Biochemical Systematics and Ecology 34 (2006) 240e250 www.elsevier.com/locate/biochemsyseco Species identity and phylogenetic relationship of the pearl oysters in Pinctada Ro¨ding, 1798 based on ITS sequence analysis Da Hui Yu a,b,*, Ka Hou Chu b a South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, Guangdong, China b Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China Received 8 April 2005; accepted 9 September 2005 Abstract Analysis of ITS 1 and ITS 2 sequences in the pearl oysters Pinctada albina, Pinctada chemnitzi, Pinctada fucata, Pinctada fu- cata martensii, Pinctada imbricata, Pinctada margaritifera, Pinctada maxima, Pinctada nigra and Pinctada radiata was carried out. Homogeneity test of substitution patterns suggests that GC contents are highest in P. margaritifera and P. maxima and chro- mosomal rearrangements occurred in P. chemnitzi. These observations indicate that P. margaritifera and P. maxima are primitive species and P. chemnitzi is a recent species. Phylogenetic analysis shows that the pearl oysters studied constitute three clades with P. margaritifera and P. maxima forming the basal clade, congruent with results revealed by the substitution pattern test. The second clade consists of P. fucata, P. fucata martensii and P. imbricata. Low genetic distances among these taxa indicate that they may be conspecific. The remaining species make up the third clade and low genetic divergence between P. albina and P. nigra suggests that they may represent the same species. The ITS 1 sequence of P. radiata in GenBank is almost identical to that of P. chemnitzi de- termined in the present study and we suspect that the specimen used for the P. radiata sequence was misidentified. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Internal transcribed spacer; Pinctada; Species identity; Phylogeny Pearl oysters in the genus Pinctada include species widely distributed in tropical and subtropical oceans and some of them are of great economic importance, being cultured for pearl production. As the shell morphology and morpho- metrics of pearl oysters vary greatly, it is difficult to classify them based on shell materials only, thereby leading to proliferation of trivial names. Ranson (1961) recognized 11 species. They are Pinctada albina (Lamarck, 1819), Pinc- tada anomioides (Reeve, 1857), Pinctada capensis (Sowerby, 1889), Pinctada chemnitzi (Philippi, 1849), Pinctada maculata (Gould, 1850), Pinctada margaritifera (Linnaeus, 1758), Pinctada martensi (Dunker, 1872) Pinctada max- ima (Jameson, 1901), Pinctada mazatlanica (Hanley, 1855), Pinctada nigra (Gould, 1850) and Pinctada radiata (Leach, 1814). However, taxonomic confusion is still prevalent in the literature and phylogenetic relationships among the species remain unclear. * Corresponding author. South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, Guangdong, China. Tel.: þ86 20 84451432; fax: þ86 20 84451442. E-mail address: [email protected] (D.H. Yu). 0305-1978/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2005.09.004 D.H. Yu, K.H. Chu / Biochemical Systematics and Ecology 34 (2006) 240e250 241 The main confusion on taxonomy involves P. martensi, P. radiata, P. albina, Pinctada fucata (Gould, 1850) and Pinctada imbricata Ro¨ding, 1798. In Japan, the most common local pearl oyster was previously named as P. martensi (Ranson, 1961) but later was renamed as P. fucata (e.g., Wada, 1982), and recognized as two subspecies, Pinctada fucata martensii representing the northern populations (e.g., Mie and Nagasaki Prefectures), and Pinctada fucata fu- cata representing the southern populations (e.g., Okinawa Prefecture). In China, the most common pearl oyster, and also the major cultivated species in southern China, was named as P. martensi (Wang, 1978) but later referred to as P. fucata martensii (Wang, 2002)orP. albina (Bernard et al., 1993). At present the taxonomic names P. fucata and P. martensi(i) are frequently used by different authors in China for this species. We use the name P. fucata for this species in China. In Australia, Hynd (1955) classified the common small pearl oyster there as P. fucata, and regarded it to be conspecific with P. radiata, the pearl oyster in East Indies and Arabian Gulf. Ranson (1961) regarded P. fucata as a junior synonym of P. radiata.YetShirai (1994) named the common small pearl oyster in Australia as P. imbricata, which has been accepted by subsequent authors (e.g., Urban, 2000; Colgan and Ponder, 2002). Beaumont and Kham- dan (1991) used the name P. radiata for the common pearl oyster in the Arabian Gulf. They examined the collections of Japanese pearl oysters (named as P. fucata, P. martensi or P. fucata martensii) in the Natural History Museum, London and found that they represent P. radiata. Based on protein electrophoresis, Colgan and Ponder (2002) found that Japanese P. imbricata (¼P. radiata, P. fucata) and Australian P. imbricata are conspecific, distinct from Austra- lian P. albina. Wada (1982) also showed that Japanese P. fucata and P. albina are distinct from each other by using protein electrophoresis. Atsumi et al. (2004) also suggested that Japanese P. fucata martensii and Chinese P. fucata are conspecific as revealed by allozyme data and breeding experiments. Thus the common pearl oysters in China, Japan, Australia and the Arabian Gulf seas may represent the same species. The phylogenetic relationship among Pinctada species is poorly understood. Jameson (1901) subdivided the spe- cies into two sections based on the criterion of the absence or presence of hinge teeth. Section I includes two big pearl oysters, P. maxima and P. margaritifera, without hinge teeth and Section II includes all the smaller pearl oysters with hinge teeth. Considering that most species in Pteriidae have oblique shell form and hinge teeth, Hynd (1955) sug- gested that species in Section II are primitive. Yet karyotypical and other evidences do not support this view. Wang (2002) indicated that P. margaritifera is the only Pinctada species without hinge teeth. We examined our shell materials and found that P. margaritifera indeed does not have any clear hinge teeth but P. maxima has a big rounded hinge-tooth-like structure on its right shell, consistent with Wang’s (2002) description. Moreover, the hinge tooth of P. fucata is not as apparent as that of Pteria penguin. Thus the absence or presence of hinge teeth may not be a good character for phylogenetic inference. On the other hand, the differences in chromosomal numbers (P. chemnitzi has 11 pairs of chromosomes; the other species including P. margaritifera and P. maxima have 14 pairs) and karyo- types suggest that P. chemnitzi is a more recent species which has diverged from its ancestor by Robertsonian trans- location (Jiang and Wei, 1986). Thus further studies using DNA markers are constructive for elucidating the taxonomic identity and phylogenetic relationship of pearl oysters. The eukaryotic nuclear ribosomal DNA (rDNA) genes, consisting of conservative genes of 18S, 5.8S and 28S, and variable internal transcribed spacers (ITSs), namely ITS 1 and ITS 2, are widely used in phylogenetic analysis (Hillis and Dixon, 1991). Because of the high level of variability, they are often used for species level phylogeny as well as species identification (Beauchamp and Powers, 1996; Remigio and Blair, 1997; Chu et al., 2001; Chen et al., 2002; Lo´pez-Pinˇo´n et al., 2002). He et al. (2005) studied variability of ITS 2 in six pearl oyster species and found that ITS 2 is appropriate for phylogenetic study of this group. In the present study we performed a comprehensive analysis of ITS 1 and ITS 2 to elucidate the phylogenetic relationship among common pearl oyster species of the genus Pinctada,as well as to address the taxonomic confusion among Australian P. imbricata, Japanese P. fucata martensii and Chinese P. fucata. 1. Materials and methods 1.1. Sample collection and DNA extraction Two specimens of Pinctada fucata martensii were collected from Mie Prefecture, Japan; three P. albina and two P. imbricata from Port Stephens, Australia; and the other species (P. fucata, P. chemnitzi, P. maxima, and P. margari- tifera) from southern China including Hong Kong (HK), Daya Bay (DB), Guangdong Province, Beibu Bay (BB), Guangxi Province, and Sanya Bay (SB), Hainan Province, respectively (Table 1). Pteria penguin from Sanya Bay 242 D.H. Yu, K.H. Chu / Biochemical Systematics and Ecology 34 (2006) 240e250 Table 1 Species examined in this study, their sampling localities and GenBank accession numbers (those with asterisks were from GenBank) Species and no. of Abbreviation Sampling locality GenBank accession nos. individuals analyzed ITS 1 ITS 2 Pinctada fucata (3) PfucCN (including Daya Bay (DB), Sanya Bay (SB) AY877512 AY877585 PfucSB, PfucDB, and Beibu Bay (BB), China (CN) AY877523 AY877586 PfucBB) AY877525 AY877592 P. fucata martensii (2) PfucJP Mie Prefecture, Japan (JP) AY877577 AY877612 AY877578 AY877616 P. imbricata (2) Pimb Port Stephens, Australia AY877571 AY877606 AY877569 AY877609 P. albina (3) Palb Port Stephens, Australia AY877498 AY877508 AY877499 P. margaritifera (2) Pmar Sanya, Hainan Island, China AY877500 AY877506 AY877502 AY877507 P. chemnitzi (2) Pche (including Daya Bay (DB) and AY877496 AY877509 PcheDB & PcheHK) Hong Kong (HK) AY877497 AY877510 P. maxima (2) Pmax Sanya, Hainan Island, China AY172345* AY877504 AY877505 P. nigra Pnig Sanya, Hainan Island, China AY192147* AY282728* AY192714* P. radiata Prad Sanya, Hainan Island, China AY144603* e Pteria penguin (3) Ptpen Sanya, Hainan Island, China AY877503 AY192715* is included as an outgroup for phylogenetic reconstruction. A small piece of adductor muscle tissue was isolated from each animal and preserved in 95% ethanol. Total DNAwas extracted according to the instructions of tissue protocol in QIAamp DNA mini kit (QIAGEN). 1.2. Polymerase chain reaction and DNA sequencing PCR products of ITS 1 with partial 18S and 5.8S rRNA gene segments and ITS 2 with partial 5.8S and 28S gene segments were amplified using polymerase chain reaction (PCR).