A molecular phylogeny of Bharal and dwarf blue sheep based on mitochondrial cytochrome b gene sequences 3

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1 49 (2) :198204, 2003 A cta Zoologica S inica b ) (, (, ) ( Pseudois nayaur) ( Pseudois schaef eri) 7 Cyt b (1 140 bp), ( Capra hircus) ( Capra ibex) ( Ovis aries) ( Ovis am mon) DNA,, :,, (Cyt b = 312 %) (813 %1211 %),,, 1 3 [ 49 (2) : , 2003 ] b A molecular phylogeny of Bharal and dwarf blue sheep based on mitochondrial cytochrome b gene sequences 3 CAO Li2Rong WAN G Xiao2Ming 33 FAN G Sheng2Guo ( School of L if e Sciences, East Chi na Normal U niversity, S hanghai , Chi na) ( School of L if e Sciences, Zhejiang U niversity, Hangz hou , Chi na) Abstract The phylogenetic relationship between the bharal or blue sheep ( Pseudois nayaur) and dwarf blue sheep ( Pseudois nayaur schaef eri) has been an open question. Total DNA was extracted and sequenced from three dried skin samples of bharal and four dwarf blue sheep from Sichuan and Qinghai provinces in China. The complete sequences were deposited in the GenBank (accession numbers : AF AF473608). Based on the complete Cyt b gene sequences (1 140 bp) obtained from these samples, the authors aligned them using the Clustal W software program. By comparing the Cyt b sequences of four other species Capra hircus, Capra ibex, Ovis am mon, Ovis aries in the family Bovidae, the divergences and base substitutions among these sequences were analyzed with the MEGA software. Accordingly, the base composition of bharal and dwarf blue sheep showed the same characters as the other bovidae members. The phylogenetic trees constructed by multiple methods (NJ and MP) supported the same topology, in which the monophyly of bharal and dwarf blue sheep was obviously demonstrated and the phylogenetic trees were in agreement with the traditional morpho2 logical data. Our molecular results show that the sequence divergence between bharal and dwarf blue sheep is lower than that between other species in the family Bovidae. However, the differences surpassed the corresponding number within in2 traspecific individuals and were in the range of sequence variation that typically distinguishes subspecies. Together with these data, the authors propose that the dwarf blue sheep ( Pseudois nayaur schaef eri) should be a subspecies of the bharal , (No ) [ This research was funded by the National Natural Science Foundation of China (No ), The Ministry of Education Foundation for Key University Teachers, TheShuGuangProject of Shanghai and Shanghai Priority Academy Discipline of Ecology] 33 (Corresponding author) E2mail : sta. net. cn,, : ν 2003 Acta Zoologica Sinica

2 2 : b 199 ( Pseudois nayaur). In addition, the divergence times between clades was discussed in accordance to the Cyt b molecular clock, which estimated an approximate divergence time between the bharal and dwarf blue sheep of 1-3 million years [ Acta Zoologica Sinica 49 (2) : , 2003 ]. Key words Bharal ( Pseudois nayaur), Dwarf blue sheep ( Pseudois nayaur schaef eri), Cyt b, Molecular phylogeny, L GA TA TGAAAAACCA TCGTTG23 ( Wang H CCTTCTCTGGTTTACAA GAC23 et al., 2000) ; Allen (1939) PCR P TC2200 Peltier Thermal cycler, ; Englemann (MJ Research, Inc. USA) (1938) Haltenorth (1963) 50 l : 95 5 min, 94 ; Groves (1978), 1 min, 42 1 min, min, 40 ; min, ( P. DNA, schaef eri), Feng et al. (2001) PCR 1 %, DNA Y ZF Y - 20, 113 b ( Sangon) UN IQ2 ( Vladimir et 10 PCR ( UN IQ210 column al., 1999 ;, 2002), PCR Products Purification Kit) ( Cibele et al., 2001 ;, 2001 ;, 1999) (Amersham Pharmacia Biotech Inc., USA),, M EGABACE 1000 sequence Analyzer mtdna b, (Amersham Pharmacia Biotech Inc. USA) b ),,, D NA, DNASTAR (DNASTAR Inc.,, ) MEGAL IGN D NA PCR DNA ( Capra ibex) ( GeneBank : AF034735) (2001) PCR ( Ovis aries) ( GeneBank : AF034730) Kocher et al. ( 1989) Irwin et al. (1991) b AF034727) MEGA L14724 H15915, H15915, ( Kumar et al., 1993), : PCR,, D YEnamic ET Dye Terminator Cycle Sequencing Kit DNA Cyt b,,, Cyt b PCR pmd182t Vector ( GeneBank ( Capra hi r2 cus) ( GeneBank : X56289 ) ( Ovis am mon ) ( GeneBank : / Kimura (1980) (22parame2

3 ter) ; DNASTAR b, ; ( 5141 ) D = 2 t (Li et al., 1981) Cyt b (01474 %) (Matthee et al., 1999), ( 1317 ) 0123 % 0101/ (1119 %) (2), ; MEGA 3 Kimura Kimura 22parameter NJ (3),, PHYL IP316 ( Felsenstein, 2001) (MP), Kimura ( Bos taurs) ( GeneBank : V00654) , ,, (Bootstrap) b DNA GeneBank, AF AF AF473608, bp Cyt b AF AF AF AF473605, 47 Kimura (22parame2, 34 ter) (NJ) (1 : A) 33, 72 % (MP) (1 : B), Cyt b, A TCG (Bos taurs) 3113 %2511 %2919 %1316 %, (Bootstrap) G ( 1), T : 3 < > C A < > G, A,,, < > C A < > T T < > G (BCL ) : BCL = 100 % C < > G (1A), BCL = 97 % (1B) ; 2 ( / 2819/ 5141),, (BCL 96 %) ; 3 (Bias) , ;, GeneBank, 305, 171 (BCL 97 %) 1 ( %) Table 1 Frequencies of base composition in Bharal and dwarf blue sheep A T C G A1 T1 C1 G1 A2 T2 C2 G2 A3 T3 C3 G3 Ai Ai Ai Ai Ai Ai Ai Ai Si Si Si Si Qh Qh b, ( Frequencies are given by codon position for cytochrome b. No. 1, 2, 3 denote the 1st, 2nd and 3 rd codon positions) Ai2, Ai3, Ai5, Ai6, Si3, Si5, Qh (Ai2, Ai3, Ai5, Ai6 denote four dwarf blue sheep samples ; Si3, Si5 denote two blue sheep samples from Sichuan Province ; Qh denotes a blue sheep sample from Qinghai Province)

4 2 : b / ( ) ( ) Table 2 Numbers of transitions/ transversions ( above diagonal) and percent difference of base transversions ( below diagonal) between different species Pseudois nayaur Capra ibex Capra hircus Ovis am mon Ovis aries Bos taurus ( Pseudois nayaur) ( Capra ibex) ( Capra hircus) ( Ovis am mon) ( Ovis aries) ( Bos taurus) 9716/ / / / / / 5 106/ / / / / / / 1 126/ / / 2819/ 5141, (Numbers of transitions/ transversions and percent difference of base transversion between Bharal and dwarf blue sheep were 2819/ 5141, , respectively) 3 Cyt b ( ) ( ) Table 3 Kimura 22parameter distance ( above diagonal) and percent difference ( below diagonal) for cytochrome b nucleotide sequences of animals examined in this study OTUs Ai2 Ai5 Ai6 Ai3 Si3 Si5 Qh ibex goat argali sheep cow Ai Ai Ai Ai Si Si Qh ibex goat argali sheep cow ibex, goat, argali, sheep, cow [ibex, goat, argali, sheep, cow denote ibex ( Capra ibex), goat ( Capra hi rcus), sheep ( Ovis aries), argali ( Ovis am mon), cow ( Bos taurus) Ai3, Ai5, Ai6, Si2, Si3 and Qh are noted in Table 1) 3 respectively ] Ai2 Ai3 Ai5 Ai6 Si2 Si3 Qh 1 (Ai2, 311 Cyt b 5134 (2), mtdna 210 ( Knight et al., 1993),,,, ( Hedges et al., 1991 ; Zhang et al., 1993),, Cyt b,

5 Cyt b Fig. 1 Molecular phylogenetic trees of Bharal and dwarf blue sheep based on cytochrome b sequences data with Bootstrap Test ( replications) (BCL), Ai2 Ai3 Ai5 Ai6 Si2 Si3 Qh 1 [ The values of bootstrap confidence level (BCL) of the nodes are indicated above the branch. Ai2, Ai3, Ai5, Ai6, Si2, Si3 and Qh are noted in Table 1 ], ( Kim et al., 1993), 312 (1 : A, B) Cyt b, (BCL ), 50 %, b 70 %, ( Kim2 ( Irwin et al., 1991) ball et al., 1999) (1),,,, (2) Kimura %1213 % ( ( ) Kimura 3), ( ) 813 %1211 % ( Marc et al., 1992) 613 % Kimura % (Schreliber et al., 1999) ( ) ; ( 218 %318 % ( 312 %) ) 014 %110 % ( ;, 017 %) 012 %017 % 218 %318 %, 312 % (3), ( 015 %) (3), (Cyt b = 312 %) 813 %1211 % ( Marc et al., 1992),, Schreliber et al. (1999) ( Groves, ( B ubal us depressicornis) 1978) (017 %111 %),, (Masuda et al., 1994 ;, Wayne et al., 1991 ; Nicole et al., 2001),, ( S ylvicapra. gri m mia) ( Felsenstein et al., 1988) ( Cyt b = 218 %) ( Cephalophus,

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