 and ITS-2, successfully identified all species in the genus. Furthermore, the secondary structure of ITS-2 RNA, especially compensatory base changes, appears complementary to classical primary sequence analysis for DNA barcoding.
  • Xing GUO,Mark P. SIMMONS,Paul Pui-Hay BUT,Pang-Chui SHAW, Rui-Jiang WANG
    J Syst Evol. 2011, 49(3): 203-212.
    The potential application of DNA barcodes of plastid (matK, trnH–psbA, petD, and rbcL) and nuclear (internal transcribed spacer (ITS) of rDNA) DNA regions was investigated for 25 Hedyotis taxa. The ITS showed the best species discrimination by resolving 23 of the species as exclusive lineages with no shared alleles between any of the 24 distinct species (H. assimilis and H. mellii are not supported as distinct species based on our molecular and morphological data). Conversely, rbcL performed the worst and only resolved 10 of the species as exclusive lineages, and 10 species with shared alleles. Using ITS has the advantage of high PCR amplification success and it provides good intra- and interspecific variation distribution patterns. The most powerful plastid markers were petD and trnH–psbA, but we could amplify and sequence trnH–psbA for only 83% of the accessions sampled. Combination of ITS and petD performed extremely well, with all 24 of the distinct species resolved as exclusive lineages and no shared alleles between any of the distinct species. We therefore recommend ITS, or a combination of ITS and petD, as the standard DNA barcode in Hedyotis, but acknowledge that there are no shared alleles between distinct species for matK and rbcL combined.
  • Jing GU, Jun-Xia SU, Ruo-Zhu LIN, Rui-Qi LI, Pei-Gen XIAO
    J Syst Evol. 2011, 49(3): 213-224.
    DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH–psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH–psbA, and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH–psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH–psbA as a candidate barcode sequence.
  • Hai-Fei YAN, Gang HAO, Chi-Ming HU, Xue-Jun GE
    J Syst Evol. 2011, 49(3): 225-236.
    DNA barcoding is a method of identifying species by analyzing one or a few short standardized DNA sequences. There are particular challenges in barcoding plants, especially for distinguishing closely related species. Hence, there is an urgent need to evaluate the performance of candidate loci for distinguishing between species, especially closely related species, to complement the rbcL + matK combination suggested as the core barcode for land plants. We sampled 48 individuals representing 12 species in Primula sect. Proliferae Pax in China to evaluate the performance of eight leading candidate barcode loci (matK, rbcL, rpoB, rpoC1, trnH-psbA, psbK-psbI, atpF-atpH, and internal transcribed spacer (ITS)). The core combination rbcL+matK gave only 50% species resolution in sect. Proliferae. In terms of intraspecies and interspecies divergence, degree of monophyly, and sequence similarity, ITS, trnH-psbA, and psbK-psbI showed good performance as single-locus barcodes. Internal transcribed spacer displayed the highest genetic divergence and best discriminatory power, both alone and in combination with rbcL+matK (83.3% species resolution). We recommend evaluating the use of ITS for barcoding in other species. Low or single copy nuclear regions would provide more sophisticated barcoding tools in the long term, even though further research is required to find suitable loci.
  • Yuan-Miao FU, Wei-Mei JIANG, Cheng-Xin FU
    J Syst Evol. 2011, 49(3): 237-245.
    Many species of Tetrastigma (Miq.) Planch. (Vitaceae) have long been used as medicinal plants in China, and some are endangered due to overexploitation. Although adulterants are often added to traditional Chinese medicines, there is no reliable or practical method for identifying them. In this study, we used four markers (rbcL, matK, trnH-psbA, and internal transcribed spacer [ITS]) as DNA barcodes to test their ability to distinguish species of Tetrastigma. The results indicated that the best barcode was ITS, which showed significant inter-specific genetic variability, and thus its potential as a DNA barcode for identifying Tetrastigma. Multiple loci provided a greater ability to distinguish species than single loci. We recommend using the combined rbcL+matK+ITS barcode for the genus. Phylogenetic trees from each barcode were compared. Analyses using the unweighted pair group method with arithmetic mean discriminated an equal or greater percentage of resolvable species than did neighbor joining, maximum likelihood, or maximum parsimony analyses. Additionally, five medicinal species of Tetrastigma, especially T. hemsleyanum, could be identified precisely using DNA barcoding.
  • Zhi-Yuan DU, Alitong QIMIKE,Chun-Feng YANG,Jin-Ming CHEN, Qing-Feng WANG
    J Syst Evol. 2011, 49(3): 246-251.
    The pondweeds (Potamogetonaceae) are among the most important plant groups in the aquatic environment. Owing to their high morphological and ecological diversity, species identification of this aquatic family remains problematic. DNA barcoding involves sequencing a standard DNA region and has been shown to be a powerful tool for species identification. In the present study, we tested four barcoding markers (rbcL, matK, internal transcribed spacer (ITS), and trnH–psbA) in 15 Potamogeton species and two Stuckenia species, representing most species of the Potamogetonaceae in China. The results show that all four regions can distinguish and support the newly proposed genera of Stuckenia from Potamogeton. Using ITS and trnH–psbA, significant interspecific genetic variability was shown. However, intraspecific genetic variability of trnH–psbA is high and so it is not suitable for barcoding in Potamogetonaceae. The ITS and matK regions showed good discrimination. However, matK was not easy to sequence using universal primers. The best performing single locus was ITS, making it a potentially useful DNA barcode in Potamogetonaceae.
  • Xiao-Guo XIANG, Jing-Bo ZHANG, An-Ming LU, Rui-Qi LI
    J Syst Evol. 2011, 49(3): 252-260.
    DNA barcoding is a method of species identification and recognition using DNA sequence data. A tiered or multilocus method has been recommended for barcoding plant species. In this study, we sampled 196 individuals representing 9 genera and 54 species of Juglandaceae to investigate the utility of the four potential barcoding loci (rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS)). Our results show that all four DNA regions are easy to amplify and sequence. In the four tested DNA regions, ITS has the most variable information, and rbcL has the least. At generic level, seven of nine genera can be efficiently identified by matK. At species level, ITS has higher interspecific p-distance than the trnH-psbA region. Difficult to align in the whole family, ITS showed heterogeneous variability among different genera. Except for the monotypic genera (Cyclocarya, Annamocarya, Platycarya), ITS appeared to have limited power for species identification within the Carya and Engelhardia complex, and have no power for Juglans or Pterocarya. Overall, our results confirmed that a multilocus tiered method for plant barcoding was applicable and practicable. With higher priority, matK is proposed as the first-tier DNA region for genus discrimination, and the second locus at species level should have enough stable variable characters.
  • Lin-Chun SHI, Jin ZHANG, Jian-Ping HAN, Jing-Yuan SONG,Hui YAO,Ying-Jie ZHU,Jia-Chun LI,Zhen-Zhong WANG,Wei XIAO, Yu-Lin LIN, Cai-Xiang XIE,Zhong-Zhi QIAN, Shi-Lin CHEN
    J Syst Evol. 2011, 49(3): 261-266.
    In 2009, the Consortium for the Barcode of Life (CBOL) recommended the combination of rbcL and matK as the plant barcode based on assessments of recoverability, sequencing quality, and levels of species discrimination. Subsequently, based on a study of more than 6600 samples belonging to 193 families from seven phyla, the internal transcribed spacer (ITS) 2 locus was proposed as a universal barcode sequence for all major plant taxa used in traditional herbal medicine. Neither of these two studies was based on a detailed analysis of a particular family. Here, Zingiberaceae plants, including many closely related species, were used to compare the genetic divergence and species identification efficiency of ITS2, rbcL, matK, psbKpsbI, trnHpsbA, and rpoB. The results indicate that ITS2 has the highest interspecific divergence and significant differences between inter- and intraspecific divergence, whereas matK and rbcL have much lower divergence values. Among 260 species belonging to 30 genera in Zingiberaceae, the discrimination ability of the ITS2 locus was 99.5% at the genus level and 73.1% at the species level. Thus, we propose that ITS2 is the preferred DNA barcode sequence for identifying Zingiberaceae plants.
  • Chun-Ying XUE, De-Zhu LI
    J Syst Evol. 2011, 49(3): 267-270.
    Gentianopsis paludosa (Hook. f.) Ma (Gentianaceae) is an important species in Tibetan folk medicine commonly used to clear away the “heat evils” and toxic materials. A survey of market samples revealed that nine adulterant species, Gentianopsis barbata, G. contorta, G. grandis, Halenia elliptica, Lomatogonium macranthum, L. rotatum, Swertia angustifolia, S. bifolia and S. erythrosticta, are often marketed as G. paludosa. Methods to distinguish G. paludosa from its adulterants are limited by current morphological and chemical methods. DNA sequence analysis of the nuclear ribosomal DNA internal transcribed spacer region (ITS) was used in the differentiation of G. paludosa from the adulterant species. The data showed that the internal transcribed spacer regions differ significantly between G. paludosa and all nine adulterant species, so that they could be easily distinguished at the DNA level.
  • Reviews
  • Ming LI, Hui CAO, Paul Pui-Hay BUT, Pang-Chui SHAW
    J Syst Evol. 2011, 49(3): 271-283.
    Herbal medicinal materials have been used worldwide for centuries to maintain health and to treat disease. However, adulteration of herbal medicines remains a major concern of users and industry for reasons of safety and efficacy. Identification of herbal medicinal materials by DNA technology has been widely applied, started from the mid-1990s. In recent years, DNA barcoding of global plant species using four standard barcodes (rbcL, matK, trnHpsbA and ITS) has been a major focus in the fields of biodiversity and conservation. These DNA barcodes can also be used as reliable tools to facilitate the identification of herbal medicinal materials for the safe use of herbs, quality control, and forensic investigation. Many studies have applied these DNA barcodes for the identification of herbal medicinal species and their adulterants. The present article reviews efforts in the identification of herbal medicinal materials using the standard DNA barcodes and other DNA sequence-based markers.