DNA Barcoding of Subtropical Forest Trees——A Study from Ailao Mountains Nature Reserve, Yunnan, China

doi:10.7677/ynzwyj201313171

Abstract

Abstract:

To evaluate how effective DNA barcoding is for the identification of subtropical forest trees, we sampled 525 individuals representing 204 species in 111 genera of 51 plant families that occur in the Ailao Mountains Nature Reserve, and tested the ability of rbcL, matK, trnHpsbA and ITS sequences to discriminate species. PCR success was over 90% for each of these four sequences, while sequencing success rate was highest for rbcL and matK (90.7% and 90.5%, respectively), followed by trnHpsbA (83.6%), and lowest for ITS (73.7%). Thus, all four sequences showed a relatively high level of applicability for subtropical forest trees that occur in the Ailaoshan Mountains Nature Reserve. Using two different “species identification” methods - BLAST and Neighbor Joining (NJ)—the highest rate of success for identification at species (68.4%-81.3%) and genus (99.0%-100%) levels was obtained using ITS when only a single region was used. When two molecular regions were used in combination, rbcL and matK correctly identified 52.8%-60.2% of species and 86.7%-90.5% of genera, while using all four regions in combination correctly discriminated 74.7%-79.6% of species. The relatively low sequencing success rate of ITS was mainly due to failure in certain groups (such as Lauraceae and Fagaceae), which play an important role in subtropical forest, suggesting that the ITS region may not be appropriate for DNA barcoding these particular plant groups.

Key words: DNA barcode, Subtropical forest trees, Ailao Mountains Nature Reserve, Species identification, ITS

摘要：

作为新一代植物志iFlora的重要组成部分，DNA条形码已经成为物种鉴定中重要且有效的方法。本研究以亚热带森林的乔木树种为研究对象，开展了DNA条形码的尝试性工作。为评估DNA条形码对鉴定亚热带森林树种的有效性，收集并研究了来自哀牢山自然保护区内51科111属中204个树种的525个乔木个体。结果显示，所选4个DNA片段（rbcL, matK, trnHpsbA和ITS）的PCR扩增成功率都超过90%；测序成功率rbcL和matK最高，分别为90.7%和90.5%，trnHpsbA次之（83.6%），ITS最低（73.5%），表明4个片段在亚热带森林乔木中都具有较好的通用性。应用BLAST与NJ Tree两种方法，对物种和属水平的鉴别成功率进行统计，发现单片段中ITS最高，分别为68.4%~81.3%和99.0%~100%，核心条码rbcL和matK组合的成功率是52.8%~60.2%和86.7%~90.5%，再与补充条码trnHpsbA和ITS联合，可以成功鉴别74.7%~79.6%哀牢山自然保护区亚热带森林中的乔木物种。由于ITS片段在亚热带森林部分重要树种类群（樟科和壳斗科等）中的测序成功率较差，所以对这些植物类群采用trnHpsbA作为DNA条形码是一个更好的选择。

关键词: DNA条形码, 亚热带森林, 哀牢山自然保护区, 物种鉴别, ITS, 乔木

CLC Number:

Q 949
Q 781

Lu Meng-Meng-, CI Xiu-Qin-, YANG Guo-Ping-, LI Jie. DNA Barcoding of Subtropical Forest Trees——A Study from Ailao Mountains Nature Reserve, Yunnan, China[J]. Plant Diversity, 2013, 35(6): 733-741.

卢孟孟1、2, 慈秀芹1、2, 杨国平1, 李捷1. 亚热带森林乔木树种DNA条形码研究——以哀牢山自然保护区为例[J]. Plant Diversity, 2013, 35(6): 733-741.

References

哀牢山自然保护区综合考察团, 1988. 哀牢山自然保护区综合考察报告集［M］. 昆明: 云南民族出版社
朱华, 闫丽春, 2009. 云南哀牢山种子植物［M］. 云南: 云南科技出版社
Abeysinghe PD, Wijesinghe KGG, Tachida H et al., 2009. Molecular characterization of Cinnamon (Cinnamomum verum Presl) accessions and evaluation of genetic relatedness of Cinnamon species in Sri Lanka based on trnL intron region, intergenic spacers between trnTtrnL, trnLtrnF, trnHpsbA and nuclear ITS［J］. Research Journal of Agriculture and Biological Sciences, 5 (6): 1079—1088
Armenise L, Simeone MC, Piredda R et al., 2012. Validation of DNA barcoding as an efficient tool for taxon identification and detection of species diversity in Italian conifers［J］. European Journal of Forest Research, 131 (5): 1337—1353
Bromberg C, Cash H, Curtis P et al., 1995. Sequencher. Gene Codes Corporation. Ann Arbor, Michigan
Burgess KS, Fazekas AJ, Kesanakurti PR et al., 2011. Discriminating plant species in a local temperate flora using the rbcL plus matK DNA barcode［J］. Methods in Ecology and Evolution, 2: 333—340
CBOL Plant Wording Group, 2009. A DNA barcode for land plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 106 (31): 12794—12797
Chase MW, Cowan RS, Hollingsworth PM et al., 2007. A proposal for a standardised protocol to barcode all land plants［J］. Taxon, 56 (2): 295—299
Chase MW, Fay MF, 2009. Barcoding of plants and fungi［J］. Science, 325 (5941): 682—683
Costion C, Ford A, Cross H et al., 2011. Plant DNA barcodes can accurately estimate species richness in poorly known floras［J］. PLoS ONE, 6 (11): e26841
Cowan RS, Chase MW, Kress WJ et al., 2006. 300000 species to identify: problems, progress, and prospects in DNA barcoding of land plants［J］. Taxon, 55 (3): 611—616
De Mattia F, Gentili R, Bruni I et al., 2012. A multimarker DNA barcoding approach to save time and resources in vegetation surveys［J］. Botanical Journal of the Linnean Society, 169 (3): 518—529
De Vere N, Rich TCG, Ford CR et al., 2012. DNA barcoding the native flowering plants and conifers of wales［J］. PloS ONE, 7(6): e37945
Denk T, Grimm GW, 2010. The oaks of Western Eurasia: traditional classifications and evidence from two nuclear marks［J］. Taxon, 59 (2): 351—366
Devey DS, Chase MW, Clarkson JJ, 2009. A stuttering start to plant DNA barcoding: microsatellites present a previously overlooked problem in noncoding plastid regions［J］. Taxon, 58 (1): 7—15
Dincǎ V, Zakharov EV, Hebert PDN et al., 2011. Complete DNA barcode reference library for a country’s butterfly fauna reveals high performance for temperate Europe［J］. Proceedings of the Royal Society B: Biological Sciences, 278 (1704): 347—355
Dolman PM, Panter CJ, Mossman HL, 2012. The biodiversity audit approach challenges regional priorities and identifies a mismatch in conservation［J］. Journal of Applied Ecology, 49 (5): 986—997
Fazekas AJ, Burgess KS, Kesanakurti PR et al., 2008. Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well［J］. PloS ONE, 3 (7): e2802
Fazekas AJ, Steeves R, Newmaster SG, 2010. Improving sequencing quality from PCR products containing long mononucleotide repeats［J］. Biotechniques, 48 (4): 277—285
GarcíaRobledo C, Erickson DL, Staines CL et al., 2013. Tropical plantherbivore networks: reconstructing species interactions using DNA barcodes［J］. PloS ONE, 8 (1): e52967
Gonzalez MA, Baraloto C, Engel J et al., 2009. Identification of Amazonian trees with DNA barcodes［J］. PLoS ONE, 4 (10): e7483
Gu J, Su JX, Lin RZ et al., 2011. Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae) ［J］. Journal of Systematics and Evolution, 49 (3): 213—224
Hebert PDN, Cywinska A, Ball SL et al., 2003. Biological identifications through DNA barcodes［J］. Proceedings of the Royal Society of London Series B: Biological Sciences, 270 (1512): 313—321
Hollingsworth PM, 2008. DNA barcoding plants in biodiversity hot spots: progress and outstanding questions［J］. Heredity, 101 (1): 1—2
Hollingsworth PM, Graham SW, Little DP, 2011. Choosing and using a plant DNA barcode［J］. PLoS ONE, 6 (5): e19254
Ivanova NV, Fazekas AJ, Hebert PDN, 2008. Semiautomated, membranebased protocol for DNA isolation from plants［J］. Plant Molecular Biology Reporter, 26 (3): 186—198
Ji Y, Ashton L, Pedley SM et al., 2013. Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding［J］. Ecology Letters, 16 (10): 1245—1257
Jin Q, Han H, Hu X et al., 2013. Quantifying species diversity with a DNA barcodingbased method: Tibetan Moth species (Noctuidae) on the QinghaiTibetan plateau［J］. PloS ONE, 8 (5): e64428
Kress WJ, Wurdack KJ, Zimmer EA et al., 2005. Use of DNA barcodes to identify flowering plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 102 (23): 8369—8374
Kress JW, Erickson DL, Jones FA et al., 2009. Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama［J］. Proceedings of the National Academy of Sciences of the United States of America, 106 (44): 18621—18626
Larkin MA, Blackshields G, Brown NP et al., 2007. Clustal W and Clustal X version 2.0［J］. Bioinformatics, 23 (21): 2947—2948
Lahaye R, van der Bank M, Bogarin D et al., 2008. DNA barcoding the floras of biodiversity hotspots［J］. Proceedings of the National Academy of Sciences of the United States of America, 105 (8): 2923—2928
Li DZ, Gao LM, Li HT et al., 2011. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants［J］. Proceedings of the National Academy of Sciences of the United States of America, 108 (49): 19641—19646
Li DZ (李德铢), Wang YH (王雨华), Yi TS (伊廷双) et al., 2012. The next generation Flora: iFlora［J］. Plant Diversity and Resources (植物分类与资源学报), 34 (6): 525—531
Liu K, Raghavan S, Nelesen S et al., 2009. Rapid and accurate largescale coestimation of sequence alignments and phylogenetic trees［J］. Science, 324 (5934): 1561—1564
Mller M, Cronk Q, 1997. Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences［J］. American Journal of Botany, 84 (7): 956—956
Pei NC (裴男才), Zhang JL (张金龙), Mi XC (米湘成) et al., 2011. Plant DNA barcodes promote the development of phylogenetic community ecology［J］. Biodiversity Science (生物多样性), 19 (3): 284—294
Roy S, Tyagi A, Shukla V et al., 2010. Universal plant DNA barcode loci may not work in complex groups: a case study with Indian Berberis species［J］. PLoS ONE, 5 (10): e13674
Sala OE, 2000. Global biodiversity scenarios for the year 2100［J］. Science, 287 (5459): 1770—1774
Sang T, Crawford D, Stuessy T, 1997. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae) ［J］. American Journal of Botany, 84 (8): 1120
Sass C, Little DP, Stevenson DW et al., 2007. DNA barcoding in the Cycadales: testing the potential of proposed barcoding markers for species identification of cycads［J］. PLoS ONE, 2 (11): e1154
Simeone MC, Piredda R, Papini A et al., 2013. Application of plastid and nuclear markers to DNA barcoding of EuroMediterranean oaks (Quercus, Fagaceae): problems, prospects and phylogenetic implications［J］. Botanical Journal of the Linnean Society, 172: 478—499
Tripathi AM, Tyagi A, Kumar A et al., 2013. The internal transcribed spacer (ITS) region and trnHpsbA are suitable candidate loci for DNA barcoding of tropical tree species of India［J］. PloS ONE, 8 (2): e57934
White TJ, Bruns T, Lee S et al., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics［J］. PCR Protocols: A Guide to Methods and Applications, 18: 315—322
Xue DU (薛达元), Jiang MK (蒋明康), 1995. Contrubutions of nature reserves in China to biodiversity conservation［J］. Journal of Natural Resources (自然资源学报), 10 (3): 286—292
Yu J, Xue JH, Zhou SL, 2011. New universal matK primers for DNA barcoding angiosperms［J］. Journal of Systematics and Evolution, 49 (3): 176—181

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