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). PCR conditions for ITS 1 amplification followed Chu et al. (2001) using primers sp-1-5 and sp-1-3. ITS 2 were amplified using the primer pair 5.8S-F (5# GCA GGA CAC ATT GAA CAT CG 3#) and 28S-R (5# CCA AGG ACG TTC TTA GCA GAA G 3#) designed in this study. PCR reaction of ITS 2 was performed in 20 mL solution containing 1.5 mM MgCl2, 0.25 mM of each nucleotide, 0.1 mM of each primer and 0.5 units of Taq DNA polymerase (Promega) with cycling program as follows: 4 min at 93 C, 35 cycles of 40 s at 93 C, 40 s at 50 C, 1 min at 72 C, and final extension for 5 min at 72 C. Before direct sequencing, PCR products of ITS 1 and ITS 2 were purified using the QIAquick gel purification kit. Double-stranded PCR products were sequenced in both directions using each of the same primer pairs as in the amplification reaction. The cycle sequencing reactions were performed using ABI PRISMÔ dRhodamine Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer’s instructions. The sequencing reaction products were purified us- ing ethanol/sodium acetate precipitation and loaded onto an ABI Prism 3100 genetic analyzer (PE Applied Biosys- tems) for analysis. Both strands could usually be read over the full length of the original PCR product.
1.3. Data analysis
ITS 1 sequences of P. maxima, P. nigra and P. radiata, and ITS 2 sequences of P. nigra and P. penguin obtained from GenBank (Table 1) were included for analysis. The preliminary multiple sequence alignment with outgroup P. penguin was made by using ClustalX 1.83 (Thompson et al., 1997). As it is difficult to align non-coding regions (Kim- ball and Crawford, 2004), we conducted several alignments by changing penalty settings to determine the optimum D.H. Yu, K.H. Chu / Biochemical Systematics and Ecology 34 (2006) 240e250 243 settings but we found no substantial impacts on the topology of the resulting phylogenetic tree with different settings. Hence we adopted the default settings for alignment. The boundaries of ITS 1 and ITS 2 were determined by excluding the partial sequences of 18S, 5.8S and 28S genes flanking ITS 1 and ITS 2 by referring to the results of local ClustalX and online Blastn alignments (Wheeler et al., 2000). Prior to phylogenetic analysis, minor adjustment to the alignment was made manually. Base composition and pairwise nucleotide substitutions were determined using PAUP* v4.0b10 (Swofford, 2003). Also using PAUP*, a chi-square (c2) test of homogeneity of base frequencies across taxa as well as partition homo- geneity test (Farris et al., 1995) for congruence of aligned ITS 1 and ITS 2 data sets were tested. For partition homo- geneity test, 1000 partition replicates were conducted with heuristic search, with 10 random additions of taxa replicates per partition replicate. The result indicated the substitution pattern between ITS 1 and ITS 2 is homogeneous (P ¼ 0.99). The homogeneity test of substitution patterns between sequences indicated by disparity index (Kumar and Gadagkar, 2001) was conducted using MEGA 3 (Kumar et al., 2004). For a robust and reliable phylogenetic analysis, we used four methods, namely neighbor joining (NJ), parsimony (MP), maximum likelihood (ML) and Bayesian approaches. For ML method, we at first conducted hierarchical likeli- hood ratio test to determine the appropriate model of evolution and parameters by using Modeltest 3.06 (Posada and Crandall, 1998) for ITS 1 and ITS 2 combined data set. Based on Modeltest results, ML tree was reconstructed using PAUP* with tree bisection reconnection (TBR) branch swapping algorithm. Bootstrapping test (Felsenstein, 1985) was conducted using 100 pseudoreplicates. NJ tree (Saitou and Nei, 1987) and MP tree were also constructed using PAUP* with bootstrap test by 1000 replicates. NJ analysis was based on the ML distances from Modeltest. Gaps/miss- ing data were pairwisely deleted in NJ method and all sites were used in MP method. Bayesian analysis was implemented using BEAST v1.1.2 (Drummond and Rambaut, 2003). GTR þ G model with four numbers of gamma categories was assumed in Bayesian approach while other settings were kept default. Markov chains were run for 5 000 000 generations with 50 000 pre-burn-in states, sampled every 1000 generations resulting in 5001 trees. The majority rule consensus tree was calculated using PAUP* after discarding the first 51 trees (which are from the 50 000 burn-in generations). Posterior probabilities were used to assess the robustness of the consensus tree. Other parameters were summed using Tracer 1.2 (Drummond and Rambaut, 2003).
2. Results
2.1. Length variation and base composition
Aligned length of ITS 1 consists of 529 sites, of which 316 were variable and 193 were parsimony informative. Aligned length of ITS 2 consists of 274 sites, of which 170 were variable and 95 were parsimony informative. The lengths and nucleotide compositions of ITS 1 and ITS 2 of the taxa are given in Table 2. P. maxima and P. margar- itifera have the longest ITS 1 (482 bp and 457 bp, respectively) but the shortest ITS 2 (210 bp and 214 bp, respective- ly), while P. chemnitzi, P. albina and P. nigra have the longest ITS 2 (248e249 bp). P. maxima and P. margaritifera also have the highest GC contents in both ITS 1 (60% and 57%) and ITS 2 (62% and 61%). P. chemnitzi and P. radiata have the lowest GC contents in ITS 1. All taxa are in GC balance with GC contents of ITS 1 and ITS 2 nearly equal as in most organisms (Torres et al., 1990). The base frequencies in both genes are differentiated across taxa (Table 2). Generally, A% ¼ T% and G% ¼ C% but frequency of A in ITS 2 is lower than that of T in P. fucata, P. fucata martensii, P. imbricata and P. penguin, and frequency of C in ITS 2 is higher than G in all taxa. Homogeneity test of base frequency of the combined data set also indicated significant differentiation across taxa (c2 ¼ 44.38, df ¼ 27, P ¼ 0.019) (Table 3). The distribution of chi square (c2) values indicates that they are mostly contributed by P. maxima, P. chemnitzi, P. margaritifera, P. radiata and P. penguin across taxa, and by bases A, C and T across bases (Table 3). For P. radiata and P. chemnitzi, the ob- served frequency of A is much higher than expected, and the observed frequencies of G and C are much lower than expected. In contrast, P. maxima and P. margaritifera have significantly lower observed frequency of T. In P. fucata, P. fucata martensii and P. imbricata, the observed frequencies of C are lower than expected, and the observed frequen- cies of A and T are a little higher than expected, showing a similar pattern as in P. chemnitzi and P. radiata but in contrast to P. maxima and P. margaritifera. These results demonstrate that P. chemnitzi and P. radiata, P. maxima and P. margaritifera are quite different from the others in base frequencies and base compositions. 244 ..Y,KH h iceia ytmtc n clg 4(06 240 (2006) 34 Ecology and Systematics Biochemical / Chu K.H. Yu, D.H.
Table 2 Base composition, GC contents and length variation of ITS 1, ITS 2 and the combined data (ITS 1 þ 2) Species A C G T G þ C Size (bp) ITS 1 ITS 2 ITS 1 þ 2 ITS 1 ITS 2 ITS 1 þ 2 ITS 1 ITS 2 ITS 1 þ 2 ITS 1 ITS 2 ITS 1 þ 2 ITS 1 ITS 2 ITS 1 þ 2 ITS 1 ITS 2 ITS 1 þ 2 PfucCN 0.25 0.19 0.22 0.24 0.28 0.26 0.27 0.24 0.26 0.24 0.29 0.26 0.51 0.52 0.52 411 230 641 PfucJP 0.25 0.19 0.23 0.24 0.28 0.26 0.27 0.24 0.26 0.24 0.28 0.25 0.51 0.53 0.52 412 227 638 Pimb 0.25 0.19 0.23 0.24 0.28 0.25 0.27 0.24 0.26 0.24 0.29 0.26 0.51 0.52 0.51 411 229 640 Pnig 0.22 0.20 0.21 0.25 0.29 0.27 0.26 0.25 0.25 0.27 0.26 0.26 0.51 0.54 0.52 410 249 659 Palb 0.24 0.20 0.22 0.26 0.29 0.27 0.25 0.25 0.25 0.25 0.26 0.26 0.51 0.54 0.52 422 248 670 Pche 0.27 0.24 0.26 0.24 0.29 0.26 0.24 0.25 0.24 0.25 0.22 0.24 0.48 0.53 0.50 448.5 248.5 697 Prad 0.26 e 0.26 0.25 e 0.25 0.24 e 0.24 0.25 e 0.25 0.49 ee 446 ee Pmar 0.21 0.17 0.20 0.29 0.32 0.30 0.29 0.29 0.29 0.22 0.22 0.22 0.57 0.61 0.58 457.5 214 671.5 Pmax 0.20 0.16 0.18 0.30 0.35 0.32 0.29 0.27 0.28 0.21 0.23 0.21 0.60 0.62 0.60 482 210 692 Ptpen 0.19 0.17 0.18 0.28 0.30 0.29 0.26 0.27 0.26 0.27 0.26 0.27 0.54 0.57 0.55 393 243 636 Mean 0.24 0.19 0.22 0.26 0.29 0.27 0.26 0.26 0.26 0.24 0.26 0.25 0.52 0.55 0.53 427.4 231.5 645.9 e 250 Table 3 Distribution of chi squares (c2) in homogeneity test of base frequency of ITS 1e2 P Taxon PfucCN PfucJP Pimb Pnig Palb Pche Prad Pmar Pmax Ptpen c2 P (df) O E c2 O E c2 O E c2 O E c2 O E c2 O E c2 O E c2 O E c2 O E c2 O E c2 A 3.72 0.10 6.84 0.33 5.28 0.20 3.77 0.10 3.37 0.08 29.66 5.74 19.02 3.69 14.41 1.41 25.02 4.12 24.72 4.37 20.14 <0.05(9) C 10.15 0.59 10.25 0.60 9.70 0.54 2.98 0.05 0.47 0.00 10.63 0.59 11.81 1.15 15.74 1.35 31.01 5.09 8.30 0.40 10.36 w0.30(9) G 0.70 0.00 1.35 0.01 0.17 0.00 3.82 0.08 9.21 0.48 13.99 1.08 9.29 0.74 18.05 1.86 16.57 1.52 2.17 0.03 5.81 w0.70(9) PT 5.74 0.21 4.75 0.14 4.24 0.11 10.57 0.69 5.37 0.17 5.04 0.15 2.07 0.04 19.38 2.27 22.56 2.98 14.24 1.29 8.07 w0.50(9) c2 0.91 1.09 0.86 0.92 0.74 7.55 5.62 6.89 13.71 6.09 44.38 w0.02(27) P (df ¼ 3) w0.80 w0.70 w0.90 w0.80 w0.90 w0.06 w0.20 w0.10 w0.01 w0.10 O: observed frequency, E: expected frequency. 246 D.H. Yu, K.H. Chu / Biochemical Systematics and Ecology 34 (2006) 240e250
The results of homogeneity test of substitution patterns between sequences show that the substitution patterns of P. chemnitzi, P. maxima and P. margaritifera are significantly different from the others (Table 4), in consistence with the patterns of GC content variation and the heterogeneity in base frequency. The substitution pattern is also significantly distinct between P. maxima and P. margaritifera. These observations imply that they have significantly high GC con- tents or have experienced chromosomal rearrangement (Kumar and Gadagkar, 2001).
2.2. Phylogeny and evolution of the pearl oysters
Modeltest shows that models selected by Akaike Information Criterion (AIC) are the best-fit models for ITS 1, ITS 2 or the combined data set because the AIC selected models produce higher likelihood values and more consistent parameters of estimated base frequencies with observed base frequencies than hierarchical likelihood ratio tests (hLRTs), supporting the viewpoint of Posada and Buckley (2004). The partition homogeneity test indicated phyloge- netic congruence between the ITS 1 and ITS 2 data sets (P ¼ 0.99). Thereby a matrix of 803 aligned base positions of combined ITS 1 and ITS 2 data set for 10 taxa with 17 individuals was compiled for phylogenetic analysis based on the AIC selected model, TVM þ G. The TVM þ G model corresponds to the GTR þ G model which is a commonly used model in phylogenetic analysis. Phylogenetic trees were reconstructed by four approaches and the ML tree is presented in Fig. 1. The length of MP tree is 693 with CI ¼ 0.90, RI ¼ 0.94, RCI ¼ 0.85 for all sites, and iCI ¼ 0.86, iRI ¼ 0.94, iRCI ¼ 0.81 for parsimony informative sites. The likelihood value ( ln L) is 3653.3 in ML tree, and 3637.5 in Bayesian tree with effective sample size of 2843.3 (siteModel alpha ¼ 1.276). Based on the branching pattern, the pearl oysters studied are separated into three clades. Clade I includes P. fucata, P. fucata martensii and P. imbricata. Clade II can be further subdivided into two subclades, with Clade IIA including P. albina and P. nigra, and Clade IIB including P. chemnitzi and P. radiata. Clade III consists of P. margaritifera and P. maxima. The branching pattern also demonstrates that most genetic var- iations are distributed among clades, and only a few are distributed between subclades or species. The topology also shows that the P. margaritifera and P. maxima clade is basal and have diverged earlier. The best-fit model based ML distances were presented in Table 5. The ML distances are 28.0e33.1% between Clade I and Clade II, 46.7e51.6% between Clade I and Clade III, 52.2e76.5% between Clade II and Clade III,
Table 4 Disparity index (upper diagonal) and probability (lower diagonal) from the homogeneity test of substitution pattern for combined data, the shaded number denote significance at 0.05 level 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1PfucSB 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.00 0.56 0.54 0.22 0.73 0.87 2.08 0.36 2PfucDB 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.06 0.04 0.56 0.54 0.23 0.68 0.81 2.11 0.40 3PfucBB 0.37 0.27 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.48 0.46 0.14 0.75 0.89 2.18 0.44 4PfucJP1 1.00 1.00 1.00 0.00 0.00 0.00 0.02 0.06 0.05 0.49 0.47 0.18 0.76 0.90 2.22 0.54 5PfucJP2 1.00 1.00 1.00 1.00 0.00 0.00 0.02 0.06 0.04 0.49 0.47 0.18 0.76 0.90 2.22 0.54 6Pimb1 1.00 1.00 1.00 1.00 1.00 0.00 0.01 0.08 0.06 0.53 0.51 0.21 0.83 0.97 2.31 0.51 7Pimb2 1.00 1.00 0.26 1.00 1.00 1.00 0.00 0.04 0.03 0.55 0.53 0.23 0.62 0.75 2.10 0.43 8Pnig 1.00 1.00 1.00 0.34 0.33 0.37 1.00 0.12 0.07 0.80 0.77 0.43 0.71 0.85 1.99 0.00 9Palb1 0.32 0.26 0.36 0.29 0.28 0.25 0.31 0.00 0.00 0.32 0.29 0.11 0.52 0.61 1.63 0.38 10Palb2 0.37 0.30 0.35 0.29 0.28 0.26 0.31 0.00 1.00 0.44 0.40 0.21 0.51 0.60 1.63 0.19 11PcheDB 0.01 0.01 0.02 0.02 0.02 0.01 0.02 0.00 0.00 0.00 0.00 0.03 1.10 1.21 2.21 2.06 12PcheHK 0.02 0.01 0.03 0.02 0.02 0.02 0.02 0.00 0.01 0.00 1.00 0.02 1.00 1.11 2.09 1.88 13Prad 0.10 0.09 0.19 0.15 0.14 0.12 0.11 0.00 0.06 0.01 0.00 0.00 0.37 0.37 0.86 1.47 14Pmar1 0.01 0.02 0.02 0.01 0.02 0.01 0.03 0.02 0.04 0.06 0.00 0.01 0.11 0.00 0.31 1.05 15Pmar2 0.01 0.01 0.00 0.01 0.01 0.01 0.02 0.01 0.03 0.03 0.00 0.01 0.11 1.00 0.28 1.17 16Pmax 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.00 0.00 2.15 17Ptpen 0.19 0.16 0.14 0.12 0.12 0.10 0.11 1.00 0.17 0.23 0.01 0.00 0.01 0.03 0.02 0.00 D.H. Yu, K.H. Chu / Biochemical Systematics and Ecology 34 (2006) 240e250 247
Pinctada fucata (SB)
P. fucata (DB)
P. fucata (BB)
P. f. martensi 1 I
100/100/96/100 P. imbricata 1
P. imbricata 2
P. f. martensi 2 100/100/100/100 1 100/88/94/100 P. albina
100/100/83/100 P. albina 2 IIA
P. nigra
100/100/100/100 P. chemnitzi (DB)
P. chemnitzi (HK) IIB 100/100/100/100
P. radiata
100/100/67/100 P. margaritifera 1
P. margaritifera 2 III 100/100/90/100 P. maxima
Pteria penguin 0.05 substitutions/site
Fig. 1. The ML tree based on combined ITS 1 and ITS 2 data sets. Numbers near the branches represent bootstrap values of neighbor joining, maximum parsimony and maximum likelihood analysis and posterior probability of Bayesian approach. and 8.7e10.2% between Clade IIA and Clade IIB. In Clade I, the intraspecific divergence (0.6e1.1%) and interspe- cific divergence (0.6e1.4%) in terms of ML distance overlapped, and are very low compared with those between clades. This suggests that P. fucata, P. fucata martensii and P. imbricata in Clade I are most likely conspecific.
3. Discussion
The plasticity of shell morphology in pearl oysters and the lack of direct comparative study of samples from dif- ferent geographical localities make the taxonomy of pearl oyster controversial. Some of the species names may be synonymous. Thus it is desirable to address this problem using molecular markers. Allozyme data have demonstrated that Japanese P. fucata martensii and Chinese P. fucata (Atsumi et al., 2004), and Australian P. imbricata and Japa- nese P. imbricata (¼P. radiata, P. fucata)(Colgan and Ponder, 2002), are conspecific. And the breeding experiments with Japanese P. fucata martensii and Chinese P. fucata resulted in very high rates of fertilization and survival (Atsumi et al., 2004), while those between P. fucata and P. chemnitzi or P. fucata and P. maxima did not result in high fertil- ization or survival rates (Wei et al., 1983; Jiang et al., 1983). Further biochemical analysis indicated that the offsprings are not hybrids but gynogens (Li et al., 1983, 1985). Limited DNA data are available on the different taxa. ITSs (ITS 1 248 D.H. Yu, K.H. Chu / Biochemical Systematics and Ecology 34 (2006) 240e250
Table 5 Pairwise ML distance based on TVM þ G model Species 1234567891011121314151617 1PfucSB e 2PfucDB 0.010 e 3PfucBB 0.006 0.006 e 4PfucJP1 0.014 0.010 0.013 e 5PfucJP2 0.014 0.010 0.013 0.009 e 6Pimb1 0.008 0.011 0.008 0.013 0.013 e 7Pimb2 0.010 0.010 0.006 0.009 0.006 0.011 e 8Pnig 0.309 0.287 0.280 0.297 0.295 0.296 0.300 e 9Palb1 0.321 0.299 0.293 0.302 0.307 0.308 0.312 0.012 e 10Palb2 0.324 0.301 0.295 0.305 0.310 0.311 0.314 0.012 0.000 e 11PcheDB 0.328 0.307 0.304 0.316 0.315 0.320 0.319 0.095 0.102 0.101 e 12PcheHK 0.328 0.307 0.305 0.316 0.315 0.320 0.320 0.095 0.102 0.101 0.003 e 13Prad 0.326 0.331 0.326 0.328 0.325 0.325 0.327 0.087 0.097 0.095 0.004 0.004 e 14Pmar1 0.496 0.490 0.506 0.504 0.508 0.516 0.508 0.522 0.536 0.538 0.596 0.597 0.679 e 15Pmar2 0.496 0.493 0.508 0.503 0.508 0.515 0.507 0.530 0.544 0.545 0.600 0.601 0.678 0.008 e 16Pmax 0.474 0.467 0.483 0.477 0.485 0.493 0.483 0.568 0.588 0.590 0.648 0.650 0.765 0.083 0.087 e 17Ptpen 1.702 1.683 1.708 1.684 1.675 1.734 1.679 1.812 1.855 1.823 1.935 1.911 2.128 1.675 1.684 1.663 e and ITS 2) appear to be candidate markers as they have evolved in concert as the other regions of rDNA, making them highly variable between species but less variable within species (Hillis and Dixon, 1991). Therefore, they are useful for inferring phylogeny among closely related taxa and have already been widely used in many animal groups (Beau- champ and Powers, 1996; Chen et al., 2002; Chu et al., 2001) including mollusks (e.g., Anderson and Adlard, 1994; Remigio and Blair, 1997; Lo´pez-Pinˇo´n et al., 2002). In this study we provide the first direct DNA evidence to show that the three taxa P. fucata, P. fucata martensii and P. imbricata are conspecific. The intraspecific and interspecific divergences overlap among these taxa and are much lower than those among the three clades. We also found that one of our ITS 2 sequence (AY877604; unpublished data) of P. fucata is entirely identical to the ITS 2 sequence (AY192712) of P. marternsi reported by He et al. (2005). In addition, these taxa are distinct from P. albina, consistent with allozyme analyses (Wada, 1982; Colgan and Ponder, 2002). It can, therefore, be concluded that the three geo- graphical (namely Australia, Japan and China) local pearl oyster populations represent the same species. For further reinforcement of this conclusion, more molecular evidences are desirable. According to priority rule of nomination, the name P. imbricata should be the correct name. Yet a recent report indicated that the Indo-Pacific ‘P. imbricata’is different from the Atlantic P. imbricata (Masaoka and Kobayashi, 2005). Therefore, the species in Indo-Pacific should be referred to as P. fucata. It is also possible that the species names P. albina and P. nigra are synonymous. Their genetic divergence is much lower than those among other pearl oysters. The two species are distinct only in the color of the shell; P. nigra has dark brown or dark purple shell while P. albina has milk white or light yellowish brown shell (Wang, 2002). In the case of P. margaritifera, it is also found that there are two color patterns, black or red type, of the gills and the non-nacreous border of inner shell in sympatric P. margaritifera in China or Polynesia where it is recognized as two subspecies, P. m. var. cumingi and P. m. var. erythraensis (Pouvreau et al., 2000). Similarly, P. albina and P. nigra may also be two subspecies. As for P. chemnitzi and P. radiata, their genetic divergence is also very low but the former is quite different from the other pearl oysters in terms of morphology. P. chemnitzi has an extended large posterior ear and a long hinge line (Hynd, 1955; Wang, 2002). Yet descriptions of the morphological features of P. radiata appear to be subjective and ambiguous, and similar to those of P. fucata martensii (Wang, 2002). The difficulty in delimiting P. radiata has resulted in a long list of synonyms. Our sequences of P. chemnitzi are consistent with those of P. chemnitzi in GenBank (Accession no. AY144600 and AY196791 for ITS 1 and ITS 2, respectively) and the morphological features of P. chemnitzi are also consistent with those described by Wang (2002), showing that the species identity of our specimen is correct. We suspect that the specimen on which the P. radiata ITS 1 sequence in GenBank (Accession no. AY144603) is based might have been misidentified, and represent P. chemnitzi. In fact, the validity of the name P. radiata is highly controversial. It may be synonymous with P. martensi, P. fucata or P. f. martensii (Beaumont and Khamdan, 1991; Urban, 2000). This study reveals that with more molecular systematic studies, the number of valid species of pearl oysters would decrease. D.H. Yu, K.H. Chu / Biochemical Systematics and Ecology 34 (2006) 240e250 249
Our phylogenetic analysis demonstrates that the two big pearl oyster species, P. maxima and P. margaritifera, rep- resent a group separated from the other pearl oysters. Jameson (1901) put the two big oysters without hinge teeth into one group and all small oysters with hinge teeth into another group. His analysis is supported by results presented here. Hynd (1955) regarded the small pearl oysters with hinge teeth as primitive, considering that most species in the family Pteriidae have hinge teeth. In contrast, high GC content suggests that P. maxima and P. margaritifera may be more primitive because high GC content is an ancestral character state (Rodrı´guez-Trelles et al., 2000). Neither does the basal position of the two big pearl oysters in the phylogenetic tree support Hynd’s (1955) opinion. On the other hand, the low GC content in P. chemnitzi suggests that it may have experienced a chromosomal rearrangement (Kumar and Gadagkar, 2001). The karyotype and chromosome numbers (2n ¼ 22) of P. chemnitzi as well as low genetic di- vergence (8.7e9.5%) between P. chemnitzi and P. nigra further suggest that P. chemnitzi might have diverged from P. nigra (2n ¼ 28, as in P. maxima, P. margaritifera and P. radiata, see Wada and Komaru, 1985) by Robertsonian trans- location (Jiang and Wei, 1986) and is a recent species. Moreover, Wang (2002) determined that only P. margaritifera does not have hinge teeth. After examining our P. maxima and P. margaritifera samples, we confirmed his finding. Therefore, hinge teeth are not a phylogenetic informative character and are of little value in phylogenetic inference of Pinctada. Hebert et al. (2003) proposed the use of mitochondrial cytochrome oxidase I (COI) as the DNA barcode for species identification. With the great contrast between inter- and intraspecific divergence in ITS sequences in Pinctada as well as in other animals, ITS sequence would be a good alternative candidate gene for this purpose. As the PCR primers of ITS are on the highly conserved regions of nuclear rRNA genes, they are more universal than COI primers. A draw- back of ITSs is that they exist in multiple copies in the genome so that there may be intragenomic divergence in some taxa, making the analysis problematic (Harris and Crandall, 2000). Particularly for the hybrid corals, the level of intra- individual divergence is nearly as high as that of intraspecific divergence, obscuring the phylogenetic signal in the data (Vollmer and Palumbi, 2004). Therefore, one has to be cautious when dealing with organisms with complicated mat- ing systems. Nevertheless, many successful applications in species identification using ITS sequence information show that ITS sequences are potentially good DNA barcodes.
Acknowledgments
We would like to thank Dr. Wayne O’Connor, Mr. Bing Yan and Mr. Youning Li for collecting pearl oyster speci- mens, and Prof. C.K. Wong for his comments on the manuscript. This work was supported by research grants from The Chinese University of Hong Kong to KHC, the National High Technology Research Development (863) Program (Project code: 2002AA603022) from the Ministry of Science and Technology, and Program (Project code: 2002B2150101) of the Science and Technology Bureau, and Program (Project code: 037148) the Natural Science Foundation of Guangdong Province, People’s Republic of China to DHY.
References
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