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Chinese Journal of Applied Entomology 2017, 54(1): 13 21. DOI: 10.7679/j.issn.2095 1353.2017.002 基于 DNA 条形码的物种界定算法比较研究 * 以北京周边地区舟蛾科为例金倩 1, 2** 刘旭 1 武春生 3 杨采青陈芬 2 1 郝梦迪 2 罗桂杰 1 张爱兵蔡卫佳 2*** 1 1.

1 Chinese Journal of Applied Entomology 2017, 54(1): DOI: /j.issn 基于 DNA 条形码的物种界定算法比较研究 * 以北京周边地区舟蛾科为例金倩 1, 2** 刘旭 1 武春生 3 杨采青陈芬 2 1 郝梦迪 2 罗桂杰 1 张爱兵蔡卫佳 2*** 摘要目的 3 jmotu ABGD GMYC 方法 C Ⅰ Cytochrome c oxidase subunit Ⅰ gene CO COX1 650 bp 3 jmotu ABGD GMYC 结果 3 ABGD 100% BEAST GMYC 64~68 67 d8tree/mpltree GMYC MOTUs jmotu 结论 ABGD BEAST GMYC 关键词 DNA GMYC ABGD jmotu The reliability of DNA barcoding as a means of identifying Notodontid moth species (Insecta: Lepidoptera, Notodontidae) in Beijing, China JIN Qian 1, 2** WU Chun-Sheng 3 CHEN Fen 1 LUO Gui-Jie 1 CAI Wei-Jia 1 LIU Xu 1 YANG Cai-Qing 2 HAO Meng-Di 2 ZHANG Ai-Bing 2*** (1. Suqian Institute of Agricultural Sciences, Jiangsu Academy of Agricultural Sciences, Suqian , China; 2. College of Life Sciences, Capital Normal University, Beijing , China; 3. Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing , China) Abstract [Objectives] To evaluate the performance of three species identification algorithms (jmotu, ABGD, GMYC) with respect to the Notodontidae (Insecta, Lepidoptera). [Methods] CO genes from 483 notodontid moth specimens collected from 10 collection sites in Northern China around Beijing were amplified using universal barcoding primers. [Results] Results obtained using each of the three algorithims differed. Using the results obtained by morphological species identification methods as a reference, the ABGD algorithm was 100% accurate. The results obtained using GMYC_BEAST were also generally consistent with those obtained by morphological methods, having a confidence interval of c.f. 67 species identified by morphological methods. However, the GMYC_d8tree/MPLtree method tended to overestimate MOTUs and the jmotu method tended to underestimate the number of species. [Conclusion] ABGD and GMYC_BEAST can * Supported projects SQCX ** First author jinhongyu2001@ 163.com *** Corresponding author zhangab2008@mail.cnu.edu.cn Received Accepted

2 14 Chinese Journal of Applied Entomology 54 reliably identify species within Notodontidae on the basis of CO gene variation. Key words Notodontidae, DNA barcoding, species delineation, GMYC, ABGD, jmotu Unsupervised jmotu Jones et al ABGD Puillandre et al GMYC Pons et al Jones 2011 Blaxter 1998 DNA MOTUs jmotu Taxonerator BLAST NW Needleman- Wunsch MOTUs Puillandre 2012 ABGD Automatic barcode gap discovery DNA DNA DNA MOTUs DNA MOTUs DNA MOTUs Pons 2006 GMYC MOTU Boykin et al jmotu ABGD GMYC Ratnasingham and Hebert 2013 Dellicour and Flot 2015 Thomsen and Willerslev 2015 Ratnasingham Hebert GMYC 89% ABGD 85.2% jmotu 74.3% 2013 ABGD 100% jmotu ABGD Athetis lepigone Phalera flavescens Loudonta dispar Besaia goddrica Dinara combusta Togarishachia albistriga C Ⅰ Cytochrome c oxidase subunitⅠgene COⅠ COX1 650 bp DNA jmotu ABGD GMYC 3 DNA 1 材料与方法 1.1 标本采集及形态鉴定

3 1 : DNA 15 Table 1 Sampling sites 表 1 采样点信息及采集数量 Geographical coordinates of sample localities and specimen numbers in this study Short title E Latitude N Longitude m Elevation Number of specimen Labagoumen LBG Wulingshan WLS Lanfang LF Baoding BD Baihuashan BHS Donglignshan DLS Songshan SS Jiufeng JF Jiangou JG Longjiazhuang LJZ DNA 提取 COⅠ 扩增及测序 3~4 100% 4 Biomed / DNA DNA -20 COⅠ DNA LCO GGTCAACA- AATCAT AAAGATATTGG-3 HCO TAAACTTCAGGGTGACCAAAAAATCA-3 Vrijenhoek 1994 COⅠ bp 25 μl PCR 2 Mastermix 12.5 μl LCO1490 HCO μl 10 μmol L DNA 2.5 μl ddh2o 9 μl PCR 94 2 min s s s min PCR 1% 700 bp PCR ABI 3130 GenBank KX KX 数据校对 COⅠ Chromas DNA Perl smkxxy.cnu.edu.cn/szll/zrjs/js/4557.htm COⅠ /.fasta BioEdit 数据分析序列分析 Perl edu.cn/szll/zrjs/js/4557.htm R A/T/C/G GC

4 16 Chinese Journal of Applied Entomology 54 / base.freq GC.content seg.sites pis {phyloch} nuc.div {pegas} titv {spider} DNA DNA DNA barcoding gap DNA DNA Perl DNA cnu.edu.cn/szll/zrjs/js/4557.htm jmotu 方法 jmotu Jones et al., 2011 MOTU MOTU 1~80 bp 97% 95% ABGD 方法 ABGD ABGD Puillandre et al ABGD abgd/abgdweb.html K2P P Prior intraspecific divergence gap X Minimum relative gap width 1.5 ABGD GMYC 模型 Ultrametric tree 1 PATHd8 jmodeltest2.2.2 Darriba et al Akaike AIC COⅠ RAxML7.0.3 ML Stamatakis 2006 PATHd8 Britton et al PATHd8 d8tree/mpltree R multi2di ML BEASTv1.8.0 Drummond and Rambaut 2007 Yule ULRC Drummond et al MCMC 10 4 jmodeltest2.2.2 Darriba et al Tracer Rambaut and Drummond 2007 Effective sample size 200 BEAST TreeAnnotator version Maximum clade credibility tree GMYC R Splits{gmyc} Single-threshold GMYC Fujisawa and Barraclough 2013 gmyc GMYC LRT Yule 2 结果与分析 2.1 分类信息和序列特征 COⅠ bp 179 A 29.70% T 38.35% C 17.15% G 14.80% A+T 68.05% G+C 31.95% 594 Conservative sites 307 Variable sites 287 Parsimony information sites % / 90.82% 0~ ~

5 1 : DNA 17 1 DNA jmotu 图 1 DNA 条形码空白区 Fig. 1 DNA barcoding gap 2.2 jmotu 划分 MOTUs 结果 Cut-off value 2 MOTU 7~36 bp 1.18%~6.06% MOTUs 51~68 图 2 分子序列差异截取值与可操作分类单元数目的关系 Fig. 2 The relationship between cut-off value and the number of MOTUs 2.3 ABGD 划分 MOTUs 结果 ABGD 0.001~0.1 P 483 Initial partition Recursive partition 3 Fig. 3 图 3 ABGD 方法划分结果 The automatic partition results of dataset by ABGD

6 18 Chinese Journal of Applied Entomology ~25 P 14 ABGD 100% 2.4 GMYC 模型划分 MOTUs 结果 JModelTest2.2.2 HKY+I+G BEAST GMYC 4 2 GMYC LRT *** Yule Million years ago, Mya 66 64~68 d8tree 2 GMYC GMYC LRT *** Yule Mya 73 69~77 MPLtree 2 GMYC GMYC LRT *** Yule Mya 73 69~77 BEAST GMYC PATHd8 GMYC PATHd8 BEAST 2 *** P< 讨论 DNA 2013 DNA DNA jmotu ABGD GMYC DNA ABGD 100% BEAST GMYC ABGD BEAST GMYC d8tree/ MPLtree GMYC MOTUs jmotu 3% 10 Hebert et al ~ ~ Overlap 1 DNA jmotu MOTU MOTU java jmotu v1.0.6 Jones et al ~100 Cut-off value MOTUs DNA DNA barcoding gap MOTUs MOTUs MOTUs 2 MOTUs 7 bp 1.18% 68 MOTUs 36 bp 6.06% 51 MOTUs DNA

7 1 : DNA 19 表 2 GMYC 模型界定物种 Table 2 GMYC delimitation in this study Morphyospecies Method a Entities b Clusters c L null d GMYC L GMYC e Likelihood ratio f BEAST *** 67 d8tree *** MPLtree *** a: BEAST/d8tree/MPLtree b: GMYC c: d: e: GMYC f: *** P<0.001 ** P<0.01 * P<0.05 a: Different ultrametric tree obtention method; b: Number of entities including single individual; c: Number of entities without single individual; d: The likelihood value of null model; e: The maximum likelihood value of GMYC model; f: Likelihood ratio test (*** P<0.001, **P<0.01, *P<0.05). 图 4 GMYC 物种界定 Fig. 4 GMYC model delimitation A. BEAST B. C. A. The ultrametric tree implemented with BEAST software. Gray line indicates transition from between-species to within-species. Gray branches represent coalescence processes. Black branches represent speciation and extinction processes; B. Relationship between time and lineages. The sharp increase in branching rate, corresponding to the transition from interspecies to intraspecies branching events; C. Relationship between time and likelihood value.

8 20 Chinese Journal of Applied Entomology 54 1 MOTUs ABGD ABGD DNA ABGD Fasta ABGD 3 P ABGD 3~5 GMYC 1 PATHd8 d8tree MPLtree 2 BEASTv1.8.0 Drummond and Rambaut 2007 BEAST GMYC BEAST GMYC PATHd8 GMYC MOTUs Talavera et al BEAST GMYC DNA ABGD BEAST GMYC DNA 3 10 GenBank/BOLD CO 参考文献 (References) Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, Vanfleteren JR, Mackey LY, Dorris M, Frisse LM, A molecular evolutionary framework for the phylum Nematoda. Nature, 392(6671): Boykin LM, Armstrong KF, Kubatko L, De Barro P, Species delimitation and global biosecurity. Evolutionary Bioinformatics, 8: 1. Britton T, Anderson C, Jacquet D, Lundqvist S, Bremer K, PATHd8-a program for phylogenetic dating of large trees without a molecular clock. Avalilable at www math su se/pathd8. Britton T, Anderson CL, Jacquet D, Lundqvist S, Bremer K, Estimating divergence times in large phylogenetic trees. Systematic Biology, 56(5): Darriba D, Taboada GL, Doallo R, Posada D, jmodeltest 2: more models, new heuristics and parallel computing. Nature Methods, 9(8): Dellicour S, Flot JF, Delimiting species-poor datasets using single molecular markers: a study of barcode gaps, haplowebs and GMYC. Systematic Biology, 64(6): Drummond AJ, Ho SY, Phillips MJ, Rambaut A, Relaxed phylogenetics and dating with confidence. PLoS Biology, 4(5):

9 1 : DNA 21 e88. Drummond AJ, Rambaut A, BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7(1): 214. Fujisawa T, Barraclough TG, Delimiting species using singlelocus data and the Generalized Mixed Yule Coalescent (GMYC) approach: a revised method and evaluation on simulated datasets. Systematic Biology, 62(5): Hebert PD, Penton EH, Burns JM, Janzen DH, Hallwachs W, Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America, 101(41): Jin Q, Zhang AB, Distance-based DNA barcoding methods for insects. Chinese Journal of Applied Entomology, 50(1): [,, DNA., 50(1): ] Jones M, Ghoorah A, Blaxter M, jmotu and Taxonerator: turning DNA Barcode sequences into annotated operational taxonomic units. PLoS ONE, 6(4): e Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S, Kamoun S, Sumlin WD, Vogler AP, Sequencebased species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology, 55(4): Puillandre N, Lambert A, Brouillet S, Achaz G, ABGD, automatic barcode gap discovery for primary species delimitation. Molecular Ecology, 21(8): Rambaut A, Drummond A, Tracer v1. 4: MCMC trace analyses tool. See bio ed ac uk/software/tracer/[pmc free article][pubmed]. Ratnasingham S, Hebert PD, A DNA-based registry for all animal species: the Barcode Index Number (BIN) system. PLoS ONE, 8(7): e Song SS, Shi ZY, Jin Q, Han HL, Lin XF, Hao MD, Zhang AB, Species identification of Noctuidae moths (Insecta: Lepidoptera) from Baoding and Langfang, Hebei, China with DNA barcoding and establishment of a local DNA barcoding library. Chinese Journal of Applied Entomology, 51(1): [,,,,,,, DNA., 51(1): ] Stamatakis A, RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22(21): Talavera G, Dincă V, Vila R, Factors affecting species delimitations with the GMYC model: insights from a butterfly survey. Methods in Ecology and Evolution, 4(12): Thomsen PF, Willerslev E, Environmental DNA an emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv., 183: Vrijenhoek R, DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3(5): Yang CH, Jin Q, Chen FQ, Wu CS, Zhang AB, Application of tree-based, distance-based and character-based DNA barcoding methods for the identification of Crambidae species (Insecta: Lepidoptera) in Baihuashan, China. Chinese Journal of Applied Entomology, 50(1): [,,,,, DNA., 50(1): ]

1 : DNA 157 classification results and a local DNA barcoding library would be useful for moth identification. Key words Lepidoptera, Noctuide, DNA bar

1 : DNA 157 classification results and a local DNA barcoding library would be useful for moth identification. Key words Lepidoptera, Noctuide, DNA bar Chinese Journal of Applied Entomology 2014, 51(1): 156 168. DOI: 10.7679/j.issn.2095 1353.2014.018 DNA 条形码技术在河北保定廊坊地区鳞翅目昆虫上的应用及小型区域数据库的构建 * 宋韶彬 1** 石志勇 1 金倩 1 韩辉林 2 刘晓枫 1 郝梦迪 1 张爱兵 1*** 1. 100048 2. 150040