28 6 ( Mollusca) COI 16S rrna ( Bivalvia) ( Anisomyaria), ( Mytilidae),, 1, [ 1 ] 1. 1, , Mytilus edulis ( M. gallprovincialis) ( M. trossulus

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1 South China Fisheries Science Vol. 6, No. 5 Oct., 2010 doi: / j. issn COI 16S rrna ,,, ( 1., ; 2., ; 3., ) : 5 DNA COI 16S rrna, ( Perna viridis) ( Septifer virgatus), ( NJ) ( MP) ( ML) ( BI), 5 [ Mytilus edulis, ( M. galloprovincialis), ( M. trossulus), ( M. coruscus), ( M. californianus) ] 2,, M. edulis, : ; I; 16S rrna; ; ; : Q 244; Q : A : ( 2010) Molecular phylogenetics of genus Mytilus based on COI and 16S rrna sequences MAO Yangli 1, CAI Houcai 2, LI Chengjiu 3, GAO Tianxiang 1 ( 1. College of Fisheries, Ocean University of China, Qingdao , China; 2. Nanji Islands National Marine Nature Reserve Administration, Pingyang , China; 3. Fisheries Seed Bureau of Liaoning, Dalian , China) Abstract: In the present study, we compare the partial sequences of mitochondrial COI and 16S rrna of five genus Mytilus to study their phylogenetic relationship. Taking Perna viridis and Septifer virgatus as outgroups, phylogenetic trees are constructed based on neighbor-joining, maximum parsimony, maximum likelihood, Bayesian inference and combined Bayesian analyses methods. The result demonstrates that Mytilus edulis, M. galloprovincialis, M. trossulus, M. coruscus and M. californianus form into two clades. Among the five species of genus Mytilus, M. californianus is the most primitive, then comes M. coruscus. M. galloprovincialis and M. edulis show very close genetic relationship, and M. coruscus is closer to M. californianus than to M. edulis, M. galloprovincialis and M. trossulus. The molecular phylogeny analysis provides theoretical reference for future comparative study on species evolution, immigration and breeding strategies of Mytilus. Key words: genus Mytilus; cytochrome oxidase I; 16S rrna; phylogeny; combined sequences analyses; divergent time : ; : : ( ) : ( ),,, com :, gaozhang@ ouc. edu. cn

2 28 6 ( Mollusca) COI 16S rrna ( Bivalvia) ( Anisomyaria), ( Mytilidae),, 1, [ 1 ] 1. 1, , Mytilus edulis ( M. gallprovincialis) ( M. trossulus) ( M. californianus), 3 ( M. coruscus) M. edulis , 95%,, [ ( Perna viridis), Bank ( 1) [ 2] TO [ 3] [ 12 ], - SHEN [ 4] DNA DNA, COI 2 2, PCR, COI COI-F ( 5 -GGTCAACAAAT,, GGTGACCAAAAAATCA-3 ) [ 13 ],, COI 16S rrna, SCARLAT DNA 100 mg ( Septifer virgatus) ] Gen- CATAAAGATATTGG) COI-R ( 5 -TAAACTTCAG- 16S rrna-ra: 5 -CGCCTGTTTAT- CAAAAACAT-3 ; 16S rrna-rb: 5 -CCGGTCT- DNA ( mtdna) GAACTCAGATCACGT-3 [ 14 ] PCR DNA, 50 L, 10 PCR [ 200 mmol L - 1 Tris-HCl, ph 8. 4; 200 mmol L - 1, KCl; 100 mmol L - 1 ( NH 4 ) 2 SO 4 ; 15 mmol L - 1 [ 5] MgCl 2 ] 5 L, dntp 200 mol L - 1, I ( cytochrome oxidase subunit I, COI) 0. 2 mol L - 1, Taq U (, COI ), DNA 20 ng, Milli-Q H 2 O 50 L [ 6], Eppendorf PCR, COI [ 7] [ 8 ] s, s, 72 [ 9] 16S rrna 60 s, ,, min, 72 7 min 16S rrna [ 10 ], 16S s, s, rrna, s, min, [ 11 ] min 10, ; DNA 100 L TE

3 5 : COI 16S rrna 29 1 GenBank Tab. 1 Sample species, sampling sites and dates, and their molecular markers and GenBank accession No. species sampling site sampling date 16S rrna abbreviation No. source COI abbreviation No. source M. galloprovincialis Qingdao M. galloprovincialis1 M. galloprovincialis2 M. galloprovincialis1 M. galloprovincialis Dalian M. galloprovincialis3 M. galloprovincialis5 M. galloprovincialis3 M. galloprovincialis5 - - M. galloprovincialis6 AY M. galloprovincialis6 AY M. coruscus Wenzhou M. coruscus1 M. coruscus2 M. coruscus1 M. coruscus Fuding M. coruscus3 M. coruscus5 M. coruscus3 M. coruscus5 - - M. coruscus6 M. coruscus10 GQ GQ472149, AF M. coruscus6 M. coruscus10 FJ495279, FJ495282, FJ FJ M. edulis - - M. edulis1 M. edulis2 AF317054, AY M. edulis1 M. edulis2 AY484747, NC_ M. trossulus - - M. trossulus1 M. trossulus3 MTU22879 MTU22881 M. trossulus1 M. trossulus2 AF242033, AF M. californianus - - M. californianus AF317057, U68770 M. californianus MCU68776 P. viridis S. virgatus - - P. viridis AB P. viridis DQ S. virgatus GQ S. virgatus AB DNA, MEGA 2. 0 [ 15 ] 2 L PCR 1% ( Ts) ( Tv), ( U = 5 V cm - 1 ) UNIQ- 10 DNA ( ) PCR ABI 3700 Ts/Tv ( parsim-informative site) PAUP * 4. 0b10 [ 16 ] COI ( chi- square test, 2 ) PAUP * 4. 0b10 Modeltest DNA, PCR 3. 7 [ 1 7] COI 16S rrna PAUP * 4. 0b10 MrBayes 3. 0 [ 18 ] 1. 3 DNAStar ( DNASTAR, Inc ), ( NJ) ( MP) SeqMan, COI ( ML) ( Bayesian Inference, BI) 3 16S rrna EditSeq, PAUP * 4. 0b10, K2P 1 COI ;, 4 ; MegAlign,, 3 1 ; 10

4 , S rrna MrModeltest 2. 0 [ 2 0], ( ), PAUP * 4. 0b10 2 4, ( 2), P, ( partition homogeneity test) [ 19 ] COI 1 2 3,, 2 Tab. 2 Partitioning strategies used in this study partitioning strategies partitioning method P 1 P 2 P 3 1 COI; 16S rrna COI 1 2 ; COI 3 ; 16S rrna P 4 COI 3 ; 16S rrna DNA 5 COI 432 bp COI 16S rrna bp 121, , M. edulis ; A T C G 26. 4% 35. 0% 16. 8% 21. 7%, A + T 61. 4%, AT / 2. 79, TC,, TA CG 3, COI 2 : 1 = , df = 63, P 2 = ; 2 = , df = 63, P = ; 3 = , df =63, P = COI S rrna 460 bp 460 bp 68, 56 ; A T C G 29. 6% 30. 6% 15. 8% 24. 0%, A + T 60. 2%, AT / 2. 53,, K TC AG,, 2 COI TA, CG COI 16S rrna M. edulis,, ( COI ) ( 16S rrna) ( COI) ( 16S rrna),, ( synon- ymous substitution), 3,, COI 143, M. edulis 1 ( amino acid substitution) R = K/2T, K, T 2, > 16S rrna, COI ( ) > 16S rrna ( ) COI COI 16S rrna 16S rrna ( 3)

5 5 : COI 16S rrna 31 3 K2-P ( : 16S rrna, : COI) Tab. 3 Average K2-P genetic distance based on 16S rrna region ( below diagonal) and COI region ( above diagonal) among different species of genus Mytilus species M. galloprovincialis M. coruscus M. edulis M. trossulus M. californianus M. galloprovincialis M. coruscus M. edulis M. trossulus M. californianus COI MP ML BI (, ) Fig. 1 Phylogenetic tree of Mytilus species constructed with neighbor-joining, maximum parsimony, maximum likelihood and Bayes analyses based on COI ( numbers on the tree represent posterior probability values and supporting values; the same case in the following figure) ( 350 ) ( Late Miocene) ( Early Pliocene) M. edulis ( Late Pleistocene) [ 21], COI 16S rrna 2. 3, M. edulis COI 16S rrna, 427 COI 16S rrna, NJ MP ML 753, BI ( 1 2), 63. 6,

6 S rrna MP ML BI Fig. 2 Phylogenetic tree of Mytilus species constructed with neighbor-joining, maximum parsimony, maximum likelihood and Bayes analyses based on 16S rrna 5 2 ( A B), A M. edulis, ; B 1, M. edulis 1, M. edulis 1 M. edulis COI 16S rrna,, 12S rrna [ 2 2] COI 16S rrna 2 16S rrna,,, COI, PAUP COI 16S rrna 2 ( partition 460 bp 16S rrna, 5 homogeneity test) [ 19 ], 2 [ 2 3],, 68, 432 bp COI P 0. 07, 2, ,, 5 COI 4 16S rrna COI 16S rrna, P 3, COI ( 3) 16S rrna

7 5 : COI 16S rrna 33 4 Tab. 4 Partitions, optimal model of sequence evolution and total number of characters in each partition partition evolutionary model number of characters in partition COI GTR + G 432 COI 1 HKY + G 144 COI 2 HKY 144 COI 3 HKY + G 144 COI 1 2 HKY + G S rrna GTR + G 469 GTR + I + G 901 Fig. 3 3 COI 16S rrna ( P 3 ) 50% ( 50% ) 50% consensus tree of Mytilus species constructed with Bayesian analysis ( strategy P 3 ) based on COI and 16S rrna sequences. Bayesian posterior probabilities are indicated above the branch ( bootstrap scores larger than 50% were shown) 2 AT DNA A + T, 61. 4% 60. 2%, G + C, 16S rrna [ 24-26] 12S rrna COI, [ 2 7] [ 2 8],

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