16S RNA. Phylogenetic relationships of Hynobiidae based on sequences of mitochondrial 16S ribo2 somal RNA gene 3

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1 50 (3) : , 2004 A cta Zoologica S inica 16S r RNA 3 1, , , , Phylogenetic relationships of Hynobiidae based on sequences of mitochondrial 16S ribo2 somal RNA gene 3 L I Yue 1,2, WU Min 13 3, WAN G Xiu2Ling 3 11College of Life Science, Zhejiang University, Hangzhou , China 21 The Key Laboratory of Conservation Genetics and Reproductive Biology for Endangered Wild Animals, Ministry of Education, Hangzhou , China 31Department of Biology, Xinjiang Normal University, Urumqi , China Abstract To study the phylogeny of Hynobiidae, we amplified DNA fragments of 470 bp 16S ribosomal RNA (16S rrna) gene on mitochondrial DNA from Ranodon sibiricus and Ranodon tsinpaensis. PCR products were cloned into PMD18 T vector after purification. These sequences were determined and deposited in the GenBank (accession numbers : A Y for Ranodon sibiricus, A Y for Ranodon tsinpaensis). By comparing the nucleotide differences of 16S ribosomal RNA sequences among L iua shihi, Pseudohynobius f lavom aculatus and B at rachuperus genus from GenBank database, we analyzed the divergences and base substitution among these sequences with the MEGA software. The molecular results support that B1 tibetanus, B1 pinchonii and B1 karlschmidti are classified into three valid species. L iua shihi has closer phylogenetic relationships to Ranodon tsinpaensis than to other species. More our results reveal that Pseudohynobius f lavom aculatus is not a synonym of Ranodon tsinpaensis. [ Acta Zoologica Sinica 50 (3) : , 2004 ]. Key words Hynobiidae, 16S ribosomal RNA, Molecular phylogeny 16 S RNA (2000), 1994 ;, 2001 ;, 1999 ; ( Hynobiidae) 7, ( B at rachuperus ), ( Hynobius ) ( L iua ) ( O ny2, 1995 ;, 1990) mtdna chodactyl us ) ( Pachyhyuabius ) ( Ranodon ) ( S alam andrella ) ( > 350 ) (Brown, 1983 ; Avise, 1994 ;,,, 2003 ;, 2002 ;, 2002 ;, 2002 ;, 2002 ;, ( Pseudohynobius ) (, 2001) 16S RNA 1983 ;, 1993 ;, 2000 ; ( Hay et , (Corresponding author). ν 2004 Acta Zoologica Sinica E2mail :

2 3 : 16S rrna 465 al., 1995) 16S rrna,, 10 Pharmacia Megabase 1000, 115, DNASTAR ( DNASTAR Inc., ( Ranodon sibi ricus),, ; 3 ( B. londongensis, A Y028719) ( R. tsinpaensis),, ( B. tibetanus, A Y ) ( B. yenyuanensis, A Y028734) ( B. ( L iua shihi, GenBank A Y ) ( Pseudohynobius f lavom aculat us, A Y028745) ( B at rachuperus pinchonii, A Y ) ( B. karlschmidti, A Y028726) ( B. londongensis, A Y ) ( B. tibetanus, A Y ) ( B. yeny uanensis, A Y ) ( B. taibaiensis, A Y028730) 16S rdna GenBank 112 D NA 50 mg ( Sam2 anus), brook et al., 1989), (Bootstrap) 1 000, DNA 113 PCR GenBank, Primer Premier 510 : S 1, 5 2CT GT T TACCAAAAA2 CA TCA TCGC23 ; S2, 5 2CC GGTCT GAACTCA2 GA TCACG23 50 l, 012 mmol/ L dn TPs, 015 mol/ L, 10 mmol/ L Tris2HCl (p H 810), 115 mmol/ L MgCl 2, 115 U Taq PUL S, 100 ng DNA 470 bp 95 5 min ; s, , 38 T C A G 60 S, S, 35 ; min PCR PTC % (, 5 l / 22/ 13) 110 % PCR PCR, , (Sangon) UNIQ10 PCR (UNIQ 10 column PCR Products Purification Kit) 1996) MegAlign GenBank ( L iua shihi A Y028746) ( P. f lavom aculat us, A Y028745) ( B. pinchonii, A Y028724) ( B. karlschmidti, A Y028726) taibaiensis, A Y028730),, MEGA211 ( Kumar et al., 2001) / ; MEGA211 Kimura 22parameter (Neighbor Joining, NJ), PHYL IP316 ( Felsenstein, 2001) Heuristic ( Maximum Parsi2 mony, MP) ( Maximum Likehood, ML) ( A ndrias davidi S r RNA 16S rrna GenBank, ( A Y ) ( A Y ) GenBank 2715 % 1715 % 3715 % , S rrna PCR PMD182T ( ), 01085, /

3 / 2-7/ 2, / 210 / ( Knight and Mindell, 1993), : 16/ 8 20/ 7 24/ 15 / 13/ 8-23/ 14, 1 / ( ) ( Kimura 22parameter,, ) Table 1 Numbers of transitions/ transversions ( lower2left matrix) and Genetic distances ( Kimura 22parameter model, transi2 tion + transversion, upper2right matrix) for different species 11 L i ua shihi 21 Ranodon tsipaensis 31 Ranodon sibiricus 41 Pseudchynobius f lavomaculat us 51 Bat rachuperus tibetanus 61 B1 pi nchonii 71 B1 karlschmi dti 81 B1 longdongensis 91 B1 taibaiensis 101 B1 yenyuanensis 111 A ndria davidianus / / 7 24/ / 9 29/ 15 19/ / 8 21/ 16 13/ 11 13/ / 6 22/ 14 14/ 9 14/ 9 1/ / 6 23/ 14 15/ 9 13/ 9 3/ 2 4/ / 8 20/ 16 13/ 9 14/ 11 4/ 4 5/ 2 7/ / 8 20/ 16 12/ 11 13/ 11 3/ 4 4/ 2 6/ 2 5/ / 5 20/ 13 13/ 8 13/ 9 2/ 2 3/ 0 5/ 0 4/ 2 3/ / 19 43/ 25 35/ 17 41/ 17 42/ 21 43/ 19 42/ 19 41/ 18 42/ 19 42/ S rrna Fu et NJ MP ML ( 1) al Song et al. (2001) ( B. Tibetanus), ( B. Pinchonii), ( B. ( B. taibaiensis ), 16S Karlschmidti) B. Pinchonii rrna, NJ (, 1986 ) 16S MP ML rrna 312, ( 1994) MP, 3, (2001) 90 % NJ 012 % %,, 61 %, 411 %, 70 % ML 610 % 715 %, 413 % -, 617 % 16S rrna 61 %,,, < 210 %, 50 % Fu et al. (2001) > 410 %,

4 3 : 16S rrna s rrna, 50 A1 NJ B1 MP C1 ML Fig11 Phylogenetic trees of Hynobiidae species based on 16s rrna sequences data Numbers above branches are percentage bootstrap supporting values (1 000 replications). A. NJ tree. B. MP tree. C. ML tree. Values less tha 50 are not shown., b ( ) (1994) (2001),, 16S rrna,, 518 % 815 % 518 %, 313,

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