50 (3) :464-469, 2004 A cta Zoologica S inica 16S r RNA 3 1,2 13 3 3 11, 310029 21, 310029 31, 830054 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 310029, China 21 The Key Laboratory of Conservation Genetics and Reproductive Biology for Endangered Wild Animals, Ministry of Education, Hangzhou 310029, China 31Department of Biology, Xinjiang Normal University, Urumqi 830054, 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 Y373459 for Ranodon sibiricus, A Y372534 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) : 464-469, 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 2003211224, 2004202226 33 (Corresponding author). ν 2004 Acta Zoologica Sinica E2mail : wumin @cls1zju1edu1cn
3 : 16S rrna 465 al., 1995) 16S rrna,, 10 Pharmacia Megabase 1000, 115, DNASTAR ( DNASTAR Inc., 1 111 2 ( Ranodon sibi ricus),, ; 3 ( B. londongensis, A Y028719) ( R. tsinpaensis),, ( B. tibetanus, A Y028743 ) ( B. yenyuanensis, A Y028734) ( B. ( L iua shihi, GenBank A Y028746 ) ( Pseudohynobius f lavom aculat us, A Y028745) ( B at rachuperus pinchonii, A Y028724 ) ( B. karlschmidti, A Y028726) ( B. londongensis, A Y028719 ) ( B. tibetanus, A Y028743 ) ( B. yeny uanensis, A Y028734 ) ( 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 ; 94 60 s, 50 144, 38 T C A G 60 S, 72 60 S, 35 ; 72 10 min PCR PTC2200 1715 % (, 5 l / 22/ 13) 110 % 1 114 PCR PCR, 01002-01020, (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 davidi2 2 211 16S r RNA 16S rrna GenBank, ( A Y373459 ) ( A Y372534 ) GenBank 2715 % 1715 % 3715 % 01256-01295, 01043-01067 16S rrna PCR 01041 - PMD182T ( ), 01085, /
66 4 50 1/ 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 1 2 3 4 5 6 7 8 9 10 11 01041 01060 01058 01058 01053 01058 01048 01055 01055 01289 16/ 8 01075 01085 01067 01062 01067 01067 01067 01065 01295 20/ 7 24/ 15 01058 01048 01043 01048 01046 01048 01046 01256 19/ 9 29/ 15 19/ 10 01050 01046 01046 01053 01050 01048 01272 18/ 8 21/ 16 13/ 11 13/ 11 01004 01011 01016 01013 01007 01285 19/ 6 22/ 14 14/ 9 14/ 9 1/ 2 01007 01011 01009 01002 01285 20/ 6 23/ 14 15/ 9 13/ 9 3/ 2 4/ 0 01018 01016 01009 01288 14/ 8 20/ 16 13/ 9 14/ 11 4/ 4 5/ 2 7/ 2 01020 01013 01279 17/ 8 20/ 16 12/ 11 13/ 11 3/ 4 4/ 2 6/ 2 5/ 4 01011 01291 19/ 5 20/ 13 13/ 8 13/ 9 2/ 2 3/ 0 5/ 0 4/ 2 3/ 2 01288 42/ 19 43/ 25 35/ 17 41/ 17 42/ 21 43/ 19 42/ 19 41/ 18 42/ 19 42/ 18 212 10 16S rrna Fu et NJ MP ML ( 1) al. 3 3. 1 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 % - 210 %,, 61 %, 411 %, 70 % ML 610 % 715 %, 413 % -, 617 % 16S rrna 61 %,,, < 210 %, 50 % Fu et al. (2001) > 410 %,
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3 : 16S rrna 469 2 (4) : 31-37.,,,, 20021, 34-41.. 48 (3) : 333-3381,,, 1993. L i ua (2) : 55. 1 1 2, :, 1 -, 2001. 1 20 2 : 53-57.,,,, 20021,,,, 2002. mtdna mtdna. 48 (4) : 487-493.,, 20011 12S rrna 24, 1990.. : 1 47 (1) : 38-441,,,,, 2002. 10 DNA 1 48 (5) : 625-6321,,,,, 20031,, 19951. mtdna. 49 1 14 (1) : 20-23. (1) : 53-60.,, 1986. 1 :, 1998.. 17 (4) : 177-180. 1 48 (1) : 131-1341.. :, 150-158., 19941 1 13 (4) : 162-165.,,,, 2000.. 19 (3) : 196-2071