Molecular Ecology Resources (2012) 12, 791–796 doi: 10.1111/j.1755-0998.2012.03152.x

DNA barcoding of six Ceroplastes species (: Coccoidea: ) from China

JUN DENG,*† FANG YU,† TONG-XIN ZHANG,‡ HAO-YUAN HU,§ CHAO-DONG ZHU,† SAN-AN WU* and YAN-ZHOU ZHANG† *The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China, †Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China, ‡Ningbo Technology Extension Center for Forestry and Specialty Forest Products, Ningbo 315012, China, §College of Life Science, Anhui Normal University, Wuhu 241000, China

Abstract Ceroplastes Gray (wax scales) is one of the genera of Coccidae, most species of which are considered to be serious economic pests. However, identification of Ceroplastes species is always difficult owing to the shortage of easily distinguishable mor- phological characters. Mitochondrial cytochrome c oxidase I (COI) sequences (or DNA barcodes) and the D2 expansion seg- ments of the large subunit ribosomal RNA gene 28S were used for accurate identification of six Ceroplastes species (C. floridensis Comstock, C. japonicus Green, C. ceriferus (Fabricius), C. pseudoceriferus Green, C. rubens Maskell and C. kunmingensis Tang et Xie) from 20 different locations in China. For COI data, low GÆC content was found in all species, averaging about 20.4%. Sequence divergences (K2P) between congeneric species averaged 12.19%, while intra-specific divergences averaged 0.42%. All 112 samples fell into six reciprocally monophyletic clades in the COI neighbour-joining (NJ) tree. The NJ tree inferred from 28S showed almost same results, but samples of two closely related species, C. ceriferus and C. pseudoceriferus, were clustered together. This research indicates that the standard barcode region of COI can effi- ciently identify similar Ceroplastes species. This study provides an example of the usefulness of barcoding for Ceroplastes identification.

Keywords: 28S, Ceroplastes, COI, DNA barcoding, DNA markers, wax scales Received 11 January 2012; revision received 21 March 2012; accepted 30 March 2012

Green, C. rubens Maskell and C. xishuangensis Tang et Xie) Introduction known in China (Tang 1991; Martin & Lau 2011). Among Identification of pest coccoids remains a big problem to them, C. floridensis, C. japonicus, C. ceriferus, C. pseudoce- many applied entomologists because most scale riferus and C. rubens are well-known polyphagous pests are small, and closely related species may be very similar infested on crops and ornamental plants around the superficially (Gullan & Kosztarab 1997). Moreover, scale world (Gimpel et al. 1974; Huang & Huang 1988; Smith insects are identified to species level by examining mor- 1986; Ben-Dov 1993; Li 1994; Xie 1998; Wang 2001). Both phological traits present in adult females, which requires immature and adult stages of C. japonicus may be diffi- the preservation of the adult cuticle, proper preparation of cult to distinguish from C. floridensis (Longo 1985; Pellizz- specimens and examination by a trained taxonomist. Even ari & Camporese 1994). Ceroplastes kunmingensis is also for adult females, variation in characters, such as the stig- very similar to C. floridensis and C. centroroseus as well as matic and dorsal setae (Gimpel et al. 1974; Gullan & C. japonicus, the main difference being unequal claw digi- Kosztarab 1997), often make species identification difficult. tules (Tang 1991). A great deal of confusion exists over Ceroplastes Gray (Hemiptera: Coccidae) contains about the correct use of the names C. ceriferus and C. pseudoce- 138 species in the world (Ben-Dov 1993; Ben-Dov & riferus. Although Kawai & Tamaki (1967) and Tamaki Hodgson 1997). There are 10 Ceroplastes species (C. actini- et al. (1969) treated C. pseudoceriferus as a different species formis Green, C. centroroseus Chen, C. ceriferus (Fabricius), from C. ceriferus, Kawai (1980) considered that C. pseud- C. floridensis Comstock, C. japonicus Green, C. kunmingen- oceriferus and C. ceriferus were synonyms. For these con- sis Tang et Xie, C. murrayi Froggat, C. pseudoceriferus troversies, Gimpel et al. (1974) even considered the possibility that the C. pseudoceriferus specimen studied by Correspondence: Yan-Zhou Zhang, Fax: 86-10-64807099; E-mail: [email protected] Kawai & Tamaki (1967) was C. ceriferus. Although the San-An Wu, Fax: 86-10-62336596; number of spiracular setae and marginal bristle-shaped E-mail: [email protected] setae were important traits, these traits were not always

2012 Blackwell Publishing Ltd 792 J. DENG ET AL. stable. Another trait, the length of caudal appendage, mer set (C1-1554F, C1-2342R) was [F] 5¢-CAGGAA was confirmed variable among conspecific individuals TAATAGGAACATCAATAAG-3¢ and [R] 5¢- ATCAATG (Wang 2001). Specimens of Ceroplastes dugesii Lichten- TCTAATCCGATAGTAAATA-3¢. We ascertained the stein have been misidentified as C. ceriferus by Gimpel positions of the primer-annealing site by comparing it to et al. (1974) (Hamon & Williams 1984). mitochondrion genome of Drosophila yakuba (Folmer et al. DNA barcoding has provided a new approach for spe- 1994). When amplifying 28S ribosomal gene, we used the cies-level identification among invertebrates (Costa et al. primers D2-3566F (5¢-TGCAGCTCTAAGTTGGTGGT-3¢) 2007; Mikkelsen et al. 2007) and vertebrates (Hebert et al. (Gillespie et al. 2005) and D2-4068 (5¢-TTGGTCCGTGTT 2004; Hajibabaei et al. 2006; Wong et al. 2009). It has pro- TCAAGACGGG-3¢) (Campbell et al. 1993). All PCRs ven promising for discriminating between closely related were processed in a 50 lL volume with 4 lL DNA tem- species across diverse insects (Hebert et al. 2003b). How- plate, 5 lL10· Buffer, 25 mM MgCl2, 2.5 mM dNTP mix- ever, DNA barcoding has been virtually unused in scale ture, 10 pmol of each primer and 1 unit of ExTaq DNA insects (Hemiptera: Coccoidea), except those by Ball & polymerase (TaKaRa Bio Inc., Otsu, Japan). PCRs were Armstrong (2007) and Park et al. (2011). More work run on Eppendorf Mastercycler Thermal Cyclers with the should be carried out to show the possibility of using following profile: an initial step of 3 min at 95 C fol- DNA barcoding for distinguishing scale insects. lowed by 35 cycles of 30 s at 94 C, 50 s at 52 C and Given the time-consuming preparation of slides and 1 min at 72 C followed in turn by final extension of the difficulty of species identification using morphologi- 10 min at 72 C. Products were visualized on 1% agarose, cal traits by nonexperts, we aimed to ascertain whether and the most intense products were sequenced bidirec- patterns of COI sequences variation are congruent with tionally using BigDye v3.1 on an ABI3730xl DNA Ana- those of morphological differentiation in revealing spe- lyzer (Applied Biosystems). Sequences were aligned in cies boundaries of the available Ceroplastes species – Bioedit (Hall 1999). Sequences divergences were calcu- C. rubens, C. floridensis, C. japonicus, C. pseudoceriferus, lated using the K2P distance model (Kimura 1980). C. kunmingensis and C. ceriferus – occurred in China Neighbour-joining (NJ) trees (Saitou & Nei 1987) of COI (Tang 1991; Ben-Dov 1993; Xie 1998; Wang 2001). and the D2 expansion segments of 28S were recon- structed using MEGA4 (Tamura et al. 2007). Parthenoleca- nium corni (Bouche´) was chosen as outgroup. Materials and methods Detailed specimen information (sequences and collec- tion information) is available in the project ‘Scale Insects Specimen sampling of China’ (project code CSI) on the Barcode of Life A total of 112 individuals were collected from various Data Systems (http://www.boldsystems.org/views/ provinces of China and preserved in 95% ethanol. Figure 1 projectmenu). The project will become publicly available provides an overview of their geographical distribution. upon the publication of this work. Details of sampling locations, host plant and date are provided in the (Table S1, Supporting information). Indi- Results viduals were identified based on morphological charac- ters and the taxonomic keys (Gimpel et al. 1974; Tang DNA sequences analyses 1991). All specimens used here were identified by San-An Wu, an experienced taxonomist of scale insects in China. The samples used in this study included 112 individuals representing six species of the genus Ceroplastes. The samples per species ranged from 4 to 36, with an average DNA extraction, amplification and sequencing of 19. We finally obtained a COI sequence fragment of Total DNA was extracted from individuals preserved in 543 bp from all Ceroplastes individuals after deleting the 95% ethanol using DNeasy Blood & Tissue Kit (Qiagen), terminal ambiguous part of the aligned data. Further- following the manufacturer’s protocols. Universal prim- more, no insertions and deletions were found in DNA ers LCO1490 and HCO2198 (Folmer et al. 1994) were sequences and no stop codons were found when translat- firstly used to amplify 658 bp of the 5¢ end of mitochon- ing amino acid, implying that all amplified sequences drial cytochrome c oxidase I (COI). However, we had the code for functional COI. All nucleotide substitutions in same difficulty in recovering this barcode region of scale the COI region constituted synonymous changes. Inter- insects as met by Kondo et al. (2008) and Park et al. estingly, despite 5.0% interspecific divergence between (2010). Only one-third of the 112 samples could be suc- the COI regions of C. ceriferius and C. pseudoceriferus, the cessfully amplified when using universal primers. Thus, translated amino acid sequences were identical. a new primer set was designed, with which we success- In the COI region, the frequency of adenine (A) and fully amplified all the rest of the samples. The new pri- thymine (T) was high (A = 40.9%, C = 14.4%, G = 6.0%,

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65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145

45

40

35

30

25

C. japonicus C. kunmingensis 20 C. rubens

C. ceriferus 0 250 500 750 KM C. pseudoceriferus C. floridensis

Fig. 1 Sampling sites in China. Detail sampling information is shown in Table S1. One hundred and twelve samples were collected from 20 different locations.

T = 38.7%). The GÆC frequency was lower at the third COI, except that samples of C. ceriferus and C. pseudoce- codon position (mean 12.9% for all individuals), than at riferus were clustered together. the first and second positions (mean 18.2% and 30.1%). The COI sequences ranged 5.0–15.6% in interspecific Discussion divergence, with a mean divergence of 12.19%. Intraspe- cific COI sequence divergences were 0–0.9%, with a mean The present study reports for the first time COI barcode divergence of 0.42% (Fig. 2a). There was no overlap sequences of six Chinese species of the genus Ceroplastes, between maximum K2P distance among conspecific indi- for the purpose of identification. Each of the six species viduals (0.42%) and minimum congeneric distance gave distinct COI sequences, distinguishing them from between C. ceriferus and C. pseudoceriferus (5.0%). Expect- conspecifics through the DNA barcode method. The edly, C. ceriferus and C. pseudoceriferus clusters showed a average intraspecies K2P distance was estimated at only minimal level of divergence, as they are closely related 0.42% whereas it was about 12.19% among congeneric species. species, in accordance to the mean value 10.09% known NJ analysis of COI sequences revealed that the 112 for the mealybugs and armoured scales (Park et al. 2011). samples were split into six haplogroups with high boot- Hence, there was a 30-fold increase in sequence variation strap values (Fig. 2a). The C. ceriferus and C. pseudocerife- among congeneric species compared to conspecific indi- rus were separated into two clusters, indicating that they viduals. A steady increase of genetic variation was were close to each other. Three species, C. kunmingensis, observed from conspecific individuals to congeneric spe- C. floridensis and C. japonicus, formed three distinct clus- cies, supporting the use of COI sequences to distinguish ters, with interspecific divergence ranging from 8.6% to the species boundaries. Although the 28S sequences lack 10.4%. sufficient variation to identify closely related species, The final alignment of 28S was 358 bp in length. The they provided some available information for distin- NJ tree of 28S (Fig. 2b) was almost identical to that of guishing species in scale insects.

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S2011-137a S2011-137b (a) S2011-137b (b) S2011-137c S2011-137c S2011-137a S2011-139b S2011-085a S2011-139a S2011-139c S2011-139c S2011-016c S2011-016b S2011-005a S2011-085a S2011-082a S2011-085c S2011-006c S2011-051b S2011-139a S2011-016a S2011-074c S2011-085b S2011-074b S2011-051a S2011-014b S2011-016c S2011-051a S2011-005a S2011-082b S2011-138b C. japonicus S2011-006a C. japonicus S2011-014b S2011-138c S2011-014a S2011-016a S2011-138c S2011-074a S2011-014c S2011-016b 100 S2011-138a S2011-082c S2011-005b S2011-138b S2011-006a S2011-085c S2011-006b S2011-005b S2011-006c S2011-014c S2011-074a 99 S2011-139b S2011-082b S2011-051b S2011-082c S2011-014a S2011-074b S2011-138a S2011-074c S2011-006b S2011-082a S2011-085b S2011-163b S2011-071a S2011-163a S2011-071b 100 C. kunmingensis S2011-163c S2011-072a 97 S2011-019b S2011-072b S2011-062b C. foridensis S2011-163b S2011-019a S2011-062b S2011-062a S2011-163c S2011-008b S2011-062a S2011-019c S2011-008a C. foridensis S2011-008a S2011-019a S2011-072b 86 S2011-008b S2011-071b C. kunmingensis S2011-019b 100 S2011-071a S2011-163a S2011-072a S2011-019c C0003a S2011-135b S2011-070a S2011-027b C0002a S2011-001a S2011-070b S2011-146a 99 C0002b S2011-141c C0002d S2011-146b C0003d S2011-022a C0003b S2011-028b C0003c S2011-068c C0002c S2011-141a S2011-076a 100 S2011-001b S2011-076b S2011-053a S2011-076c S2011-022b S2011-030a S2011-001c S2011-030b S2011-135a S2011-030c S2011-141b C. pseudoceriferus & C. ceriferus 98 S2011-003a S2011-135c WHd C. rubens S2011-022c S2011-142c S2011-027c S2011-036b S2011-028a WHb S2011-068b WHc S2011-146c S2011-046b S2011-068a S2011-036a S2011-053b S2011-058b S2011-027a S2011-003b 87 S2011-066a S2011-129b S2011-066b S2011-142a S2011-010a1 WHa S2011-010a2 S2011-058a 85 S2011-010b1 S2011-128a S2011-010b2 S2011-142b C0002a S2011-129a C0002d S2011-128b C0003c S2011-003c C0003a S2011-046a 86 C0003b 94 S2011-027b C0002b S2011-027c C0003d 100 S2011-027a C. pseudoceriferus S2011-070a S2011-068c S2011-070b S2011-068a 81 C0002c S2011-068b S2011-003c S2011-010a1 S2011-076b S2011-010b1 S2011-003b S2011-010a2 100 S2011-128a S2011-010b2 99 WHc 90 S2011-066a S2011-142b 96 S2011-066b S2011-036b S2011-028a S2011-128b C. rubens 92 S2011-028b S2011-129a 99 S2011-053a S2011-030a S2011-053b S2011-142a S2011-135a WHb S2011-135c S2011-076a S2011-135b C. ceriferus 87 S2011-058b S2011-141a S2011-058a S2011-146b S2011-129b S2011-001a S2011-003a S2011-146c S2011-142c S2011-001b S2011-046b S2011-022a S2011-076c S2011-141c S2011-046a S2011-022b S2011-036a S2011-022c WHa S2011-141b S2011-030c S2011-001c S2011-030b S2011-146a WHd S2011-114 S2011-114 outgroup outgroup 100 S2011-122 100 S2011-122

0.02 0.01

Fig. 2 Neighbour-joining tree of six species of the genus Ceroplastes COI sequences (a) and 28S sequences (b), using Kimura-2-parame- ter distance. Bootstrap values for each haplogroup are calculated in MEGA4.0 with 1000 replicates and species are shown in the right column. Less than 80% values are hidden.

2012 Blackwell Publishing Ltd DNA BARCODING OF SIX CEROPLASTES SPECIES 795

The low CÆG content (20.4%) of COI genes of the genus SIBS, Chinese Academy of Sciences, Shanghai), Hu Li (Guizhou Ceroplastes was similar to mealybugs and armoured University, Guiyang), Xian Li (Forestry Protection Station of scales (14.9% in mealybugs and 18.2% in armoured Chengdu, Sichuan), Qiang Shen (Forestry Protection Station of Yuyao, Ningbo), Xiu-Hao Yang (Forestry Protection Station of scales, Park et al. 2011). The low GÆC frequency was often Guangxi, Nanning), Ying-Jie Zhang (Yunnan Agricultural Uni- found at the third codon position (12.9%), and less pro- versity, Kunming), Hai-Bin Li and Nan Nan (Beijing Forestry nounced at the first and second positions (18.2% and University, Beijing), Ying Wang (Northeast Forestry University, 30.1%). The same low GÆC value also was found in the Harbin). Thanks to Dr. Douglas Chesters and three anonymous barcode region of other scale insects. The average GÆC reviewers for their valuable comments and suggestions, which content was 20.4%, significantly lower than the typical significantly contributed to improving the quality of the study. value of 27.7–39.5% in several other orders of insects (Hebert et al. 2003a). Further study may explain this References phenomenon. Although the universal primers LCO1490 and Ball SL, Armstrong KF (2007) Using DNA Barcodes to Investigate the Taxon- omy of the New Zealand Sooty Beech Scale . DOC research & devel- HCO2198 (Folmer et al. 1994) are broadly used in DNA opment series 287. Science and Technical Publishing, Department of barcoding in endopterygote insects, they frequently fail Conservation, Wellington, New Zealand, 14 pp. in some groups (Marcus et al. 2009), particularly in scale Ben-Dov Y (1993) A Systematic Catalogue of the Soft Scale Insects of the World insects (Kondo et al. 2008; Park et al. 2011). To overcome (Homoptera: Coccoidea: Coccidae). Sandhill Crane Press, Gainesville, Flor- ida, 536 pp. this shortcoming of the universal primers LCO1490 and Ben-Dov Y, Hodgson CJ (1997) Soft Scale Insects their Biology, Natural HCO2198, we designed a new set of primers which could Enemies and Control. World Crop Pests, Vol. 7A, Elsevier, Amsterdam, eliminate nontarget DNA interference such as the endo- 452 pp. Campbell BC, Steffen-Campbell JD, Werren JH (1993) Phylogeny of the parasites of soft scales (especially endoparasitoid wasps), Nasonia species complex (Hymenoptera: Pteromalidae) inferred from and make it easy to amplify any stage of the wax scales. an internal transcribed spacer (ITS2) and 28S rDNA sequences. Insect Using the new primer set, we succeeded in amplifying Molecular Biology, 2, 225–237. the barcoding region of other scale insects including Costa FO, deWaard JR, Boutillier J et al. (2007) Biological identification through DNA barcodes: the case of Crustacea. 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Nucleic Acids Sympo- This project was supported by the National Natural Science sium Series, 41, 95–98. Foundation of China (NSFC grant no. 31071950, 31071949), by Hamon AB, Williams ML (1984) The soft scale insects of Florida (Homop- Chinese Academy of Sciences (KSCX2-YW-NF-02) and partially tera: Coccoidea: Coccidae). In: of Florida and Neighboring by Project of the Department of Science and Technology of China Land Areas, 194 pp. Fla. Dept. of Agric. & Consumer Serv. Div. Plant (2008FY210500), Ningbo Science and Technology Bureau Ind., Gainesville, Florida. (2009C10004). The work was also supported by a grant (No. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003a) Biological identifi- O529YX5105) from the Key Laboratory of Zoological Systematics cations through DNA barcodes. Proceedings of the Royal Society of London Series B: Biological Sciences 270 and Evolution of the Chinese Academy of Sciences. We are , , 313–321. Hebert PDN, Ratnasingham S, DeWaard JR (2003b) Barcoding life: grateful to the following persons who helped us to collect Ceropl- Cytochrome c oxidase subunit I divergences among closely related spe- astes samples: Guo-Hua Huang (Hunan Agricultural University, cies. Proceedings of the Royal Society of London Series B: Biological Sciences, Changsha), Shao-Bin Huang (Guangdong Forestry Vocational 270, S96–S99. Technology College, Guangzhou), Jian-qin Wu (The Administra- Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of tive Bureau of Tianbaoyan National Nature Reserve of Yong’an, birds through DNA barcodes. PLoS Biology, 2, e312. Yong’an), Kai-Ju Wei (Youxi No.1 Middle school of Fujian Prov- ince, Youxi), Hong-Liang Li (Institute for Nutritional Sciences,

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