The Complete Mitochondrial Genome of the Chinese Giant Salamander, Andrias Davidianus (Amphibia: Caudata)

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Gene 311 (2003) 93–98 www.elsevier.com/locate/gene

The complete mitochondrial genome of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata)

Peng Zhang, Yue-Qin Chen*, Yi-Fei Liu, Hui Zhou, Liang-Hu Qu

Key Laboratory of Gene Engineering of the Ministry of Education, Biotechnology Research Center, Zhongshan University, Guangzhou 510275, P.R. China

Received 15 October 2002; received in revised form 7 February 2003; accepted 25 March 2003 Received by M. Schartl

Abstract The mitochondrial genome of the Chinese giant salamander Andrias davidianus was isolated using the long-and-accurate polymerase chain reaction (LA PCR) method. The sequencing work adopted the shotgun strategy accompanying with seven internal primers to cover the gaps where overlapping clones were not available. The entire mtDNA sequence is 16,503 bp long, with a gene content of 13 protein-coding, two ribosomal RNA and 22 transfer RNA genes, and order identical to that observed in most other vertebrates except for an additional 318 bp non-coding sequence between tRNA-Thr and tRNA-Pro genes. In order to carry out molecular phylogenetic analyses, all 13 protein sequences deduced from whole mitochondrial genomes for eight vertebrate species (six amphibians, two lobe-finned fishes) were combined to a single data set. This data set was refined by the program Gblocks using a stringent parameter setting and then subjected to MP, ML and NJ analyses. Thus phylogenetic relationships among living amphibians were discussed. q 2003 Elsevier Science B.V. All rights reserved.

Keywords: Mitochondrial genome; Long-and-accurate polymerase chain reaction; Non-coding region; Phylogeny

1. Introduction evolutionary relationships in phylogenetic analyses. To date, the complete mitochondrial genomes reported in In recent years, mitochondrial DNA (mtDNA) has been vertebrates consist of about 70 species of mammals, 32 widely used as a useful marker system in numerous species of birds and reptiles, and 116 species of ﬁshes. phylogenetic analyses of vertebrate relationships because However, as an important group of vertebrates, the of its maternal mode of inheritance and relative lack of amphibian lineage does not get enough attention. Only recombination (Saccone et al., 1999; Arnason et al., 2002). ﬁve complete mitochondrial genome sequences from Moreover, phylogenetic analyses among amphibians based amphibians are known (Xenopus laevis, Roe et al., 1985; on mitochondrial data have proven to be successful (Hay Typhlonectes natans and Mertensiella luschani, Zardoya et al., 1995). However, individual mitochondrial genes may and Meyer, 2000; Zardoya and Meyer, 2001; Rana nigromaculata Ranodon sibiricus show a poor performance in recovering the phylogenetic , Sumida et al., 2001; , Zhang et al., 2003). relationships among divergent vertebrate lineages (Zardoya The Chinese giant salamander, Andrias davidianus, and Meyer, 1996a). Hence, the complete mitochondrial belonging to the family Cryptobranchidae, is the biggest genome is expected to provide more reliable estimations of amphibian species in the world. This family has a combined Asian/North American distribution but contains only three Abbreviations: ATP6, ATPase subunit 6; ATP8, ATPase subunit 8; COI-III, cytochrome c oxidase subunit I-III; CSB, conserved sequence species (Andrias davidianus in China, Andrias japonicus in block; Cytb, cytochrome b; D-loop, displacement loop; H, strand, heavy Japan, Cryptobranchus alleganiensis in North Americ). In strand; L, strand, light strand; LA-PCR, long-and-accurate polymerase the past half century, numerous studies based on morpho- chain reaction; NADH1-4L, NADH dehydrogenase subunit 1-4L; PCR, logical characteristics have considered this family as a polymerase chain reaction; tRNA, transfer ribonucleic acid. * Corresponding author. Tel.: þ86-20-8411-2399; fax: þ86-20-8403- primitive group within the Caudata (Regal, 1966; Edwards, 6551. 1976). This conclusion depends mainly on three primitive E-mail address: [email protected] (Y.Q. Chen). characteristics (external fertilization; non fusion of the

94 P. Zhang et al. / Gene 311 (2003) 93–98 angular and prearticular bones of the lower jaw; large with the restriction enzymes HindIII, HaeIII, Eco RI, and number of chromosomes, including some small ones). MspI, respectively. Digest products were then mixed and Recently, salamander fossils from late Jurassic to early separated by electrophoresis in 1% w/v TAE (40 mM Tris– Cretaceous from north China (Dong and Wang, 1998; Gao acetate; 1 mM EDTA) agarose gels. Gel slices containing and Shubin Neil, 2001) suggest an initial radiation in Asia fragments ranging from 200 bp to 2 Kb were excised and for the basal salamanders. Moreover, there is a skeleton DNA purified using a Gel Extract Purification System similarity between these basal salamanders and the (QIAGEN, Germany). The purified DNA was incubated Cryptobranchidae. Therefore, the family Cryptobranchidae with Klenow DNA polymerase (Takara) and T7 DNA received more and more attentions on the salamander’s polyribonucleotide kinase (Takara) to generate blunt ends origin and phylogeny study. To understand the mitochon- andthenclonedintotheSma I site of pUC18. The drial genome structure of this family, we sequenced the fragments-library was screened using PCR with the P47 complete mitochondrial DNA from the Chinese giant sala- and P48 universal primers. Only the recombinant plasmids mander Andrias davidianus, which is the representative carrying fragments ranging from 200 bp to 2 Kb were of this family. We also hope this sequence can add to selected for the sequencing. A total number of 78 clones the understanding of the phylogenetic relationships among were obtained. the three living amphibian orders and the origin of Sequencing of the recombinant plasmids was performed lissamphibians. with an automatic DNA sequencer (Applied Biosystems, 377) using the Big Dye Deoxy Terminator cycle-sequencing kit (Applied Biosystems) and the P47 and P48 universal 2. Materials and methods primers. Cloned mtDNA fragments covered approximately 14.7 Kb of the A. davidianus mtDNA genome. Seven 2.1. Salamander sample, DNA isolation, PCR, and shotgun specific primers (details upon request) were designed to sequencing cover the regions where overlapping clones were not available, using the purified LA-PCR products as DNA template The Andrias davidianus sample was obtained from the for the sequencing reaction. The sequences obtained from Longsheng Chinese giant salamander breeding center in each sequencing reaction averaged 450 bp in length and Guangxi province, China. Total DNA was purified from the each sequence overlapped the next contig by ,150 bp. liver of a single female Chinese giant salamander (Andrias davidianus) specimen using the standard Proteinase K 2.2. sequence analysis method. The isolated DNA was then used for PCR amplification. Two partial sequences for the 12S-16S gene DNA sequences were analyzed using the software and COII gene were determined first by amplifying the gene Lasergene version 5.0 (DNASTAR). Contig assembly was fragments using polymerase chain reaction (PCR) with performed with the program Seqman (part of Lasergene). primers sets: RSL2 (50-CTGGGATTAGATACCCCAC- Protein-coding, rRNA, and tRNA genes were identified by TATGC-30) and 16S-R (50-CTCCGGTCTGAACTCA- comparison with the corresponding known sequences of GATCA CGT-30); RSL4 (50-CAATAGGCCACCAATGA- other amphibian taxa, including Ranodon sibiricus (Zhang TACTGAA-30)andRSL3R(50-GGTATAAAG CTG- et al., 2003), Typhlonectes natans (Zardoya and Meyer, TGGTTTGCTC-30). Based on these sequences, two pairs 2000), and Mertensiella luschani (Zardoya and Meyer, of primers were designed to amplify the rest of the entire 2001). The complete nucleotide sequence for Andrias mitochondrial DNA presumed to have a circular confor- davidianus mtDNA reported in this article is deposited in mation by long-and-accurate PCR (LA-PCR). The primers EMBL database under the accession number AJ492192. had the following sequences: MD16SP (50-TCTTATCGA- CGAATGGGTTTACG-30), MD12SR (50-TGGTGAGGTT- 2.3. phylogenetic analyses TATCGGGTATTGT CG-30), MDCOIIR (50-GGACTG- CCCAAGAATGAAGAACA-30) and MDCOIIP (50-CCG To further confirm the phylogenetic relationships among GCCGACTAAACCAAACTACATT-30). frogs, salamanders and caecilians, another five amphibian The LA-PCR was carried out in a 500 ml reaction complete mitochondrial genomes were retrieved from mixture containing 353 ml sterile distilled water, 50 ml GenBank from the following vertebrate species: clawfrog 10 £ LA PCR buffer (TaKaRa), 80 ml dNTP (2.5 mM), Xenopus laevis (M10217, Roe et al., 1985), frog Rana 5 ml of each primer (10 mM), 2 ml 10 units of LA Taq nigromaculata (AB043889, Sumida et al., 2001), sala- polymerase, and 5 ml of template using a MJ Model mander Mertensiella luschani (AF154053, Zardoya and PTC-100 thermal cycler. Cycling conditions were 30 cycles Meyer, 2001), salamander Ranodon sibiricus (AJ419960, at 98 8C for 15 s, 55 8C for 1 min, and 68 8C for 15 min. Zhang et al., 2003), caecilian Typhlonectes natans Two amplified products (10, 5 Kb) covering the rest region (AF154051, Zardoya and Meyer, 2000). The following of the entire mitochondrial DNA were obtained. lobe-finned fish species were used as outgroup taxa in this Mixtures of the two amplified products were cleaved study: coelacanths Latimeria chalumnae (U82228, Zardoya 中国科技论文在线 http://www.paper.edu.cn P. Zhang et al. / Gene 311 (2003) 93–98 95

Fig. 1. Gene organization of the A. davidianus mitochondrial genome. tRNA genes are denoted by the single letter amino acid code. The genes encoded by the L strand are marked in dark color. NC, non-coding region. and Meyer, 1997), lungfish Protopterus dolloi (L42813, evolution model for mitochondrial proteins and a four rate Zardoya and Meyer, 1996b) for it is essential to include approximated gamma distribution of among-site rate closely related outgroup species to root the basal relation- heterogeneity (10,000 puzzling steps). The gamma distri- ships of a phylogeny correctly. Multiple alignments were bution parameter alpha was estimated from data set. Protein prepared for all 13 putative protein sequences using Clustal maximum-parsimony and NJ tree reconstructions (sites W(Thompson et al., 1994) at default setting. To avoid unweighted) were performed using the algorithms artificial errors in refining alignment, we used Gblocks implemented in Phylip 3.6 (Felsenstein, 2002). In protein (Castresana, 2000) to extract regions of defined sequence NJ analysis, the assumption of rate heterogeneity among conservation from the alignment and generate a single file of sites (G distribution) was employed and the alpha parameter concatenated conserved regions. Because these sequences value was used that estimated by TREE-PUZZLE. Non- are highly divergent, we decided to use a stringent para- parametric bootstrapping analyses (Felsenstein, 1985) were meter setting (minimum number of sequences for a con- based on 100 replicate data sets. served position: 6; minimum number of sequences for a flanking position: 8; maximum number of contiguous non- conserved positions: 5; minimum length of a block: 30) to 3. Result and discussion extract conserved regions that contain more reliable phylogenetic signals. An alignment of 2,908 positions was 3.1. General features of Andrias mtDNA obtained. Maximum likelihood tree estimation was performed using the TREE-PUZZLE program (Strimmer The complete nucleotide sequence of the L strand of and von Haeseler, 1996) under the mtREV24 sequence A. davidianus mtDNA was determined to be 16,503 bp long. 中国科技论文在线 http://www.paper.edu.cn 96 P. Zhang et al. / Gene 311 (2003) 93–98

Two rRNA genes, 22 tRNA genes, 13 protein-coding genes and a control region were identiﬁed by comparison with corresponding known sequences of other amphibian taxa, including Xenopus laevis, Typhlonectes natans, and Mer- tensiella luschani. The organization of the mitochondrial genes and non-coding regions is identical to that of ﬁshes and higher vertebrates (Fig. 1). Remarkably, a long non- coding spacer region (318 bp) between tRNA-Thr and tRNA-Pro genes which can be observed in another salamander Mertensiella luschani is also present (see discussion below). The overall base composition of the L strand of A. davidianus mtDNA is 31.9% A, 32.6% T, 21.1% C, and 14.4% G. The mitochondrial genome composition in A. davidianus is rather AT-rich like other vertebrates. Fig. 2. Putative stem-loop structures in the right portions of the unusual non-coding regions for the three salamanders, Ranodon sibiricus, Andrias dividianus, Mertensiella luschani. 3.2. Non-coding sequences

The displacement (D)-loop region of A. davidianus is the functions of such stem-loop structure remain completely largest non-coding sequence in the mitochondrial genome unknown so far. of this species and is located between the tRNA-Pro and tRNA-Phe genes. The complete nucleotide sequence of 3.3. Ribosomal and transfer RNA genes this region is 771 bp long. In the right domain adjacent to tRNA-Phe, conserved sequence blocks homologous to In the A. davidianus mitochondrial genome the 12S and CSB-1 (50-GACATA-30), CSB-2 (50-CAAACCCCCCTAC- 16S rRNA genes are 917 and 1579 bp long, respectively. CCCCC-30), and CSB-3 (50-TGTCAAACCCCTAAACCA- The size is similar to those of Ranodon sibiricus (938 and 30) have been identiﬁed at positions 16,197, 16,311, and 1600 bp, respectively). As in other vertebrates these rRNA 16,360. A possible copy of the terminal-associated genes are situated between the tRNA-Phe and tRNA-Leu sequences (TAS) was found close to the 50 end of the (UUR) and are separated by the tRNA-Val. control region at position 15,783. A sequence block similar 22 tRNA genes were identiﬁed from the mitochondrial to H-strand origin of replication in frog (Sumida et al., 2001) genome of A. davidianus by comparison with homologues is located in the central domain at position 16,067. As in most vertebrates, the putative origin of L-strand replication (OL)ofA. davidianus mitochondrial genome is Table 1 located in the WANCY region. This region is 36 bp long Genetic code and codon usage in the A. davidianus mitochondrial genome and has the potential to fold into a stem-loop secondary Codon Count Codon Count Codon Count structure with 11 bp in the stem and ten nucleotides in the 0 0 loop. The 5 -GCCGG-3 conserved motif (Hurst et al., 1999) TTT Phe 184 CCA Pro 100 GAT Asp 49 is also found at the base of the stem within the tRNA-Cys TTC Phe 68 CCG Pro 11 GAC Asp 23 gene. TTA Leu 272 ATC Thr 61 GAA Glu 65 A non-coding spacer between the tRNA-Thr and tRNA- TTG Leu 38 ACC Thr 73 GAG Glu 24 CTT Leu 103 ACA Thr 129 TGT Cys 21 Pro genes in Mertensiella luschani (320 bp) and Ranodon CTC Leu 44 ACG Thr 9 TGC Cys 13 sibiricus (156 bp) is also found in A. davidianus (318 bp). CTA Leu 109 GCT Ala 67 TGA Trp 92 To get more information about these non-coding regions, CTG Leu 31 GCC Ala 96 TGG Trp 20 structural and comparative analyses were performed. Firstly, ATT IIE 260 GCA Ala 97 CGT Arg 18 ATC GCG CGC these regions contain no obvious ORFs and BLAST searches IIE 82 Ala 6 Arg 15 ATA IIE 205 TAT Tyr 99 CGA Arg 34 of this region produced no obvious matches. Thus these ATG Met 41 TAC Tyr 28 CGG Arg 4 regions seem not to be derived from any protein-coding GTT Val 70 TAA End 11 AGT Ser 31 sequences. Further analyses reveal that these non-coding GTC Val 23 TAG End 2 AGC Ser 31 blocks all contain repeated sequences. In the Andrias and GTA Val 69 CAT His 62 AGA End 0 GTG Val 21 CAC His 37 AGG End 1 Mertensiella, these regions contain two almost identical TCT Ser 76 CAA Gin 79 GGT Gly 60 repeated sequences (,100 bp) in the two ends. While in the TCC Ser 40 CAG Gin 15 GGC Gly 61 Ranodon, there are three 17-bp tandem repeats in the left TCA Ser 102 AAT Asn 95 GGA Gly 59 portion. Remarkably, the right portions of this region in the TCG Ser 7 AAC Asn 43 GGG Gly 43 three salamanders all have the potential to fold into a stem- CCT Pro 60 AAA Lys 80 CCC Pro 25 AAG Lys 4 loop secondary structure (Fig. 2). However, the biological 中国科技论文在线 http://www.paper.edu.cn

P. Zhang et al. / Gene 311 (2003) 93–98 97

Fig. 3. Phylogenetic relationships among living amphibian orders inferred from mitochondrial protein data set that combines all 13 protein sequences deduced from complete mitochondrial genomes. The data set was analyzed with (A) ML and MP (left and right numbers along branches, respectively) and (B) NJ methods. Lobe-finned fishes are used as outgroup taxa. Numbers shown represent bootstrap values with 100 replicates (MP and NJ) or quartet puzzling support values with 10,000 steps (ML). of the luschan’s salamander (Mertensiella luschani) 15 nucleotides, within what is found in most vertebrates (Zardoya and Meyer, 2001) and by their ability to fold (4 , 17 nucleotides). into putative secondary structures. These tRNA genes range in size from 66 to 75 bp and have the same gene 3.5. Phylogenetic reconstruction organization as in other amphibians. All of the 22 tRNAs can be folded into typical cloverleaf secondary structures The stringent parameter setting in Gblocks yielded an with the same anticodon usage as reported in other alignment of 2,908 amino acid sites. Of these 1625 are vertebrates. constant and 641 are parsimony informative. The ML and MP analyses produced the same topologies (Fig. 3A) but 3.4. Protein-coding genes with minor difference in the NJ analysis (Fig. 3B). Most basal nodes within amphibians were well resolved in the All 13 protein-coding open reading frames (ORFs) ML tree. However, inconsistency and poor performance in generally found in other vertebrates are also present in resolving basal nodes in the MP and NJ analyses suggests A. davidianus mitochondrial genome with the same these approaches might be unsuitable to resolve distantly organization. The codon usage in the 13 protein-coding phylogenetic relationships. genes of Andrias mtDNA is given in Table 1. The mtDNA ML, MP, and NJ trees all suggested a close relationship of A. davidianus has a strong bias against the use of ‘G’ at between frogs and salamanders (Fig. 3). Reliability of this the third codon position (39.6% A; 18.5% C; 7.3% G; result was confirmed by high quartet puzzling support 34.6% T), which is typical of vertebrates. At the second values (100%) in protein ML analyses and moderate boot- codon position pyrimidines are over-represented compared strap support values in MP and NJ analyses (69 and 86%, with purines (T þ C ¼ 67.6%). respectively). The extant amphibian relationships have still All these genes begin with an ATG start codon except for been debated in many morphology- and molecule-based COI, which initiates with GTG. As in other vertebrates, the studies so far. In morphological and paleontological aspects, A. davidianus mtDNA also uses TAA, TAG, and AGR as most studies support a sister-group between frogs and stop codons. Of these stop codons, the TAA is the most salamanders (Batrachia) (Duellman and Trueb, 1986; Trueb prevalent one. Five genes (NADH2, COI, ATP8, NADH3, and Cloutier, 1991), but there are also opponents (Carroll NADH4L, and NADH5) use the TAA stop codon whereas and Holmes, 1980; Carroll, 1988) advocating a close phylo- five genes (COII, ATP6, COIII, and Cytb) appear to end in genetic relationship between salamanders and caecilians. In an incomplete stop codon of TNN. The TAG stop codon is molecular aspect, molecular studies based on nuclear and observed in the NADH1 and NADH4. The NADH6 that is mitochondrial rRNA data have concluded caecilians as the encoded by the L-strand of the mtDNA ends with AGG. sister taxon of salamanders (Hedges et al., 1990; Feller and Two cases of reading-frame overlap on the same strand are Hedges, 1998), whereas another analysis based on complete found in A. davidianus mtDNA. The ATPase 8 and ATPase mitochondrial genomes (Zardoya and Meyer, 2001) resulted 6 genes share ten nucleotides, as in birds and other in a contrary answer. Several recent studies have pointed out amphibians (with the exception of caecilians, which have using short sequence data set may produce misleading only seven nucleotides). The NADH4 and NADH4L genes results in phylogenetic reconstructions among distantly overlap by seven nucleotides, as in all other chordates. related taxa (Cummings et al., 1995; Zardoya and Meyer, In addition, the NADH5 and NADH6 genes, which are 1996a). Therefore, larger data set such as complete mito- located on the opposite strands, have an overlap of chondrial genomes will be required to confidently resolve 中国科技论文在线 http://www.paper.edu.cn

98 P. Zhang et al. / Gene 311 (2003) 93–98 most questions dealing with major phylogenetic events. genet sp. Nov.) from the early Cretaceous of western Liaoning However, the difficulty in resolving amphibian phylogeny province, China. Vertebrata Palasiatica, 159–172. using complete mitochondrial genomes is that some fast- Edwards, J.L., 1976. Spinal nerves and their bearing on salamander phylogeny. J. Morphol. 148 (3), 305–328. evolving species display a long branch on an unrooted tree, Feller, A.E., Hedges, S.B., 1998. Molecular evidence for the early history of which often results in a long-branch attraction (LBA) arti- living amphibians. Mol. Phylogenet. Evol. 9, 509–516. fact. This problem is due to mutational saturation (Philippe, Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using 2000). To avoid LBA, there are two main approaches sug- the bootstrap. Evolution 39, 783–791. gested by Philippe (2000): (1) adding new species or gene Felsenstein, J., 2002. 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