Differences Between Pagrus Major and Pagrus Auratus Through Mainly Mtdna Control Region Analysis
Total Page：16
File Type：pdf, Size：1020Kb
FISHERIES SCIENCE 2000; 66: 9–18 Original Article Differences between Pagrus major and Pagrus auratus through mainly mtDNA control region analysis Kazuo TABATA1* AND Nobuhiko TANIGUCHI2 1Hyogo Prefectural Fisheries Research Institute, Akashi, Hyogo 674-0093 and 2Faculty of Agriculture, Tohoku University, Sendai, Miyagi 980-8578 Japan SUMMARY: The magnitude of intraspecific and interspecific genetic differentiation in Pagrus major collected from two Japanese areas, the East China Sea (ECS) and the South China Sea (SCS), and Pagrus auratus, collected from Australia (AUS) and New Zealand (NZ), was estimated using restric- tion fragment length polymorphism (RFLP) analysis and DNA direct sequencing of the mtDNA control region. The RFLP haplotypic diversities in P. major samples were high (0.88–0.93); in contrast, these diversities were relatively lower (0.58–0.65) in P. auratus samples. The relative relationships among samples that resulted from RFLP analysis were almost the same as those from DNA direct sequenc- ing, except that values from the former were less sensitive and were one-third to one-fifth lower than those from the latter. A significant heterogeneity was observed in the distribution of RFLP haplotypes between samples from P. auratus and P. major, and between samples from AUS and NZ. The dif- ference of the nucleotide substitution by direct sequencing in the control region between P. auratus and P. major was 3.48%. Based on the substitution rate, the division time between samples from P. auratus and P. major was assumed to be 2–6 million years ago. With regard to morphological aspects, there was a significant difference in the bump between NZ and ECS samples, although there were no other significant external morphological differences. From these results, we suggest that the rela- tionship between these ‘species’ is at the level of a subspecies. Accordingly, P. major might be renamed P. auratus major and P. auratus renamed P. auratus auratus. KEY WORDS: DNA direct sequence, fish population genetics, mtDNA control region, Pagrus auratus, Pagrus major, red sea bream, RFLP, snapper. INTRODUCTION Southern Hemisphere of Australian and New Zealand waters.3 In 1962, Akazaki suggested that P. major was a The red sea bream Pagrus major is distributed in adjacent geographical variety of P. auratus.4 Further, in 1990, waters of Japan, the East China Sea and the South China Paulin proposed that these species should be redescribed Sea. The snapper Pagrus auratus is found throughout sub- as a single species named Pagrus auratus (P. auratus is the tropical to warm and temperate waters of southern main- senior synonym of P. major), based on results of his land Australia and New Zealand. Pagrus auratus was first morphological study5 and an electrophoretic study by described by Bloch and Schneider in 1801.1 The scien- Taniguchi et al.6 in which it was concluded that diversi- tific name of this species has been changed twice fication of isozyme genes in specimens from New Zealand since Günther classified specimens from New Zealand and Japan were ‘less diversified than the species level’. In and Australia as Pagrus unicolor and those from Japan as spite of Paulin’s conclusions, P. major has been treated as P. major.2 Fowler stated that the ‘two so-called species’ an independent species in Japan.7 We think that this is were largely accepted based on their geographical sepa- due to the lack of more detailed genetic, morphological ration, and he provisionally recognized them within the and cross information. genus Chrysophrys: C. major in the Northern Hemi- The purpose of the present study is to clarify the sphere of Indo-Chinese-Japanese and C. auratus in the relationships between P. major and P. auratus using mitochondrial DNA control region analysis [both restriction fragment length polymorphism (RFLP) and *Corresponding author: Tel: 0789418601. Fax: 078 9418604. Email: DNA direct sequencing], which is more sensitive than [email protected] allozyme analysis and also by use of morphometric Received 17 August 1998. analysis of the head bump. 10 FISHERIES SCIENCE K Tabata and N Taniguchi The nucleotide sequence data reported in this ences were calculated using the method described in pre- paper will appear in the DDBJ/EMBL/GenBank nucleo- vious papers.8,14–16 tide sequence databases with the accession numbers For the DNA direct sequencing analysis, nucleo- AB012718 (Chrysophrys major) and AB012719 (Pagrus tide sequence divergence (mean nucleotide substitu- auratus). tion between individuals) was calculated with Arlequin version 1.1 (University of Geneva, Geneva, 17 MATERIALS AND METHODS Switzerland). Net nucleotide sequence divergence, within (dx, dy) Collection of fish and extraction of the DNA and between (dxy) two samples, was calculated following the method of Nei and Li.15 Net nucleotide sequence divergence (dA) was calculated using the following Samples of snapper from New Zealand were obtained 15 from two Japanese wholesalers, who had imported the fish equation: as frozen fish from New Zealand in 1996 (sample names: ddddAx()xy, =-+yxy()2 New Zealand 1, New Zealand 2). Samples from Australia were transported as frozen fish from New South Wales in 1997. Samples from the South China Sea were obtained External morphology directly by one of the authors at the Island of Nanau, Guangzhou Province, China, in 1997. Samples from External morphological measurements were performed the East China Sea were obtained from a trawl fishery at on 15 individuals of the snapper from New Zealand Nagasaki, Japan in 1997. Two samples from areas off (average fork length, 295 mm) and on 15 individuals of mainland Japan were obtained from the Kitan Channel the red sea bream from the East China Sea (average fork (Hyogo Prefecture) by trawling in 1995 (sample name: length, 335 mm). The 24 morphological parameters Japan 1), and from the Japan Sea (Hyogo Prefecture) by selected by Akazaki4 were measured according to the pro- set net in 1995 (sample name: Japan 2). The average fork tocol of Nakabo.7 Furthermore, the bump phase was length of sampled fish is shown in Table2. The DNA investigated by soft X-ray. The angle contained between extraction method is described in a previous paper.8 the line bound between the front edge and the top of the supraoccipital against the line bound between the front Amplification of mtDNA and sequencing edge of the supraoccipital and the upper front of the first defective interneural spine was measured (Fig. 1). This For the polymerase chain reaction (PCR)-RFLP analy- measurement was carried out using the TV Image Proces- sis, amplification of the specific region, thermal cycling sor EXCEL (Nippon Avionics, Tokyo, Japan). In the parameters, digestion by restriction endonucleases, and present study, this angle is referred to as the bump index. electrophoretic method were identical to those pre- 8–10 viously described. RESULTS The primers used for DNA direct sequencing were PRO L15924 and H16498, which amplify a part of tRNA Haplotypic diversity from RFLP analysis and the left domain of the mtDNA control region.11–13 Thermal cycling parameters were the same as those for A total of 66 mtDNA control region haplotypes were the PCR-RFLP analysis. For the Japanese (Japan 1, 2) identified in the red sea bream and the snapper sam- and the East China Sea samples, mtDNA of 20 individ- ples from Japan, China, Australia, and New Zealand uals selected at random per sample were amplified. For (Table1). the South China Sea sample, this analysis was not per- formed. For the Australian and New Zealand samples, mtDNA of 17 individuals selected at random per sample were amplified. After amplified DNA fragments had been purified using Microcon-100 (Amicon, Beverly, MA, USA), they were sequenced on automated sequencers (ABI 373S or 377; Perkin-Elmer, Foster, CA, USA) with amplifica- tion primers using a Taq DyeDeoxy Terminator cycle sequencing kit (Perkin-Elmer). DNA data analysis Fig. 1 Lateral head view of Pagrus major and Pagrus auratus For the PCR-RFLP analysis, the haplotypic diversity, taken by a soft X-ray. The angle (the bump index) was mea- nucleotide sequence divergence, and geographic differ- sured using a TV Image Processor EXCEL (Nippon Avionics). Differences between P. major and P. auratus in mtDNA 11 Table1 Distribution of the red sea bream and the snapper mtDNA control region haplotypes in seven samples from Japan, China, and Australasia Hap. type no. Hap. type J1 J2 ECS SCS AU N1 N2 1 1 BAAAAA 1 2 2 BAAAAB 1 3 3 BAAAAC 23 19 13 13 4 5 BAAAAE 8 6 2 2 5 6 BAAAAF 1 2 1 6 7 BAAAAG 1 7 11 BAAABC 4 1 3 8 13 BAAABE 1 1 2 9 59 BAAAHC 1 2 10 131 BAACAC 2 11 139 BAACBC 1 12 193 BABAAA 6 6 7 16 13 194 BABAAB 13 13 10 11 14 195 BABAAC 2 2 1 15 196 BABAAD 1 1 1 16 198 BABAAF 1 1 2 17 199 BABAAG 3 2 4 3 1 18 202 BABABB 2 5 5 7 19 203 BABABC 1 20 205 BABABE 33 33 41 21 206 BABABF 1 22 207 BABABG 4 2 23 212 BABACD 2 3 2 24 220 BABADD 1 2 2 2 25 242 BABAGB 2 2 26 246 BABAGF 1 27 250 BABAHB 1 28 253 BABAHE 1 29 255 BABAHG 1 30 258 BABBAB 5 3 4 31 259 BABBAC 1 32 261 BABBAE 3 33 263 BABBAG 1 34 269 BABBBE 5 2 6 35 317 BABBHE 1 36 387 BACAAC 3 1 1 37 389 BACAAE 1 38 578 BADAAB 3 1 39 579 BADAAC 1 40 580 BADAAD 1 4 41 642 BADBAB 1 2 42 644 BADBAD 2 4 3 1 43 770 BAEAAB 2 2 4 1 44 961 BADBAI 3 1 2 1 45 972 BABABO 2 46 974 BADAAL 1 47 975 BADBAM 1 48 976 BABAIB 2 1 49 977 BADBAK 1 50 978 BAAAKE 1 51 979 BAFAAB 1 52 980 BAAAJE 1 53 981 BADBAN 1 54 982 BAAAJC 1 55 983 BADAAN 1 56 985 BABABP 1 57 991 BABBLE 1 58 1003 AAAAAC 2 2 12 FISHERIES SCIENCE K Tabata and N Taniguchi Table1 Continued Hap.