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2007; 73: 1042–1049

Complete mitochondrial DNA sequence, gene organization and genetic variation of control regions in Parargyrops edita Junhong XIA,1 Kuaifei XIA,2 Jinbo GONG1 AND Shigui JIANG1* 1

South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, and 2South China Institute of Botany, Chinese Academy of Sciences, Guangzhou 510650, China ABSTRACT: The complete nucleotide sequence of the mitochondrial genome of Parargyrops edita was determined by gene amplification using long polymerase chain reaction and direct sequencing techniques. The genome with 16 640 bp contained 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes). The content and order of genes was identical to those in typical teleosts. The major non-coding region of 964 bp in length with several conserved sequence features were identified as the control region (CR). Both COI and ND4 began with a GTG start codon, which is unusual in other fishes. The genetic variation of the CR from 27 wild samples was evaluated. A total of 536 bp consensus sequence of CR was aligned and 26 unique haplotypes were identified. The haplotype diversity (h) was estimated to be 0.997 (standard deviation [SD] 0.011) and nucleotide diversity (p) was 0.014 (SD 0.008) for the sample collected from coastal waters of Guangdong Province. It showed a very high level of genetic variation in the sample and also suggested that mtDNA CR sequence analysis can be use to evaluate the genetic diversity and genetic structure of P. edita. KEY WORDS: yellowtail sea bream, Parargyrops edita, mitochondrial genome, genetic variation.

INTRODUCTION The yellowtail sea bream Parargyrops edita, a member of the family Sparidae comprising 33 genera and 110 species,1 is distributed widely in China, Korea, Japan, Vietnam, and Indonesia. Parargyrops edita is one of the most valuable and popular fish resources in China.2–5 Because of the differences in habitat environment factors and the geographic isolation caused by the Leizhou Peninsula, P. edita sampled from the Beibu Gulf were evidently morphologically divergent from those of the Taiwan Strait. Parargyrops edita also differentiated in reproductive periods, which suggested that P. edita in the Beibu Gulf belongs to a special local population in Chinese waters.5,6 Because of overfishing, the annual catch decreased after the 1980s, indicating a strong decline in the number and biomass of older fishes (spawning stock).5,7,8 Consequently, there is much interest in the genetic structure of P. edita with respect to stock identification, management, and conservation. *Corresponding author: Tel: 86-20-8419-5176. Fax: 86-20-8445-1442. Email: [email protected] Received 11 December 2006. Accepted 23 April 2007.

© 2007 Japanese Society of Fisheries Science

Mitochondrial DNA (mtDNA) has become very useful in population genetics and evolutionary studies because of its small size, its high abundance in the cell, its maternal inheritance, and its evolutionary rate.9 Although Yang and Jiang10 investigated the taxonomic status of P. edita by the random amplified polymorphic DNA (RAPD) technique, there were few mtDNA sequence data for P. edita. Until now only two complete mitochondrial DNA (mtDNA) molecules, that of the Pagrus major (GenBank AP002949)11 and Pagrus auriga (GenBank NC005146) have been represented. As an attempt to explore the population genetic structure and taxonomic status of P. edita, we determine the complete mtDNA sequence and evaluate the genetic variation of control regions (CRs) from the individuals of the P. edita in this study.

MATERIALS AND METHODS Samples and DNA extraction In total 27 specimens of P. edita were collected by fishers from the Pacific coast (Shenzhen) of doi:10.1111/j.1444-2906.2007.01434.x

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Guangdong Province, China, and tissues for DNA extraction were immediately preserved at -70°C until analysis of mtDNA. Total genomic DNA was extracted from the muscle tissue using an animal genomic DNA Kit (V-Gene, Hangzhou, China) following the manufacturer’s protocol.

Strategy of mtDNA amplification and sequencing To perform long polymerase chain reactions (PCRs), three primers (14658R, 9895R, and 7465F, Table 1) were designed using the Oligo 6 primer analysis software (Molecular Biology Insights, Cascade, CO, USA), according to its primer search protocol based on the conserved regions of the reported complete mitochondrial DNA sequences of P. major and P. auriga aligned by Clustal X soft-

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ware.12 The amplicon of the primer combination (14658R, 7465F) is approximately 7 kb. In addition, the control region (CR) and 16S rRNA gene part sequences were amplified and sequenced using the available universal primer combinations (16086F and 16621R,13 and 16S-F and 16S-R,14 respectively, Table 1). In other regions, primers designed on the basis of the above three determined mtDNA sequences were used to initiate DNA synthesis. The total genomic DNA was always used as PCR templates. Initial sequencing primers were the same as those for long PCR, and those subsequently used were designed with reference to the previously determined sequences by primer walking. Long PCR amplifications were carried out in 50-mL PCR volumes containing 200 ng genomic DNA, 1 ¥ PCR buffer, 100 mmol of each dNTPs, 0.3 mmol forward primer, 0.3 mmol reverse primer,

Table 1 PCR primers used to amplify Parargyrops edita mitochondrial gene fragments

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

PCR primers

Gene

407F 850R 2137F 16S-F 2191F 2300F 16S-R 2300W2R-F 4400F 6990R 7465F 7500R 8200F 7465WF2 7465WF3 7465WF4 9895R 12000F 12202R 12000WF1 12000WF2 13900F 14658R 14724F b286F 15149R 15000F 1019R 16086F 16000R 16621R

12S rRNA 12S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA ND1 ND2 COI COII COII ATP8 COIII ND3 ND4 ND4 ND4 tRNALeu(CUN) ND5 ND5 ND6 tRNAGl u tRNAGl u CytB CytB CytB CytB CytB + tRNAThr CR CR

Sequence (5′-3′) TGTTATACGCATCCGAAGGT TTTCCGCTTACTGCTAAATC CGCCTGCCCTGTGACTATATGTT GGTAGGGCAATCACTTCTCT GTAGCGCAATCACTTGTCTTTTA ATGAAACCTGCCCTAATGT TAGCGGCTGGACCATTAGGA TCTCCTGTGTACAATTATCT AAGTTATCCAAGGCGTTGAC AAGGAGGTGGGCAACCAT TCACAACTAGGATTTCAAGATGC GGAGACTGTTGCCACGATAATGT CCTAAAGTCATAGCCCATACT GAACATTCCAAGGCCACCACAC TCCCACAAATAAGTCCCGACTA CCGCCAAATGATTGTGACCTAC CTATGTGGTATGCGTGTGCTTGG GCCCTTCATCTTCTCCCTTTAG GACCAACGGATGAGCTGTTA ACCGCAAACAACATATTCCA TGGAAGCCTGGCCCTGACAG GCGAAATACGGAGAAGGATTAG AACAACGGTGGTTTTTCAAG GACTTGAAAAACCACCGTTG CTTCACATCGGGCGAGGACT CCTCAGAAGGATATTTGTCCTC GTAGACAACGCCACCTTAACTC ATGGATCTTCAACGGGCATTC TTAGTATGGTGACAATGCAT ATGTTCCGTCCTCAAAGATTGCAC GACACCATTAACTTATGCAA

Anchor sequences 407–426 848–867 2067–2089 2119–2138 2120–2142 2318–2336 2558–2577 3326–3345 4400–4419 6969–6986 7203–7225 7309–7331 8032–8052 8991–9012 9728–9749 10420–10441 11250–? 11679–11700 11901–11920 12373–12392 13104–13123 14236–14257 14352–14371 14350–14369 14677–14696 14795–14816 14899–14920 15410–15430 ?–15536 15918–15941 16167–16186

F, in the direction of mitochondrial replication; R, in reverse direction. Position of anchor sequence refers to P. edita mt genome position. ?, lack of anchor sequence at the end of primer. Gene identification: ND#, NADH subunit #; CO#, cytochrome oxidase subunit #; ATP#, ATPase subunit #; CytB, cytochrome B; 16S rRNA, large ribosomal subunit (lrRNA); 12S rRNA, small ribosomal subunit (srRNA); CR, control region.

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and 2.5 U of LATaq DNA polymerase (TaKaRa, Dalian, China). PCR was done on a gradient thermal cycler (Biometra, Göttingen, Germany) with the cycling profile of one denaturation step for 3 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 10 min at 72°C. The final step was a prolonged extension of 10 min at 72°C. Amplification production of 500 bp-3kb was performed in 50-mL aliquots of mixture containing 100 ng genomic DNA, 1 ¥ ExTaq PCR buffer, 200 mmol dNTPs, 0.6 mmol of each primer, and 2 U ExTaq according to the manufacturer’s instructions (TaKaRa). The cycling profile was one denaturation step for 3 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at a primer-specific annealing temperature, and 1–3 min at 72°C. The final step was a prolonged extension of 10 min at 72°C. The PCR products were finally sequenced directly using an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) after purification using a PCR clean up kit (V-gene). Sequence analyses Sequence data were assembled and analyzed using Clustal X12 and Seaview15 software. Gene contents, base compositions, and codon usages were obtained by MEGA3.116 software. Coding regions were identified using searches for open reading frames, including start and stop codons, and aligned with those of P. major and P. auriga using the NCBI BLASTX program (http://www.ncbi.nlm. nih.gov/BLAST/Blast.cgi). Transfer RNAs were identified with the help of the program tRNA-scan (http://www.genetics.wustl.edu/eddy/tRNAscanSE).17 Ribosomal RNAs were identified by alignment with the 16S and 12S rRNAs of P. major and P. auriga. The complete mtDNA sequence of P. edita has been deposited (GenBank EF107158). Users of this sequence are kindly requested to refer to the present paper and not only to the accession number of the sequence. In addition, haplotype diversity and nucleotide diversity of individual CR were evaluated using Arlequin 3.018 software.

The organization and location of the genome accorded with the pattern found in P. major and P. auriga mtDNAs (Fig. 1). Most genes were encoded on the H-strand, except for the ND6 and eight tRNA genes (Q, A, N, C, Y, S, E, and P). The total length of mtDNA in P. edita was near to that of P. auriga (16 628 bp), and 391 bp shorter than that of P. major (17 031 bp). The difference is because of the additional long fragment of the control region in P. major (Table 3). The organization of genes in mtDNA was the same in P. edita, P. auriga, and P. major.

Protein-coding genes As in other bony fishes, there were 13 proteincoding genes in the mtDNA genome of P. edita (Table 2). The pairs of genes ATP8–ATP6, ATP6– COIII, ND4L–ND4, and ND5–ND6 overlapped 10, 1, 7, and 4 bp, respectively. COI and ND4 began with a GTG start codon, similar to that of P. major,11 but others began with an ATG start codon. The phenomenon was unusual compared with other fishes, e.g. Japanese anchovy Engraulis japonicus,19 P. auriga, and Emmelichthys struhsakeri,20 in which only COI starts with GTG. Open reading frames of P. edita ended with TAA (ND1, ND2, ATP8, ATP6,

E ND6

L S

The total length of the mtDNA molecule of P. edita is 16 640 nucleotides (nt). The mitochondrial genome included the small (12S) and large (16S) subunits of ribosomal RNA genes (rRNAs), 22 tRNA, 13 usual protein-coding genes, and a CR between the tRNAPro and tRNAPhe genes (Table 2). © 2007 Japanese Society of Fisheries Science

CytB F

CR

H

srRNA

V

ND4 lrRNA ND4L R

ND3

G

L ND1

COIII ND2

COII

I Q

COI M

K DS

Genome content and base composition

T P

ND5

K

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AT P6 AT P8

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Y C N

A

W

Fig. 1 Gene organization of the Parargyrops edita mitochondrion, 16 640 bp. Arrows indicate orientation (i.e. sense and anti-sense strand) of the parent template for RNA transcripts. Gene names are given in Table 1. Twenty-two tRNA genes are shown as single-letter database codes for clarity.

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Table 2 Location of features in the Parargyrops edita mitochondrial genome Position no. Features Phe

tRNA 12S rRNA tRNAVa1 16S rRNA tRNALeu(UUR) ND1 tRNAIle tRNAGln tRNAMet ND2 tRNATrp tRNAAl a tRNAAsn tRNACys tRNATyr COI tRNASer(UCN) tRNAAsp COII tRNALys ATP8 ATP6 COIII tRNAGly ND3 tRNAArg ND4L ND4 tRNAHis tRNASer(AGY) tRNALeu(CUN) ND5 ND6 tRNAGlu CytB tRNAThr tRNAPro CR

Anticodon

From

To

Size+ (bp)

GAA

1 69 1021 1093 2789 2862 3842 3911 3981 4051 5097 5167 5237 5346 5412 5483 7037 7110 7191 7882 7957 8115 8798 9583 9655 10004 10075 10365 11746 11815 11889 11962 13797 14319 14392 15533 15607 15677

68 1020 1092 2788 2861 3836 3911 3981 4050 5097 5166 5235 5309 5411 5481 7045 7107 7182 7881 7955 8124 8798 9582 9654 10003 10074 10371 11745 11814 11882 11961 13800 14318 14387 15532 15606 15676 16640

68 952 72 1696 73 975 70 71(L) 70 1047 70 69(L) 73(L) 66(L) 70(L) 1563 71(L) 73 691 74 168 684 785 72 349 71 297 1381 69 68 73 1839 522(L) 69(L) 1141 74 70(L) 964

TAC TAA GAT TTG CAT TCA TGC GTT GCA GTA TGA GTC TTT

TCC TCG

GTG GCT TAG

TTC TGT TGG

Codon Start

Stop

ATG

TAA

ATG

TAA

GTG

AGG

ATG

T-

ATG ATG ATG

TAA TAA TA-

ATG

T-

ATG GTG

TAA T-

ATG ATG

TAG TAA

ATG

T-

Intergenic nucleotide 0 0 0 0 0 +5 -1 -1 0 -1 0 +1 +36 0 +1 -9 +2 +8 0 +1 -10 -1 0 0 0 0 -7 0 0 +6 0 -4 0 +4 0 0 0 0

L, the gene is encoded on the L-strand; ‘+’ numbers correspond to the nucleotides separating different genes. Negative numbers indicate overlapping nucleotides between adjacent genes.

ND4L, and ND6), AGG (COI), and TAG (ND5) The remainder had an incomplete stop codon T (COII, ND3, ND4, and CytB), where the TAA appeared to be created by post-transcriptional polyadenylation. The length of 13 protein-coding genes (11 442 bp) and the overall A + T base composition value for the 13 genes (54.3%) were very similar to others reported for Sparidae (Table 3). As expected, the average amino acid identity of 13 protein genes between species was larger than their average nucleotide identity. The nucleotide sequences of all genes were more similar between P. edita and P. major than between P. edita and P. auriga

(Table 3). At the second codon position, pyrimidines (68.3%) are overrepresented compared with purines (31.7%); however, a bias against G (8%) at the third codon position was noted in the mt genome of P. edita, similar to those in other teleost fishes (Table 4). Small differences of codon usage and relative synonymous codon usage in mitochondria of 13 protein-coding genes were detected among species. However, the relative synonymous codon usages of stop codons UAA(*), UAG(*), and AGG(*) in P. edita were much lower than in the other two species, but with an opposite bias in codon UGA(W) and codon UGG(W) (Table 5). The © 2007 Japanese Society of Fisheries Science

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Table 3 Comparison of Sparidae mitochondrial DNA characteristics Mitochondrial DNA

Parargyrops edita

13 protein coding genes Length (bp) A + T (%) Nucleotide identity (SD %) Amino acid identity (SD %) 12S rRNA Length (bp) A + T (%) Nucleotide identity (%) 16S rRNA Length (bp) A + T (%) Nucleotide identity (%) 22 tRNAs Length (bp) A + T (%) Nucleotide identity (SD %) Putative CR Length (bp) A + T (%) Nucleotide identity (%)

Pagrus auriga

Pagrus major

11 442 54.3 100 (0.0) 100 (0.0)

11 451 54.6 87 (2.6) 95 (2.9)

11 440 54.2 91 (1.6) 97 (2.9)

952 51.6 100

952 51.4 96

953 51.9 97

1 696 54.2 100

1 697 53.8 94

1 695 53.5 96

1 556 54 100 (0.0)

1 560 54.5 95 (2.5)

1 556 54.8 97 (2.9)

964 62.1 100

948 61.2 71

1 354 61.1 74†

† Additional long fragment of the control region in P. major was omitted in the calculation as it resulted in a much lower value than expected.

Table 4 Base composition of 13 protein-coding genes of the Parargyrops edita mitochondrial genome

First Second Third Total

T

C

A

G

21.7 40.7 26.0 29.5

27.6 27.6 34.3 29.8

24.7 18.1 31.7 24.8

25.9 13.6 8.0 15.9

differences might reflect that different organisms for which selection acts on their codon usage can have different preferred codons, as reported in Ikemura.21 Transfer RNA genes and ribosomal RNA genes Twenty-two tRNA genes with a size range 66–75 nt interspersed between the rRNA and proteincoding genes were detected in the P. edita mitochondrial genome. The pairs of genes tRNAIle– tRNAGln, tRNAGln–tRNAMet, ND2–tRNATrp, and COI–tRNASer(UCN) overlapped 1, 1, 1, and 9 bp, respectively (Table 2). Comparisons of the 22 mitochondrial tRNA genes with the others reported in Sparidae indicated similar lengths and base compositions (Table 3). The encoded tRNA can fold into the cloverleaf structure characteristic of tRNA (data not shown). © 2007 Japanese Society of Fisheries Science

The 12S and 16S rRNA genes were 952 and 1696 nt long, respectively. They were conserved either in A + T composition or sequence length, compared to other Sparidae (Table 3) and similar to those observed for other teleosts. The location of two rRNA genes were also conserved, as in other vertebrates, between the tRNAPhe and tRNALeu(UUR) genes, and were separated by the tRNAVa1 gene.

Non-coding sequences The putative CR with a length of 964 bp and overall A + T base composition value of 62.1% was the major non-coding sequence in P. edita mtDNA (Table 3). It is located between the tRNAPro and tRNAPhe genes. Several conserved sequence blocks22 CSB-D, CSB-2 and CSB-3 have been identified. The sequence features were very similar to those in P. auriga, but the length of the two species were far less than that of P. major as suggested above (Table 3). The evolution and function of the additional long fragment of the control region in P. major are currently unavailable. It appears that new Sparidae mt genomes and more genetic studies were needed to address this problem clearly. Except for the CR sequence, an additional total of 64 bp non-coding sequences were detected in the mtDNA genome of P. edita. The 36-bp fragment of origin of light strand replication, as in most

8.8 9.8 7.3 1.4 14.8 12.5 11.1 4.4 12.2 8.3 7.1 4.8 6.5 4.2 5.1 1.2 4.1 4.5 0.5 0.1 2.2 5.9 5.9 1.6 3.2 5.5 5.2 0.7 1.5 4.3 6.1 1.6

(0.95) (1.05) (0.85) (0.16) (1.72) (1.46) (1.29) (0.51) (1.32) (0.91) (0.77) (1.00) (1.54) (1.00) (1.19) (0.27) (0.95) (1.05) (0.16) (0.03) (0.53) (1.47) (1.57) (0.43) (0.73) (1.27) (1.76) (0.24) (0.53) (1.47) (1.58) (0.42)

Parargyrops edita 9.7 9.3 7.2 1.8 13.4 11.8 12.8 3.8 12.7 8.0 7.9 4.2 5.5 4.7 5.1 1.2 4.1 4.7 0.6 0.1 2.8 5.3 6.7 0.8 3.2 5.6 5.3 0.8 1.6 4.2 5.8 1.8

(1.02) (0.98) (0.85) (0.21) (1.58) (1.40) (1.51) (0.45) (1.23) (0.77) (1.31) (0.69) (1.33) (1.14) (1.23) (0.30) (0.93) (1.07) (3.20) (0.40) (0.70) (1.30) (1.78) (0.22) (0.73) (1.27) (1.75) (0.25) (0.56) (1.44) (1.54) (0.46)

Pagrus auriga 9.2 9.3 6.6 1.8 13.9 13.5 11.7 4.2 12.2 8.8 7.4 4.4 5.4 4.8 4.9 1.6 4.3 4.2 0.3 0.1 2.2 6.2 6.0 1.5 3.3 5.5 5.1 0.8 1.7 4.1 6.4 1.4

(1.00) (1.00) (0.77) (0.21) (1.62) (1.56) (1.36) (0.48) (1.16) (0.84) (1.25) (0.75) (1.29) (1.14) (1.18) (0.39) (1.01) (0.99) (2.67) (0.67) (0.52) (1.48) (1.59) (0.41) (0.75) (1.25) (1.71) (0.29) (0.59) (1.41) (1.64) (0.36)

Pagrus major UCU(S) UCC(S) UCA(S) UCG(S) CCU(P) CCC(P) CCA(P) CCG(P) ACU(T) ACC(T) ACA(T) ACG(T) GCU(A) GCC(A) GCA(A) GCG(A) UGU(C) UGC(C) UGA(W) UGG(W) CGU(R) CGC(R) CGA(R) CGG(R) AGU(S) AGC(S) AGA(*) AGG(*) GGU(G) GGC(G) GGA(G) GGG(G)

Codon 4.1 5.1 5.8 0.8 4.2 6.9 5.0 0.6 4.7 8.8 7.5 0.5 4.0 13.2 8.8 1.1 1.0 1.1 8.0 1.2 0.8 1.2 3.4 0.4 0.8 3.5 0.0 0.1 3.5 5.6 6.3 3.2

(1.23) (1.53) (1.74) (0.23) (1.00) (1.66) (1.20) (0.15) (0.87) (1.65) (1.39) (0.09) (0.59) (1.95) (1.30) (0.16) (0.96) (1.04) (2.81) (1.00) (0.86) (1.25) (3.43) (0.39) (0.23) (1.04) (0.00) (0.08) (0.74) (1.21) (1.36) (0.69)

Parargyrops edita 3.8 5.1 5.1 0.8 2.9 7.6 4.8 1.2 4.5 8.8 7.7 0.5 5.3 12.5 9.0 0.8 0.7 1.4 7.8 1.4 0.9 1.4 3.1 0.6 0.8 3.5 0.0 0.1 2.9 6.0 7.0 2.8

(1.21) (1.60) (1.60) (0.24) (0.71) (1.84) (1.17) (0.28) (0.83) (1.64) (1.43) (0.10) (0.77) (1.81) (1.30) (0.12) (0.67) (1.33) (1.70) (0.30) (0.62) (0.92) (2.05) (0.41) (0.24) (1.11) (0.00) (0.40) (0.62) (1.28) (1.49) (0.61)

Pagrus auriga

3.8 5.2 5.5 1.0 3.5 7.5 4.9 0.7 5.2 8.5 7.5 0.3 5.4 11.8 8.3 1.2 0.6 1.5 8.0 1.2 1.0 1.0 3.5 0.3 0.6 3.6 0.0 0.1 2.7 6.5 5.9 3.5

(1.17) (1.59) (1.66) (0.30) (0.84) (1.80) (1.19) (0.17) (0.97) (1.57) (1.40) (0.06) (0.81) (1.76) (1.24) (0.18) (0.57) (1.43) (1.75) (0.25) (0.68) (0.68) (2.42) (0.21) (0.19) (1.10) (0.00) (0.67) (0.58) (1.39) (1.28) (0.75)

Pagrus major

All frequencies are averages over 13 protein-coding genes (average codon number for one gene is 293). Relative synonymous codon usage is given in parentheses following the codon frequency. The codon with large difference in relative synonymous codon usages among species is indicated in italics. Vertebrate mitochondrial code table was used in calculation. *, stop codon.

UUU(F) UUC(F) UUA(L) UUG(L) CUU(L) CUC(L) CUA(L) CUG(L) AUU(I) AUC(I) AUA(M) AUG(M) GUU(V) GUC(V) GUA(V) GUG(V) UAU(Y) UAC(Y) UAA(*) UAG(*) CAU(H) CAC(H) CAA(Q) CAG(Q) AAU(N) AAC(N) AAA(K) AAG(K) GAU(D) GAC(D) GAA(E) GAG(E)

Codon

Table 5 Codon usages of mitochondria 13 protein-coding genes

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Fig. 2 Stem–loop secondary structure in origin of L strand replication.

vertebrates, is located in a cluster of five tRNA genes (WANCY region) between the tRNAAsn and tRNACys. As expected, this region has the potential to fold into a stable stem-loop secondary structure that is generally characteristic of the origin of light strand replication with 13 bp in the stem and 12 bp in the loop (Fig. 2). The left eight non-coding regions were located between the protein-coding gene and tRNA gene, and between tRNA genes with a range of 1–8 bp, respectively (Table 2). Genetic variation in mtDNA CR of Parargyrops edita The CR of P. edita can be amplified by the universal primer combination 16621R and 16086F. The genetic variation of the CR from 27 wild samples of P. edita was evaluated. In total 536 bp consensus sequences of CR were aligned and 26 unique haplotypes were defined. The haplotype diversity (h) estimated to be 0.997 (standard deviation [SD] 0.011) and nucleotide diversity (p) was 0.014 (SD 0.008) for the sample. It showed a very high level of genetic variation in the population compared to the genetic diversities of other Sparidae fishes (e.g. Diplodus puntazzo (h = 0.988, P = 0.041) and Diplodus sargus (h = 0.700, P = 0.008).23 This study suggested that mtDNA CR sequence analysis can be use to evaluate the genetic diversity and genetic structure of P. edita populations and the complete mtDNA sequence data can provide important information for phylogenetic studies in sparid species. ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (No. 30500376) and talent foundation of the Chinese Academy of Fishery Sciences. REFERENCES 1. Orrell TM, Carpenter KE. A phylogeny of the fish family Sparidae (porgies) inferred from mitochondrial sequence data. Mol. Phylogenet. Evol. 2004; 32: 425–434.

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