Genes Genet. Syst. (2004) 79, p. 151–163 Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring , Buergeria buergeri (family )

Naomi Sano1, Atsushi Kurabayashi1, Tamotsu Fujii2, Hiromichi Yonekawa3, and Masayuki Sumida1* 1Institute for Biology, Hiroshima University, Higashihiroshima, Hiroshima 739-8526, Japan 2Department of Health Science, Hiroshima Prefectural Women’s University, Hiroshima 734-8558, Japan 3Department of Laboratory Science, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan

(Received 22 March 2004, accepted 26 May 2004)

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typ- ical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organ- izations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid , indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mito- chondrial genes also supported the gene rearrangement pathway.

Key words: Buergeria buergeri, complete mitochondrial genome, gene rearrange- ment, Japanese bell-ring frog, ND5 gene

1981; Roe et al., 1985; Tzeng et al., 1992; Boore, 1999). INTRODUCTION Although minor rearrangements have been reported for Vertebrate mitochondrial DNA (mtDNA) is a closed cir- marsupials (Pääbo et al., 1991), birds (Desjardins and cular molecule. The genome organization is highly com- Morais, 1990, 1991; Quinn and Wilson, 1993; Mindell et pact and the genome is approximately 16 kbp in length al., 1998), reptiles (Kumazawa and Nishida, 1995; Quinn (Wolstenholme, 1992). This genome typically contains and Mindell, 1996; Macey et al., 1997), lampreys (Lee and 37 genes for 2 ribosomal (r)RNAs, 22 transfer (t)RNAs Kocher, 1995), and teleost fishes (Miya and Nishida, and 13 proteins, and a long noncoding region called the 1999; Inoue et al., 2001; Miya et al., 2001), most of these D-loop that includes signals required for regulating and cases involve only a few rearrangements of tRNA genes initiating mtDNA replication and transcription (Wolsten- and/or the D-loop region. holme, 1992; Janke et al., 1994). Mitochondrial gene arrangements have attracted the Mitochondrial gene arrangements are generally con- attention of evolutionary biologists as novel phylogenetic served in vertebrates. All 37 genes are arranged in the markers (Smith et al., 1993; Kumazawa and Nishida, same relative order in almost all vertebrate species from 1995; Quinn and Mindell, 1996; Macey et al., 1997; Boore teleost fishes to eutherian mammals (Anderson et al., and Brown, 1998; Boore, 1999; Kurabayashi and Ueshima, 2000). The complete mtDNA sequences have Edited by Toshihiko Shiroishi been published for only six amphibian species, including * Corresponding author. E-mail: [email protected] a caecilian (Zardoya and Meyer, 2000), three sala-

152 N. SANO et al. manders, (Zardoya et al., 2003; Zhang et al., 2003a, b), ity to the family Ranidae (true frogs), and the genus Buer- and two anurans, the clawed frog Xenopus laevis (Roe et geria is regarded as the most basal group in the former al., 1985) and the Japanese pond frog Rana nigromacu- (Channing, 1989; Jiang et al., 1987; Liem, 1970; Richards lata (Sumida et al., 2001). The mitochondrial gene and Moore, 1998; Wilkinson and Drewes, 2000; Wilkinson arrangements of five of these six amphibian species are et al., 2002). identical with those of typical vertebrates. However, in In this report, we present the first data on the complete the R. nigromaculata mtDNA, the positions of four tRNA mtDNA sequence of a rhacophorid frog and describe fea- genes (tRNA-Leu(CUN), tRNA-Thr, tRNA-Pro, and tures of the genome. The evolutionary implications of tRNA-Phe) differ from those of typical vertebrates (see our findings are also discussed. Fig. 3). The same gene arrangement is also found in other ranid frogs so far investigated (Rana catesbeiana, MATERIALS AND METHODS Yoneyama, 1987; Rana limnocharis, Macey et al., 1997; Rana porosa, Sumida et al., 2000). A lack of available DNA sources. Bell-ring frogs (Buergeria buergeri) were information on the mitochondrial genomic organization of collected from Ota River, Hiroshima prefecture, western other anuran family members makes it difficult to deter- Japan. The total genomic DNAs were extracted from a mine whether this unique arrangement is shared by other clipped toe of a living frog using the DNeasy Tissue Kit anuran groups besides the ranids. (QUIAGEN) according to the manufacturer’s protocol. In order to elucidate various aspects of mitochondrial gene rearrangement, we determined the complete PCR and sequencing. The total length of B. buergeri mtDNA sequence of the bell-ring frog Buergeria buergeri, mtDNA was amplified by polymerase chain reaction a representative of the family Rhacophoridae (tree frogs). (PCR), beginning with partial mitochondrial segments Rhacophoridae is generally considered to have close affin- and finishing with the remaining mtDNA region. Two

Fig. 1. Sequencing and cloning strategy for Buergeria buergeri mtDNA. Localizations and directions of primers used in the LA-PCR amplification and DNA sequencing are denoted by arrowheads. The sequences of these primers are available from the www site, http://home.hiroshimau.ac.jp/~amphibia/syukeisei/usedprimers.html. LA-PCR products are shown as bold lines below the gene map. Cloned restriction fragments and their lengths are indicated above the gene map.

Table 1. Primers used for PCR amplification in the present study. Primer name Sequence (5’-3’) F20N7a TGAATCGGGGGCCAACCAG R16b ATAGTGGGGTATCTAATCCCAGTTTGTTTT FR16c AAAACAAACTGGGATTAGATACCCCACTAT R51d GGTCTGAACTCAGATCACGTA F70e GGGTATCCCAGTGGTGCAGCCGCTACTAAT R71e CGAATGTCTTGTTCGTCATTGAGGTTATGA ND5Fow1e TTYATHGGHTGRGARGGVGTNGG R16M1c GGGTATCTAATCCCAGTTTG Positions with mixed bases are labeled with their IUB codes: H = A / T / G, N = A / T / G / C, R = A / G, V = A / G / C, Y = C / T. aDesigned based on Sumida et al. (2001), Roe et al. (1985), Zardoya and Meyer (2000) and Zardoya and Meyer (1997). bSumida et al. (1998). cModified from Sumida et al. (1998). dSumida et al. (2002). ePresent study.

Mitochondrial genome of rhacophorid frog 153 partial mitochondrial segments were amplified from B. employed to sequence almost all portions of the mtDNA buergeri total DNA by long-and-accurate PCR (LA-PCR) (Fig. 1). The sequencing was performed using 373A and using two primer sets (F20N7 and R16; FR16 and R51) 3100-Avant automated DNA sequencers (ABI) with DYE- (Fig. 1 and Table 1). PCR mixtures were prepared using namic ET Terminator cycle sequencing reagent a TaKaRa LA Taq™ Kit as recommended by the manu- (Amersham). Sequencing primers for internal portions facturer (TaKaRa). LA-PCR reactions consisted of an of the long PCR fragment were designed by cloning five initial denaturation at 94°C for 1 min, 30 cycles of dena- restriction fragments (see Fig. 1) into pUC 118 E. coli vec- turation at 94°C for 20 sec plus annealing and extension tor and sequencing them. The D-loop region was diffi- at 60°C for 3 min, and a final extension at 72°C for 10 cult to sequence by primer walking due to an abundance min. The resultant PCR fragments were electrophore- of lengthy tandem repeat units. To determine the pre- sed on a 0.7% agarose gel, and the DNAs were purified cise sequence of this region, a series of deleted subclones from excised pieces of gel using Wizard SV Gel and the was made from the clones of an Sma I/EcoR I-digested PCR Clean-UP System (Promega) and used for sequenc- fragment (see Fig. 1) using the Exonuclease III deletion ing (see below). The remaining mtDNA fragment was method (Henikoff, 1987). The nucleotide sequence of the amplified with the primer set F70 and R71 (Fig. 1 and B. buergeri mtDNA was deposited in the DDBJ database Table 1) designed based on the ND5 gene and 16S rRNA accession number AB127977. gene sequences determined above. LA-PCR reactions consisted of 1 cycle of 1 min at 94°C, 14 cycles of 20 sec Phylogenetic analysis. For the phylogenetic analysis, at 98°C followed by 20 min at 68°C, 17 cycles of 20 sec at we created an alignment dataset using CLUSTAL W 98°C followed by 20 min 20 sec at 68°C, and 1 final cycle (Thompson et al., 1994). The data set consisted of all 37 of 10 min at 72°C. The amplified mtDNA fragment of mitochondrial gene sequences from 9 vertebrates, includ- approximately 16.5 kbp was then purified using ing 7 , 1 coelacanth (Latimeria chalumnae), MicroSpin™ S-300 HR Columns (Pharmacia Biotech) and and 1 lungfish (Protopterus dolloi). The latter two were used for the sequencing reaction. used as outgroups. The alignment was checked by eye, The entire mtDNA genome of B. buergeri was sequenced and all positions with gaps and ambiguous sites were from both strands. The primer walking method was excluded. Based on the alignment data (12,865 nucle-

Fig. 2. Organization of the B. buergeri mitochondrial genome. All protein-coding genes are encoded by the H strand, with the exception of ND6, which is encoded by the L strand. Transfer RNA genes are represented by the standard one-letter amino acid code and those encoded by the H and L strands

are shown outside and inside the circle, respectively. L1, L2, S1, and S2 denote tRNA-Leu(CUN), tRNA-Leu(UUR), tRNA-Ser(UCN), and tRNA-Ser(AGY), respectively. Genes are abbreviated as in Table 2, except for A6 and A8, which denote ATPase6 and ATPase8, respectively.

154 N. SANO et al.

Table 2. Location of features in the mitochondrial DNA of Buergeria buergeri. Position Codon Gene From To Size(bp) Strand Start Stop ND5 1 1791 1791 H ATG AGA tRNA-Leu(CUN) 1814 1885 72 H tRNA-Thr 1888 1957 70 H tRNA-Pro 1958 2026 69 L tRNA-Phe 2028 2095 68 H 12S rRNA 2096 3022 927 H tRNA-Val 3023 3091 69 H 16S rRNA 3092 4665 1574 H tRNA-Leu(UUR) 4666 4739 74 H ND1 4737 5700 964 H ATT T tRNA-Ile 5701 5771 71 H tRNA-Gln 5772 5842 71 L tRNA-Met 5842 5911 70 H ND2 5912 6949 1038 H ATT TAG tRNA-Trp 6948 7018 71 H tRNA-Ala 7019 7088 70 L tRNA-Asn 7089 7161 73 L

OL 7162 7190 29 H tRNA-Cys 7188 7251 64 L tRNA-Tyr 7252 7318 67 L COI 7323 8873 1551 H ATA AGG tRNA-Ser(UCN) 8865 8935 71 L tRNA-Asp 8936 9003 68 H COII 9004 9690 687 H ATG AGA tRNA-Lys 9694 9765 72 H ATPase8 9766 9930 165 H ATG TAA ATPase6 9921 10602 682 H ATG T COIII 10603 11386 784 H ATG T tRNA-Gly 11387 11455 69 H ND3 11453 11795 343 H ATA T tRNA-Arg 11796 11864 69 H ND4L 11865 12149 285 H ATG TAA ND4 12143 13508 1366 H ATG T tRNA-His 13509 13577 69 H tRNA-Ser(AGY) 13578 13644 67 H ND6 13649 14140 492 L ATG AGG tRNA-Glu 14142 14209 68 L Cytb 14214 15383 1170 H ATG TAA D-loop 15384 19959 4576 – Tandem repeat 15467 16251 785 – TAS 16332 16345 14 –

OH 17181 17244 64 – CSB-1 17371 17398 28 – CSB-2 17460 17477 18 – CSB-3 17532 17546 15 – Tandem repeat 17651 19885 2235 – Each region was identified by homology with known vertebrate mitochondrial genomes. Position 1 indicates the first nucleotide of the 5’ end of the ND5 gene. ATPase6 and 8 indicate subunits 6 and 8 of adenine triphosphatase; COI-III, cytochrome c oxidase subunits I-III; Cytb, cytochrome b apoenzyme; ND1-6 and ND4L, subunits 1-6 and 4L of nicotinamide adenine dinucleotide dehydrogenase; TAS, termination associated sequence; CSB: conserved sequence block; OH and OL, the replication origins of the H- and L-strands.

Mitochondrial genome of rhacophorid frog 155 otide sites), we reconstructed a phylogenetic tree by the RESULTS maximum likelihood (ML) method. The tree construc- tion was performed with PAUP* ver. 4.10b (Swofford, Genome composition. The complete nucleotide sequence 2001). The best-fit model of DNA substitution was esti- of the mitochondrial genome of B. buergeri was mated using ModelTest ver. 3.06 (Posada and Crandall, determined. Though the genome was extremely long– 1998) and a general-time-reversible + gamma + invariant 19,959 bp, the longest among all vertebrate mtDNAs so (GTR + G + I, G = 1.0444, I = 0.3276) model was proposed far examined, B. buergeri mtDNA included only 37 typical under AIC consideration. mitochondrial genes, 13 protein genes, 2 rRNA genes and

Table 3. Comparisons of lengths (bp) of mitochondrial genes of amphibians. Anura Caudata Gymnophiona Gene Buergeriaa Ranab Xenopusc Mertensiellad Ranodone Andriasf Typhlonectesg D-loop 4576 2425 2134 922 799 771 1630 12S rRNA 927 933 945 921 938 917 934 16S rRNA 1574 1588 1631 1567 1600 1579 1571 ATPase6 682 682 681 683 684 682 683 ATPase8 165 165 168 168 168 168 165 COI 1551 1539 1555 1560 1551 1551 1554 COII 687 688 688 688 690 688 691 COIII 784 784 781 784 786 784 784 Cytb 1170 1143 1140 1141 1143 1141 1141 ND1 964 973 972 970 972 970 961 ND2 1038 1038 1038 1036 1044 1039 1032 ND3 343 340 342 347 351 351 343 ND4 1366 1360 1384 1378 1373 1375 1375 ND4L 285 285 297 297 297 297 297 ND5 1791 1795 1815 1815 1824 1812 1788 ND6 492 501 513 516 519 519 516 Complete 19,959 17,804 17,553 16,650 16,418 16,503 17,005 aPresent study. bSumida et al. (2001). cRoe et al. (1985). dZardoya et al. (2003). eZhang et al. (2003a). fZhang et al. (2003b). gZardoya and Meyer (2000).

Fig. 3. Comparison of mitochondrial gene arrangements among amphibians. Arrows indicate the rearranged homologous genes. Closed and shaded boxes indicate the genes whose positions vary among anurans. Genes are abbreviated as in Table 2 and Fig. 2.

156 N. SANO et al.

Fig. 4. Putative secondary structures of B. buergeri mitochondrial tRNA genes. The cloverleaf structures of 22 tRNA genes identified in B. buergeri mtDNA are shown. Watson-Crick base pairing is indicated by solid bars (–), and the G-T pairs usually observed in animal mtDNAs are shown with plus marks (+).

Mitochondrial genome of rhacophorid frog 157

22 tRNA genes (Fig. 2 and Table 2), most of which were mtDNA is similar to those of other vertebrates. similar in length to their counterpart genes in other amphibians. However, length differences between Buer- Gene arrangement. The gene arrangement of B. geria and other amphibians were observed in the Cytb buergeri mtDNA diverged from that of typical vertebrates and ND6 genes, and especially in the D-loop region (Table (Fig. 3). Specifically, the ND5 gene and four tRNA genes 3). The large genome size of B. buergeri mtDNA was due (tRNA-Leu(CUN), tRNA-Thr, tRNA-Pro and tRNA-Phe) to the accumulation of lengthy repetitive sequences into were located between the D-loop and the 12S rRNA the D-loop region (see the section about the noncoding gene. The tRNA cluster upstream of the 12S rRNA gene region). was also identified in the Japanese pond frog, Rana nigro- The base composition of the complete B. buergeri maculata (Sumida et al., 2001) (Fig. 3) and other known mtDNA was A: 29.9%, T: 30.5%, G: 14.7%, and C: 24.8%. ranid frogs (Yoneyama, 1987; Macey et al., 1997; Sumida The slightly high A + T content (60.4%) of B. buergeri et al., 2000). However, the rearrangement of the ND5

Fig. 5. Schematic diagram of the B. buergeri mtDNA control (D-loop) region. Dark hatched squares show the tandem repeat sequences and the numbers (bp) under the map represent the lengths of the tandem repeats. Abbreviations are follows: TAS, termi-

nation associated sequence; OH, H-strand origin of replication; CSB, conserved sequence block.

Table 4. Nucleotide sequences of conserved segments and tandem repeat units in the D-loop region of B. buergeri.

Segment Sequence

Repeat unit TMYTMYYATGTAYAATCAGCATATATCTATGTYCTTCT (5’-side) 1st-20th

21th .AC.ACT.....T...... ------

TAS ACAT TAAACCCTTT

OH AGTTTTTTTTGGGGGGGGGGTTTCACCAGCATTGGTCAGAGTGATGCCACTCTGAGTTGCTTAA

CSB-1 C A TA T AAATGAATGCTAGTCGGACATAA

CSB-2 T A CC C CCCCCTTTACCCC

CSB-3 T T AT C T TAATACCCC

Repeat unit YARTAAATAAAAGTCTTATTTTTAAACTAYCTCTGCACTAAGCTCAATAACAAATTTAGARCTGGCACGGYGCGCTTAAARARGGGCTTCATTTTAATCATTTTAATCAT T TT A (3’-side) 1st- 4th

5th-23rd T.G...... A...... YCTC...... R...... YAR.G.TG.....GT.....Y...-AR......

IUB codes: H = A / T / G, M = A / C, N = A / T / G / C, R = A / G, V = A / G / C, Y = C / T. Dots indicate nucleotides identical between the 1st-20th and 21th repeat units in the 5’-side, and between the 1st- 4th and 5th-23rd repeat units in the 3’-side. Underlined letters show the alternative deleted nucleotides in each repeat unit. Dashes indicate gap sites introduced in the sequence to optimize the alignment.

Fig. 6. Putative secondary structures in the L-strand replication origins of anuran mtDNAs. The pentanucleotides predicted to be involved with the transition of RNA to DNA synthesis are boxed. The sequences and secondary structures of R. nigromaculata and X. laevis were quoted from Sumida et al. (2001) and Roe et al. (1985), respectively. 158 N. SANO et al. gene has not been seen in other amphibians sequenced ATG or ATA initiation codon (the exceptions, ND1 and previously. ND2, started with an ATT codon) (Table 2). Four pro- tein-coding genes of B. buergeri (COI, COII, ND5 and Protein-coding genes. All of the 13 protein-coding ND6) ended with the AGR (AGA and AGG) stop codon genes found in other were also present in the B. characteristic of vertebrate mtDNAs. Four protein buergeri mitochondrial genome. The codon usage of B. genes (ATPase8, Cytb, ND2, and ND4L) stopped with the buergeri mtDNAs was identical to that of the other usual TAR codon. The remaining 5 genes (ATPase6, vertebrates. All but two of these genes started with an COIII, ND1, ND3 and ND4) had an incomplete stop

Fig. 7. Maximum-likelihood (ML) tree based on all the 37 mitochondrial genes (12,856 nucleotide sites). The following parameters were used for ML analysis: the proportion of invariable sites (I) = 0.3276, the gamma distribution shape parameter (α) = 1.0444, empir- ical base frequencies (A: 0.3130; C: 0.2577; G: 0.1502; T: 0.2791), and substitution rates ([A-C] = 2.0504, [A-G] = 4.3039, [A-T] = 2.3986, [C-G] = 0.4303, [C-T] = 8.5382, [G-T] = 1). The scale bar shows the genetic distance calculated using the above parameter values. The values of internal branches are the bootstrap values (1,000 replicates). The accession numbers for the sequences used were as follows: Rana nigromaculata (AB043889; Sumida et al., 2001), Xenopus laevis (M10217; Roe et al., 1985), Mertensiella luschani (AF154053; Zardoya and Meyer, 2001), Ranodon sibiricus (AJ419960; Zhang et al., 2003a), Andrias davidianus (AJ492192; Zhang et al., 2003b), Typhlonectes natans (AF154051; Zardoya and Meyer, 2000), Latimeria chalumnae (coelacanth; U82228; Zardoya and Meyer, 1997) and Protopterus dolloi (lungfish; L42813; Zardoya and Meyer, 1996). The latter two were used as outgroups. Arrows indicate the ancestral lineages in which the gene rearrangements occurred (see text). Mitochondrial genome of rhacophorid frog 159 codon, a single stop nucleotide T, where the post tran- Phylogenetic relationships of amphibians. We ana- scriptional polyadenylation could produce a complete lyzed the phylogenetic relationship among amphibians TAA stop codon (Ojara et al., 1980). (Anura, Caudata and Gymnophiona) using the long nucle- otide sequences of all the mitochondrial genes. The Ribosomal RNA genes. The lengths of the 12S and resultant ML tree (-lnL = 85787.4062) is shown in Fig. 16S rRNA genes in the B. buergeri mitochondrial genome 7. The robustness of our result was confirmed by high (927 and 1,574 bp, respectively) were similar to those of bootstrap support (=>95) of all nodes. Each amphibian the corresponding genes in other amphibians (Table 3) order in our tree split into an independent branch. Gym- and vertebrates. Our sequence showed only minor dif- nophiona was the first to branch away, and Caudata and ferences (99% similarity) from the partial nucleotide Anura formed a monophyletic group. In the order sequences of the 12S (507 bp) and 16S rRNA (1,383 bp) Anura, Xenopus laevis (family ) was the first to genes of B. buergeri (previously reported by Wilkinson et branch away, and Rana nigromaculata (Ranidae) and B. al., 2002). buergeri (Rhacophoridae) formed a sister group.

Transfer RNA genes. Twenty-two tRNA genes were DISCUSSION identified in the mitochondrial genome of B. buergeri by comparison with homologues of other amphibians and by Mitochondrial genome size and repeat sequences their ability to fold into a putative secondary structure in noncoding region. The genome size of vertebrate (Fig. 4). Twenty-one of the 22 tRNAs could be folded into mtDNAs ranges from 15,181 bp in the tuatara (Rest et the canonical cloverleaf secondary structure, while tRNA- al., 2003) to 18,978 bp in the deep-sea gulper eel (Inoue Ser(AGY), with an unpaired dihydrouridine (D) arm, could et al., 2003). The Buergeria buergeri mtDNA, whose not. The unpaired D-arm in tRNA-Ser(AGY) is a common sequence is reported here, has the largest genome size feature of metazoan mtDNAs (Wolstenholme, 1992). (19,959 bp) among the vertebrates investigated so far. Vertebrate mtDNAs of large sizes (>18 kbp) generally Noncoding region. A major noncoding region of 4,576 have multiple long noncoding portions. For example, the bp was found in the B. buergeri mtDNA between the Cytb mtDNAs of two eel species (Eurypharynx pelecanoides, and ND5 genes (Fig. 2). This region was thought to cor- 18,978 bp, Inoue et al., 2003 and Conger myriaster, 18,705 respond to the D-loop region as it contained several com- bp, Inoue et al., 2001) contain three noncoding regions, ponents characteristic of the D-loop region: apparent and those of several bird species (18 kbp ~ 18.7 kbp) pos- homologues of the termination-associated sequence sess two noncoding regions (Haring et al., 2001). These

(TAS), an H-strand origin of replication (OH), and three additional noncoding regions are the chief factor respon- conserved sequence blocks (CSB-1, CSB-2 and CSB-3) sible for the increased genome sizes. On the other hand, (Fig. 5 and Table 4). Although these notable structural B. buergeri mtDNA contains only a single long noncoding features were conserved in the D-loop region of the B. region, corresponding to the D-loop. This region con- buergeri mtDNA, the region was extremely long (4,576 tains two series of long repeat units (Fig. 5) that consid- bp) compared to those in other vertebrates, including erably expand the genome. It is interesting that relatively amphibians (Table 3). This unusually large size was due long repetitive sequences in the D-loop region are also to the presence of two distinct tandem repeat units iden- found in all reported anuran species, particularly in tified in the 5’- and 3’-sides of the D-loop region (Fig. ranids (1.2 kbp in R. porosa and 1.0 kbp in R. nigromac- 5). The 5’-side repeated region consisted of 20 repeat ulata; Sumida et al., 2000) and in rhacophorids (at least units of 38 bp and one incomplete repeat unit of 22 bp. 2.0 kbp in Polypedates leucomystax, 1.3 kbp in Rhacoph- The 3’-side repeated region consisted of 4 repeat units of orus schlegelii; Kurabayashi et al., in preparation). The 105 bp and 19 repeat units of 96 bp (Table 4). tendency for the repetitive sequences to accumulate in The putative origin of L-strand replication (OL) of the the D-loop region might be a feature of ranid and rha- B. buergeri mitochondrial genome was located between cophorid mtDNAs. tRNA-Asn and tRNA-Cys in the WANCY tRNA gene clus- ter (Fig. 2). The putative OL was 29 nucleotides in Phylogenetic relationships of amphibians based on length and the sequence had the potential to fold in a complete mitochondrial gene sequence. No complete stem-loop secondary structure with a stem formed by 9 consensus has yet been reached on the phylogenetic rela- paired nucleotides and a loop of 9 nucleotides (Fig. 6). A tionships among the three orders of living amphibians “GACGG” sequence was present at the base of the stem (Trueb and Cloutier, 1991b; Carroll et al., 1999). Most region in B. buergeri (Fig. 6). This pentanucleotide is morphological studies support a close relationship very similar to the “GCCGG” sequence motif involved in between anurans (frogs) and caudates (salamanders and the transition from RNA to DNA synthesis of the human newts) generally referred to as the “Batrachia hypothesis” mtDNA system (Hixson et al., 1986). (Trueb and Cloutier, 1991a). On the other hand, several 160 N. SANO et al. molecular studies based on nuclear and mitochondrial brates by rearrangements of the positions of four tRNA rRNA data have supported the notion that caecilians are genes and the ND5 gene (Fig. 3). Gene rearrangement a sister taxon of salamanders (Larson and Wilson, 1989; in animal mtDNA is generally believed to take place Hedges et al., 1990; Hay et al., 1995; Feller and Hedges, through tandem duplication of gene regions as a result of 1998). In attempts to resolve this conflict, larger data slipped strand mispairing, followed by multiple deletions sets, i.e., complete mitochondrial genomes, have been of redundant genes (Moritz and Brown, 1986, 1987; employed in several phylogenetic studies, and the results Moritz et al., 1987; Boore and Brown, 1998). According supported the former hypothesis (Zardoya and Meyer, to the gene arrangement mechanism, at least two dupli- 2001; Zardoya et al., 2003, Zhang et al., 2003b). Further- cation-deletion events through an intermediate arrange- more, our phylogenetic analysis (Fig. 7) using the com- ment would be needed to generate the B. buergeri gene plete mitochondrial gene sequence data of B. buergeri also order from that of typical vertebrates (see Fig. 8). In confirmed the monophyly of anurans and caudates with addition, taking into account the parsimonious principle, sufficient statistical significance (BP = 95). two rearrangement pathways are possible, as shown in It has been broadly accepted that the Pipidae is one of Fig. 8. In these hypothetical pathways, the first step, the basal families in anurans and that Ranidae and Rha- the duplication of the region between the tRNA-Leu and cophoridae are members of a derived anuran group, the D-loop in the typical gene order of vertebrates, is com- suborder (e.g., Duellman, 1975; Ford and mon. However, the intermediate arrangements differ Cannatella, 1993). Our phylogenetic tree also supported from each other due to differences in the deleted genes. this traditional phylogenetic relationship. Regarding these pathways, it is remarkable that the gene order of the intermediate arrangement in hypothesis A Gene rearrangement pathway in anuran mtDNAs. (i.e., D-loop, tRNA-Leu, tRNA-Thr, tRNA-Pro, tRNA-Phe, The present study showed that the gene order of Buerge- 12S) is identical to that found in ranid frogs. This ria buergeri mtDNA differs from that of typical verte- strongly suggests that the pathway illustrated by

Fig. 8. Possible gene rearrangement pathways in anuran mtDNAs. Two hypothetical pathways postulated from the duplication-dele- tion rearrangement model are illustrated. Duplicated regions are indicated by thick lines and the deleted genes and D-loop are indi- cated by asterisks. Genes are abbreviated as in Table 2 and Fig. 2. The intermediate gene order in hypothesis A is the same as that in ranid frogs. Mitochondrial genome of rhacophorid frog 161 hypothesis A occurred during anuran evolution. This 13839012) from the Ministry of Education, Culture, Sports, Sci- rearrangement pathway is also consistent with the ence and Technology, Japan (to M. Sumida) and a Grant-in-Aid phylogenetic relationships of anurans. Rearrangements for the Extensive Research Program of Hiroshima Prefectural Government (to T. Fujii). of four tRNAs appear to have occurred in a common ancestral lineage of ranids and rhacophorids after the pipid branching, and the rearrangement of the ND5 REFERENCES gene appears to have taken place after ranid divergence Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L., (Fig. 7). Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, Phylogenetic implications on the mitochondrial R., and Young, I. G. (1981) Sequence and organization of the gene arrangement. In the present report, we showed human mitochondrial genome. Nature 290, 457–465. Boore, J. L., and Brown, W. M. (1998) Big trees from little that the previously known arrangement of four tRNA genomes: mitochondrial gene order as a phylogenetic tool. genes in ranids is also shared by a rhacophorid frog, B. Curr. Opin. in Genet. and Dev. 8, 668–674. buergeri, and we suggest that the rearrangements of the Boore, J. L. 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