Differences Between Pagrus Major and Pagrus Auratus Through Mainly Mtdna Control Region Analysis

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 restriction fragment length polymorphism (RFLP) analysis and DNA direct sequencing of the mtDNA control region. The RFLP haplotypic diversities in P. major samples were high (0.88Ð0.93); in contrast, these diversities were relatively lower (0.58Ð0.65) in P. auratus samples. The relative relationships among samples that resulted from RFLP analysis were almost the same as those from DNA direct sequencing, 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 difference 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 2Ð6 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 relationship 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 performed. 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 amplification 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. type no. Hap. type J1 J2 ECS SCS AU N1 N2 59 1199 AABAAG 1 60 1205 AABABE 2 61 1255 AABAHG 25 23 62 1578 AADAAB 1 63 1582 AADAAF 7 2 64 1590 AADABF 1 65 1986 AABALG 1 1 66 3001 DABBME 1 Total 95 93 74 71 52 72 78

Composite haplotypes reﬂect digestions with the following restriction endonucleases (left to right): Hae III, Hha I, Hinf I, Msp I, Taq I, and Rsa I. Haplotypes are numbered by ranging in alphabetic order the digestion types of each restriction endonuclease in principle to ease the treat- ment by computer. J1, J2, Japan; ECS, East China Sea; SCS, South China Sea; AU, Australia; N1, N2, New Zealand.

Table2 Fork length of sample, sample size analyzed, number of and ratio of haplotype appeared, haplotypic diversity, and 95% appearing probability of haplotype in Pagrus major (Japan, East and South China Sea) and Pagrus auratus (Australia and New Zealand) Japan4 East China Sea South China Sea Australia New Zealand4 Sample size analyzed 94 (93–95) 74 71 52 75 (72–78) No. haplotype1 29.5 (28–31) 25 19 10 8.5 (8–9) Ratio of haplotype2 0.314 (0.301–0.326) 0.338 0.268 0.192 0.113 (0.111–0.115) Haplotypic diversity 0.913 (0.905–0.920) 0.927 0.878 0.581 0.648 (0.633–0.662) Proportion haplotype at 95%3 0.032 (0.031–0.032) 0.040 0.041 0.056 0.039 (0.042–0.038) Fork length (mm) (82–118) 335 130–150 295

1 Number of haplotype appeared. 2 Ratio of haplotype appeared; number appeared/sample size analyzed. 3 Proportion haplotype at 95%; proportion of haplotype in which at least one individual is detected at 95% conﬁdence from random sample of n individuals.41 4 Average (range) in two samples.

The number of mtDNA control region haplotypes China Sea were either not significant or were minor. As identified from the seven geographical areas is as follows: regards to the South China Sea sample, slight but sig- 19 in the South China Sea, 25 in the East China Sea, nificant (P > 0.05) differences were found in comparison 10 in Australia, 8.5 (mean) in the two samples from with the Japanese samples. However, between the South New Zealand, and 29.5 (mean) in the two samples from China Sea and the Australian/New Zealand samples sig- Japan (Table2). The ratio of haplotypes (haplotype nificant differences were found to a level of P < 0.001. number per sample size) was 0.268–0.326 in Japan and Moreover, there were significant differences (P < 0.001) China, 0.192 in Australia, and 0.113 (mean) in New between the samples from Australia and New Zealand as Zealand (Table2). well. Specimens from the two New Zealand samples were Haplotypic diversity in each sample is shown in Table not significantly different. 2. Values for the red sea bream were high (0.878–0.927). In contrast, values for the Australian (0.581) and New Zealand (mean: 0.648) snapper were lower than the red Nucleotide sequence divergence from RFLP sea bream. A dendrogram of the samples based on the UPGMA clustering15 of the net per cent nucleotide sequence Geographic occurrence of RFLP haplotypes divergence is shown in Fig. 2a. Per cent nucleotide sequence divergence between and Results of the c2 randomization method16 based on the within samples and net per cent nucleotide sequence frequency of RFLP haplotypes between the specimens divergence between samples are shown in Table4. The from the seven areas from Japan, China, Australia, and value within the Australian sample (0.40%) was the New Zealand are shown in Table3. Differences between lowest, but the values within the samples from New the two Japanese samples and the sample from the East Zealand, Japan, and China were approximately 1%. Differences between P. major and P. auratus in mtDNA 13

Table3 Heterogeneity certiﬁcation by Chi-squared randomization test (Monte Carlo technique) between natural populations of Pagrus major (1–4) and Pagrus auratus (5–7) 1234567 1 Japan 1 - - + ++ ++ ++ 2 Japan 2 0.712 - + ++ ++ ++ 3 East China Sea 0.652 0.230 -++++++ 4 South China Sea 0.039 0.028 0.268 ++ ++ ++ 5 Australia 0.000 0.000 0.000 0.000 ++ ++ 6 NZ 1 0.000 0.000 0.000 0.000 0.000 - 7 NZ 2 0.000 0.000 0.000 0.000 0.000 0.112

Above the diagonal: The range of the P value of c2 randomization test. ++ P < 0.01, + 0.01 < P < 0.05, - P < 0.05. Below the diagonal: P value of c2 randomization test (permutation: 1000).

Table4 Per cent nucleotide sequence divergence by restriction fragment length polymorphism analysis of natural samples of Pagrus major (1–4) and Pagrus auratus (5–7) 1 234567 1 Japan 1 1.050 1.036 1.037 1.018 1.344 1.607 1.549 2 Japan 2 -0.007 1.035 1.026 1.010 1.390 1.625 1.575 3 East China Sea -0.006 -0.009 1.036 0.994 1.376 1.594 1.546 4 South China Sea 0.032 0.031 0.015 0.922 1.316 1.540 1.485 5 Australia 0.617 0.671 0.657 0.654 0.402 0.967 0.800 6 NZ 1 0.578 0.603 0.572 0.575 0.262 1.008 0.961 7 NZ 2 0.572 0.606 0.576 0.572 0.147 0.005 0.904

Above the diagonal: between samples. Diagonal (bold): within samples. Below the diagonal: net value between samples.

The net values between samples are here classiﬁed into three groups. A group having low net values (–0.01–0.03) is the following: between the Japanese samples, between the two Japanese samples and the samples from the East China Sea and the South China Sea, between the sample from the East China Sea and the South China Sea sample, and between the New Zealand samples. A group having high net values (>0.57) is between the samples from the Northern Hemisphere and the Southern Hemisphere. A group having mid net values (0.14–0.26) is between the Australian sample and New Zealand samples.

Nucleotide sequence divergence based on DNA direct sequencing

A comparison with the sequences from carp18 and Japanese ﬂounder13 veriﬁed that the sequences from the red sea bream and the snapper in the present study con- Fig. 2 A dendrogram based on the per cent net nucleotide PRO sequence divergence calculated from (a) restriction fragment tained the tRNA gene and the left domain of the length polymorphism analysis and from (b) DNA direct control region (Fig. 3). One out of 50 sites in the sequencing between Pagrus major and Pagrus auratus. Targeted tRNAPRO gene were variable (2.0%), and 116 out of 487 region is the mtDNA control region. sites in the control region were variable (23.8%). 14 FISHERIES SCIENCE K Tabata and N Taniguchi

Fig. 3 Sequence for 537 b.p. a part of tRNAPRO (No. 1–50) and the left domain of mtDNA control region (No. 51–537) from Pagrus major (consensus, V1, V2 and V3) and Pagrus auratus (NZ and NZ-V). V, variation.

Results of the nucleotide sequence divergence within Table5 Per cent nucleotide sequence divergence based on and between samples are shown in Table5, and a DNA direct sequencing of P major (1–3) and P auratus (4–5) dendrogram based on the net values is given in Fig. 2b. The relationships between samples obtained from this 1 2345 method were approximately the same as the results obtained from the RFLP analysis (Table4; Fig. 2a). 1 Japan 1 2.795 2.674 2.751 6.438 5.975 However, the values obtained from direct sequencing 2 Japan 2 -0.054 2.661 2.639 6.420 5.980 were three- to ﬁve-fold higher than those obtained from 3 East China Sea 0.012 -0.033 2.684 6.324 5.874 the RFLP analysis. This was due to the fact that the 4 Australia 3.918 3.967 3.860 2.245 3.666 detection ability of direct sequencing is higher than that 5 NZ 1 3.044 3.116 2.999 1.010 3.067 of the RFLP analysis. Besides, the direct sequencing targeted only variable regions, while the RFLP analysis Above the diagonal: between samples. Diagonal (bold): within targeted regions containing no variable regions. samples. Below the diagonal: net value between samples. Differences between P. major and P. auratus in mtDNA 15

Table6 Morphological comparisons between Pagrus major from the East China Sea and Pagrus auratus from New Zealand New Zealand East China Sea t-test P Average Max. Min. n Average Max. Min. n Bump index** 7.30 1.261* 15 3.85 0.808* 14 8.3438 0.000 Fork length (L) mm 294.9 6.53* 15 335.5 11.81* 15 Fork L/Body (B) L 0.87 0.89 0.84 15 0.88 0.95 0.85 15 0.1568 0.876 BL 1 1 1 15 1 1 1 15 Head (H) L/BL 3.08 3.20 2.99 15 3.17 3.33 2.97 15 0.0153 0.988 Body depth/BL 2.20 2.36 2.08 15 2.32 2.57 2.17 15 0.0018 0.999 Body width/BL 5.41 6.30 4.85 15 5.13 5.58 4.48 15 0.0455 0.964 Pectoral ﬁn L/BL 2.84 3.58 2.63 15 2.91 3.42 2.61 14 0.4343 0.668 Snout L/HL 2.28 2.40 2.16 15 2.14 2.30 1.93 15 0.0004 1.000 Upper jaw L/HL 2.71 2.83 2.57 15 2.59 2.81 2.33 15 0.0132 0.990 Interorbital width/HL 3.06 3.52 2.72 15 2.80 3.03 2.67 15 0.0002 1.000 Eye diameter/HL 4.01 4.34 3.66 15 3.72 4.56 3.13 15 0.0284 0.978 Dorsal spine IIII L/HL 2.39 2.71 2.14 13 2.07 2.47 1.70 13 0.0009 0.999 Anal spine II L/HL 3.35 4.10 2.91 14 3.19 3.65 2.72 15 0.2083 0.837 Anal spine III L/HL 3.32 3.78 2.95 15 3.38 4.58 2.84 15 0.6552 0.518 Pelvic ﬁn L/HL 1.48 1.67 1.30 15 1.50 1.66 1.33 14 0.5591 0.581 No. dorsal spines 12.00 12 12 15 12.00 12 12 15 0.0000 1.000 No. dorsal soft rays 10.00 10 10 15 10.00 10 10 15 0.0000 1.000 No. anal spines 3.00 3 3 15 3.00 3 3 15 0.0000 1.000 No. anal soft rays 8.00 8 8 15 8.00 8 8 15 0.0000 1.000 No. pectoral soft rays 14.87 15 14 15 14.86 15 14 14 0.9434 0.354 No. lateral line scales 53.93 55 53 15 55.20 57 53 15 0.0080 0.994 No. scales above lateral line 7.00 7 7 15 7.00 7 7 15 0.0000 1.000 No. gill rakers (upper) 8.00 9 7 15 8.07 9 7 15 0.7165 0.480 No. gill rakers (lower) 10.14 11 9 14 9.87 10 9 15 0.1685 0.867 No. gill rakers (total) 18.21 20 17 14 17.93 19 17 15 0.2907 0.773

* Standard deviation. ** Bump index (See Fig. 1 and text.) II, III, and IIII indicate the second, third, and fourth spine, respectively.

External morphology between Australian and New Zealand snappers were clear (3.48% and 1.01%, respectively). However, the Results of the external morphological measurements and differences between the East China Sea and Japanese the bump index of snappers from New Zealand and the samples were not so clear. It has been suggested that such red sea breams from the East China Sea are shown a genetic divergence might be caused by isolation due in Table6 with the results of a statistical test (t-test) to a geographical barrier. In this respect, the distance between samples. The external morphological differ- between the Japanese coast and the South China ences of each trait between the New Zealand snapper Sea and that between Australia and New Zealand is, in and the East China Sea red sea bream were not sig- both cases, approximately 1300 km. However, the fol- nificant. However, the bump index was significant lowing geographical differences are apparent between (P < 0.0001); that is, the outgrowth (bumps) of the these two sea areas: the former has a continuous con- snapper were larger than those of the red sea bream. tinental shelf, while Australia and New Zealand are divided by a continental slope and a deep sea. In several marine species it has been demonstrated that the Tasman DISCUSSION Sea, for instance, is a partial barrier to gene flow. For example, the jackass morwong Nemadactylus macropterus Nucleotide sequence divergence shows allozymic and mtDNA differentiation across the Tasman Sea.19 Similarly, P. auratus shows allozymic The differences of nucleotide sequence divergence (net differentiation.20 mean nucleotide substitution) by DNA direct sequenc- A comparison of restriction analysis and sequencing ing in the mtDNA control region between the Southern for interspecific sequence variation of mtDNA in several Hemisphere Australian/New Zealand snapper and the salmonid species revealed that the sequence divergence Northern Hemisphere Japanese/Chinese red sea bream, from sequencing was two- to three-fold higher than the and those within the Southern Hemisphere samples values from restriction analysis.21 The present study 16 FISHERIES SCIENCE K Tabata and N Taniguchi

similarly found such a difference in detection ability Japanese red sea bream, Taniguchi et al. concluded that between direct sequencing and restriction analysis. Ac- isozyme genes had a low diversification, a level equiva- cordingly, when data are compared, it is important that lent to a subspecies status.6 all the data were obtained by the same method. The difference in the bump index between the red sea According to Ovenden, the intraspecific mtDNA bream and the snapper was significant, although no sig- sequence divergence by restriction analysis in marine nificant differences were found when comparing other fishes ranges from 0.2 to 2.2%.22 Ovenden concluded external morphological characteristics. The latter results that it is more common for marine than for terrestrial are in agreement with Akazaki4 and Paulin.5 The differ- species to have a low divergence (i.e. <1.0%), although ence in the bump between the red sea bream and the two menhaden species, Brevoortia tyrannus and Brevoor- snapper could be presented in a quantitative manner. tia partronus have high diversities. Gold and Richardson Even though this difference may not be clear in younger documented that the intraspecific mtDNA sequence fish, it becomes progressively clearer in older fishes since divergence by restriction analysis of seven marine spe- the bump of the snapper increases with fish growth.29,30 cies, including estuarine-dependent, reef-associated, To summarize our results, genetic differences at the and pelagic species, was in the range 0.06–0.57%.23 The DNA level were distinguishable between the red sea restriction analysis of the D-loop region yielded the fol- bream and the snapper. Judging from the RFLP and lowing data: 1.87% in the armorhead Pseudopentaceros sequencing analysis, however, these genetic differences wheeleri9 and 0.47% in Evynis japonica (Tabata K, unpubl. were low when considered at an interspecific level, but data). In contrast, sequencing of the D-loop region has were high at an interpopulational level. Moreover, sig- given the following results: 4.33% in Japanese flounder nificant differences were observed in the bump phase, Paralichthys olivaceus,13 3.8% in swordfish Xiphias gladius,24 although no other morphological differences could be and approximately 3% in ayu Plecoglossus altivelis,25 an established. amphidromous species. The intraspecific mtDNA sequence divergences of the red sea bream and the snapper analyzed from restriction analysis in this study Substitution rates and division time were 0.9–1.1% and 0.4–1.0%, respectively, and 2.7–2.8% and 2.3–3.1%, respectively, from sequencing. These Estimates of mtDNA substitution rates contained in the values might be average values. gene-coded region range from 0.3 to 0.7% per million The interspecific sequence divergences of mtDNA by years in ectotherms,31,32 to 1–2% per million years in ter- restriction analysis in marine, estuarine, and catadro- restrial animals.33,34 However, estimates of control region mous species were in the range 3.7–13%,26 and the values substitution rates ranged from 0.5% per million years in for salmonids in the range 2.7–7.5%.27 It should be noted cetaceans,35,36 to 21% per million years in birds.37 Thus, that these are not net values. The interspecific diver- the variation in the control region is large and depends gences (1.3–1.6% as the value contained intraspecific on the species and other factors such as metabolic value, 0.57–0.67% as a net value) between the red sea rates.31,38 Since the nucleotide substitution between the bream and the snapper obtained from restriction analy- red sea bream and the snapper obtained from the control sis are considerably lower than the interspecific values region was approximately twice that obtained from the in some other marine species and in salmonids. For D- cytochrome b region (Tabata K, unpubl. data), the sub- loop sequencing, the interspecific sequence divergences stitution rate from the control region may be assumed to between salmonids are documented as 3.8–10.0% by be 0.6–1.4% per million years. Accordingly, the division Shedlock et al.28 We should note, however, that the time between the red sea bream and the snapper sample number per species of this data28 is only 1 or 2. would be assumed to be before 2–6 million years Only when there are low intraspecific diversity, will these (3.48%/0.6–1.4%/million years). However, if we take data be effective. We think that comparison with the net into account that red sea bream is a warm-water species, value will be appropriate when comparing the red sea the substitution rate may be slightly higher. bream data having a high intraspecific diversity. The Akazaki presented the following theory for the interpopulational sequence divergence from the D-loop historical dispersion of the Sparidae:39 the Sparidae sequencing between clades of swordfish is 2.7%,24 and diverged from an ancestral of Perciformes several ten the values between amphidromous ayu and landlocked million years between the Cretaceous to the Paleocene, ayu are 0.24–0.53%, all as net values.25 The interspecific and, during this period, six subfamilies which included divergences (3.0–4.0% as net) between the red sea bream the Pagrinae evolved. The center of distribution of and the snapper obtained from sequencing were less the Sparidae is considered to be the Old Mediterranean than the lowest level of salmonid interspecific values Sea. The global dispersion of coastal fish generally and slightly higher than the interpopulational values of occurs associated with continental drift. The dispersion swordfish but much higher than the interpopulational of Pagrinae to the western Pacific Ocean occurred over values in ayu. In addition, as regards the isozyme data a very long period of time because global continental between the Australian/New Zealand snapper and the drift did not occur in this area. The genus Pagrus that Differences between P. major and P. auratus in mtDNA 17

dispersed to western Pacific Ocean divided into north 4. Akazaki M. Studies on the spariform fishes anatomy, phylogeny, and south groups through time. ecology and taxonomy. Misaki Mar. Biol. Inst. Kyoto Univ. Spec. When the division between the north Pacific popula- Rep. 1962; 1: 1–368 (in Japanese). tion and the south Pacific population of Pagrus occurred 5. Paulin CD. Pagrus auratus, a new combination for the species 2–6 million years ago, it is assumed that the present known as ‘snapper’ in Australasian waters (Pisces. Sparidae). NZ. J. Mar. Freshwater Res. 1990; 24: 259–265. Australasian populations were separated from the major 6. Taniguchi N, Fujita M, Akazaki M. Genetic divergence and sys- population. Low haplotypic diversity in Australasian tematics in sparid fish from Japan. In: Ueno T, Arai R, Taniuchi populations would then be caused by a bottleneck effect T, Matsuura K (eds). Indo-Pacific Fish Biology: Proceedings of the when these were separated from the major population. 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