DNA Barcoding of Six Ceroplastes Species (Hemiptera: Coccoidea: Coccidae) from China
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Molecular Ecology Resources (2012) 12, 791–796 doi: 10.1111/j.1755-0998.2012.03152.x DNA barcoding of six Ceroplastes species (Hemiptera: Coccoidea: Coccidae) 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 insects 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 Ó 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%, Ó 2012 Blackwell Publishing Ltd DNA BARCODING OF SIX CEROPLASTES SPECIES 793 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.