Biodiversity Science ›› 2017, Vol. 25 ›› Issue (10): 1095-1104.doi: 10.17520/biods.2017164

• Original Papers: Plant Diversity • Previous Article     Next Article

Comparison of species resolution rates of DNA barcoding for Chinese coastal halo-tolerant plants

Ya’nan Wei1, Xiaomei Wang1, Pengcheng Yao1, Xiaoyong Chen2, Hongqing Li1, *()   

  1. 1 School of Life Sciences, East China Normal University, Shanghai 200241
    2 School of Ecological and Environmental Sciences, East China Normal University, Shanghai 200241
  • Received:2017-06-02 Accepted:2017-08-30 Online:2018-05-05
  • Li Hongqing

Halo-tolerant plants compose a huge group of plants with unique ecological and economical value. Little is known about their DNA barcoding speciality. In this study, 562 samples of coastal halo-tolerant plants (including 53 families, 97 genera and 116 species) were collected from 10 coastal provinces, ranging from Liaoning to Hainan. Three chloroplast DNA markers (matK, rbcL and trnH-psbA) and one nuclear DNA marker (ITS) were amplified and sequenced. Primer universality and sequence availability of each locus were examined and species resolution rates were tested. When considering sequence availability, matK and trnH- psbA were among the best. But the primer universality of ITS was marginally worse than expected. The all-to-all BLASTn searches indicated that the species resolution rate of ITS was the highest (73.36%), followed by those of matK (64.03%), trnH-psbA (61.21%) and rbcL (46.41%). Phylogenetic trees (NJ trees) indicated that the species resolution rate of matK was the highest (82.3%), but no reliable NJ tree based on trnH-psbA could be acquired because of unequal sequence length. NMDS and PCoA results demonstrated that both chloroplast DNA markers and nuclear DNA markers should be considered when conducting coastal plant DNA barcoding studies. Based on the above results, we suggest that the combination of ITS + matK should be regarded as the barcode for halo-tolerant plants in Chinese coastal regions. In total, the 1939 newly acquired sequences in this study lay the foundation for a DNA barcode database of costal halo-tolerant plants.

Key words: DNA barcoding, coastal region, halo-tolerant plants, species resolution rate, ITS, matK

Table 1

Summary of the sequencing success rate and length of candidate barcoding fragments"

Species resolution rate
Sequence availability
successfully sequenced
Fragment length (bp)
ITS 481 85.59% 106 304-632
matK 502 89.32% 116 611-721
rbcL 465 83.04% 109 371-650
trnH-psbA 491 87.36% 116 142-907

Fig. 1

Species resolution rate from all to all BLASTn searches"

Fig. 2

Species resolution rate of 15 genera which contain more than one species"

Fig. 3

Species resolution rate based on the phylogenetic method"

Fig. 4

Multivariate analysis of non-metric multidimensional scaling for the four barcode markers NMDS. Stress value: 0.011."

[1] Adoukonou-Sagbadja H, Wagner C, Ordon F, Friedt W (2010) Reproductive system and molecular phylogenetic relationships of fonio millets (Digitaria spp., Poaceae) with some polyploid wild relatives. Tropical Plant Biology, 3, 240-251.
[2] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410.
[3] Alves TLS, Chauveau O, Eggers L, de Souza-Chies TT (2014) Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus. Molecular Ecology Resources, 14, 324-335.
[4] Bafeel SO, Arif IA, Al-Homaidan AA, Khan HA (2012) Assessment of DNA barcoding for the identification of Chenopodium murale L. (Chenopodiaceae). International Journal of Biology, 4, 66-74.
[5] Barthet MM, Hilu KW (2007) Expression of matK: functional and evolutionary implications. American Journal of Botany, 94, 1402-1412.
[6] Baumel A, Ainouche ML, Levasseur J (2001) Molecular investigations in populations of Spartina anglica C. E. Hubbard (Poaceae) invading coastal Brittany (France). Molecular Ecology, 10, 1689-1701.
[7] Bera B, Das S, Mukherjee K (1993) Morphological studies on three cytotypes of Chenopodium album L. of Lower Gangetic Plains, West Bengal, India. Phytomorphology, 43(1-2), 93-103.
[8] Braukmann TWA, Kuzmina ML, Sills J, Zakharov EV, Hebert PDN (2017) Testing the efficacy of DNA barcodes for identifying the vascular plants of Canada. PLoS ONE, 12, e0169515.
[9] Burgess KS, Fazekas AJ, Kesanakurti PR, Graham SW, Husband BC, Newmaster SG, Percy DM, Hajibabaei M (2011) Discriminating plant species in a local temperate flora using the rbcL+matK DNA barcode. Methods in Ecology and Evolution, 2, 333-340.
[10] Burland TG (2000) DNASTAR’s lasergene sequence analysis software.In: Methods in Molecular Biology (ed. Walker JM), pp. 71-91.Humana Press, New Jersey.
[11] CBOL Plant Working Group (2009) A DNA barcode for land plants. Proceedings of the National Academy of Sciences, USA, 106, 12794-12797.
[12] Chase MW, Cowan RS, Hollingsworth PM, van den Berg C, Madriñán S, Petersen G, Seberg O, Jørgsensen T, Cameron KM, Carine M, Pedersen N, Hedderson TAJ, Conrad F, Salazar GA, Richardson JE, Hollingsworth ML, Barraclough TG, Kelly L, Wilkinson M (2007) A proposal for a standardised protocol to barcode all land plants. Taxon, 56, 295-299.
[13] Chase MW, Salamin N, Wilkinson M, Dunwell JM, Kesanakurthi RP, Haidar N, Savolainen V (2005) Land plants and DNA barcodes: short-term and long-term goals. Philosophical Transactions of the Royal Society: Biological Sciences, 360, 1889-1895.
[14] Chen XL, An SQ, Li GQ, Cheng XL, Zhang JL, Shen BQ (1999) The economic salt-tolerant plant resources on the coastal zone of China. Journal of Nanjing Forestry University, 23(4), 81-84. (in Chinese with English abstract)
[陈兴龙, 安树青, 李国旗, 程晓莉, 张纪林, 沈邦勤 (1999) 中国海岸带耐盐经济植物资源. 南京林业大学学报(自然科学版), 23(4), 81-84.]
[15] China Plant BOL Group (2011) Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proceedings of the National Academy of Sciences, USA, 108, 19641-19646.
[16] Chu GL, Sergei LM, Steven EC (2003) Chenopodiaceae. In: Flora of China (eds Wu ZY, Raven PH), pp. 351-414. Science Press, Beijing & Missouri Botanical Garden Press, St. Louis.
[17] Costion C, Ford A, Cross H, Crayn D, Harrington M, Lowe A (2011) Plant DNA barcodes can accurately estimate species richness in poorly known floras. PLoS ONE, e26841.
[18] Cuénoud P, Savolainen V, Chatrou LW, Powell M, Grayer RJ, Chase MW (2002) Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. American Journal of Botany, 89, 132-144.
[19] De Vere N, Rich TC, Ford CR, Trinder SA, Long C, Moore CW, Satterthwaite D, Davies H, Allainguillaume J, Ronca S, Tatarinova T, Garbett H, Walker K, Wilkinson MJ (2012) DNA barcoding the native flowering plants and conifers of Wales. PLoS ONE, 7, e37945.
[20] De Vere N, Rich TC, Trinder SA, Long C (2015) DNA barcoding for plants. In: Methods in Molecular Biology (ed. Walker JM), pp. 101-118.Humana Press,New Jersey.
[21] Dong W, Liu J, Yu J, Wang L, Zhou S (2012) Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding. PLoS ONE, 7, e35071.
[22] Ebihara A, Nitta JH, Ito M (2010) Molecular species identification with rich floristic sampling: DNA barcoding the pteridophyte flora of Japan. PLoS ONE, 5, e15136.
[23] Gedan KB, Altieri AH, Bertness MD (2011) Uncertain future of New England salt marshes. Marine Ecology Progress, 434, 229-237.
[24] Gonzalez MA, Baraloto C, Engel J, Mori SA, Pétronelli P, Riéra B, Roger A, Thébaud C, Chave J (2009) Identification of Amazonian trees with DNA barcodes. PLoS ONE, 4, e7483.
[25] Hamilton MB, Braverman JM, Soria-Hernanz DF (2003) Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in new world species of the Lecythidaceae. Molecular Biology and Evolution, 20, 1710-1721.
[26] Hebert PD, Cywinska A, Ball SL (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B: Biological Sciences, 270, 313-321.
[27] Hollingsworth PM (2011) Refining the DNA barcode for land plants. Proceedings of the National Academy of Sciences, USA, 108, 19451-19452.
[28] Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS ONE, 6, e19254.
[29] Ito Y, Ohi-Toma T, Murata J, Tanaka N (2010) Hybridization and polyploidy of an aquatic plant, Ruppia (Ruppiaceae), inferred from plastid and nuclear DNA phylogenies. American Journal of Botany, 97, 1156-1167.
[30] Khalaf MA, Kochzius M (2002) Changes in trophic community structure of shore fishes at an industrial site in the Gulf of Aqaba, Red Sea. Marine Ecology Progress Series, 239, 287-299.
[31] Kress WJ, Erickson DL, Jones FA, Swenson NG, Perez R, Sanjur O, Bermingham E (2009) Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proceedings of the National Academy of Sciences, USA, 106, 18621-18626.
[32] Kress WJ, Garcia-Robiedo C, Uriarte M, Erickson DL (2015) DNA barcodes for ecology, evolution, and conservation. Trends in Ecology and Evolution, 30, 25-35.
[33] Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH (2005) Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences, USA, 102, 8369-8374.
[34] Kruskal JB (1964) Nonmetric multidimensional scaling: a numerical method. Psychometrika, 29, 115-129.
[35] Li DZ, Zeng CX (2015) Prospects for plant DNA barcoding. Biodiversity Science, 23, 297-298. (in Chinese)
[李德铢, 曾春霞 (2015) 植物DNA条形码研究展望. 生物多样性, 23, 297-298.]
[36] Li HQ, Chen JY, Wang S, Xiong SZ (2012) Evaluation of six candidate DNA barcoding loci in Ficus (Moraceae) of China. Molecular Ecology Resources, 12, 783-790.
[37] Lin P (2006) Marine Higher Plant Ecology. Science Press, Beijing. (in Chinese)
[林鹏 (2006) 海洋高等植物生态. 科学出版社, 北京.]
[38] Liu J, Yan HF, Ge XJ (2016) The use of DNA barcoding on recently diverged species in the genus Gentiana (Gentianaceae) in China. PLoS ONE, 11, e0153008.
[39] Liu J, Yan HF, Newmaster SG, Pei N, Ragupathy S, Ge XJ (2015) The use of DNA barcoding as a tool for the conservation biogeography of subtropical forests in China. Diversity and Distributions, 21, 188-199.
[40] Lucas C, Thangaradjou T, Papenbrock J (2012) Development of a DNA barcoding system for seagrasses: successful but not simple. PLoS ONE, 7, e29987.
[41] Molnar JL, Gamboa RL, Revenga C, Spalding MD (2008) Assessing the global threat of invasive species to marine biodiversity. Frontiers in Ecology and the Environment, 6, 485-492.
[42] Munir U, Perveen A, Qamarunnisa S (2015) The utility of rbcL and matK regions for DNA barcoding analysis of the genus Suaeda (Amaranthaceae) species. Pakistan Journal of Botany, 47, 2329-2334.
[43] Newmaster SG, Fazekas AJ, Ragupathy S (2006) DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach. Canadian Journal of Botany, 84, 335-341.
[44] Newmaster SG, Fazekas AJ, Steeves RAD, Janovec J (2008) Testing candidate plant barcode regions in the Myristicaceae. Molecular Ecology Resources, 8, 480-490.
[45] Olmstead RG, Palmer JD (1994) Chloroplast DNA systematics: a review of methods and data analysis. American Journal of Botany, 81, 1205-1224.
[46] Pang XH, Liu C, Shi LC, Liu R, Liang D, Li H, Cherny SS, Chen SL (2012) Utility of the trnH-psbA intergenic spacer region and its combinations as plant DNA barcodes: a meta-analysis. PLoS ONE, 7, e48833.
[47] Parmentier I, Duminil J, Kuzmina M, Philippe M, Thomas DW, Kenfack D, Chuyong GB, Cruaud C, Hardy OJ (2013) How effective are DNA barcodes in the identification of African rainforest trees? PLoS ONE, 8, e54921.
[48] Pei N, Chen B, Kress WJ (2017) Advances of community-level plant DNA barcoding in China. Frontiers in Plant Science, 8, 225.
[49] R Core Team (2012) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.
[50] Rana TS, Narzary D, Ohri D (2012) Molecular differentiation of Chenopodium album complex and some related species using ISSR profiles and ITS sequences. Gene, 495, 29-35.
[51] Sandilyan S, Kathiresan K (2012) Mangrove conservation: a global perspective. Biodiversity and Conservation, 21, 3523-3542.
[52] Schwarzbach AE, Ricklefs RE (2000) Systematic affinities of Rhizophoraceae and Anisophylleaceae, and intergeneric relationships within Rhizophoraceae, based on chloroplast DNA, nuclear ribosomal DNA, and morphology. American Journal of Botany, 87, 547-564.
[53] Seebens H, Gastner MT, Blasius B (2013) The risk of marine bioinvasion caused by global shipping. Ecology Letters, 16, 782-790.
[54] Short F, Carruthers T, Dennison W, Waycott M (2007) Global seagrass distribution and diversity: a bioregional model. Journal of Experimental Marine Biology and Ecology, 350, 3-20.
[55] Steane DA (2005) Complete nucleotide sequence of the chloroplast genome from the Tasmanian blue gum, Eucalyptus globulus (Myrtaceae). DNA Research, 12, 215-220.
[56] Sugita M, Shinozaki K, Sugiura M (1985) Tobacco chloroplast tRNALys (UUU) gene contains a 2.5-kilobase-pair intron: an open reading frame and a conserved boundary sequence in the intron. Proceedings of the National Academy of Sciences, USA, 82, 3557-3561.
[57] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731-2739.
[58] Tatusova TA, Madden TL (1999) BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiology Letters, 174, 247-250.
[59] Triest L, Sierens T (2010) Chloroplast sequences reveal a diversity gradient in the Mediterranean Ruppia cirrhosa species complex. Aquatic Botany, 93, 68-74.
[60] Trivedi S, Aloufi AA, Ansari AA, Ghosh SK (2016) Role of DNA barcoding in marine biodiversity assessment and conservation: an update. Saudi Journal of Biological Sciences, 23, 161-171.
[61] Vega AS, Rua GH, Fabbri LT, Rúgolo de Agrasar ZE (2009) A morphology-based cladistica analysis of Digitaria (Poaceae, Panicoideae, Paniceae). Systematic Botany, 34, 312-323.
[62] Wang WQ(2013) Salt-tolerant Plant Resources of Coastal Areas of South China. Xiamen University Press,Xiamen. (in Chinese)
[王文卿 (2013)南方滨海耐盐植物资源.厦门大学出版社, 厦门.]
[63] Waycott M, Duarte CM, Carruthers TJB, Orthd RJ, Dennison WC, Olyarnik S, Calladine A, Fourqurean JW, Heck KL, Hughes AR, Kendrick GA, Kenworthy WJ, Short FT, Williams SL (2009) Accelerating loss of seagrasses across the globe threatens coastal ecosystems. Proceedings of the National Academy of Sciences, USA, 106, 12377-12381.
[64] White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ eds. PCR protocols: A guide to methods and applications.San Diego:Academic Press. 315-322.
[65] Yamashiro T, Fukuda T, Yokoyama J, Maki M (2004) Molecular phylogeny of Vincetoxicum (Apocynaceae-Asclepiadoideae) based on the nucleotide sequences of cpDNA and nrDNA. Molecular Phylogenetics and Evolution, 31, 689-700.
[66] Yan HF, Liu YJ, Xie XF, Zhang CY, Hu CM, Hao G, Ge XJ (2015) DNA barcoding evaluation and its taxonomic implications in the species-rich genus Primula L. in China. PLoS ONE, 10, e0122903.
[67] Yang JB, Wang YP, Moeller M, Gao LM, Wu D (2012) Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae). Molecular Ecology Resources, 12, 267-275.
[68] Yu J, Xue JH, Zhou SL (2011) New universal matK primers for DNA barcoding angiosperms. Journal of Systematics and Evolution, 49, 176-181.
[69] Zhao KF, Li FZ (1999) Halophytes in China. Science Press, Beijing. (in Chinese)
[赵可夫, 李法曾 (1999) 中国盐生植物: 科学出版社, 北京.]
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