Complete Nucleotide Sequence and Gene Rearrangement of the Mitochondrial Genome of Occidozyga Martensii

c Indian Academy of Sciences

RESEARCH ARTICLE

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of Occidozyga martensii

EN LI1, XIAOQIANG LI1, XIAOBING WU1∗,GEFENG1, MAN ZHANG1, HAITAO SHI2, LIJUN WANG2 and JIANPING JIANG3

1College of Life Sciences, Anhui Province Key Laboratory for Conservation and Exploitation of Biological Resource, Anhui Normal University, Wuhu 241000, People’s Republic of China 2College of Life Sciences, Hainan Normal University, Haikou 571158, People’s Republic of China 3Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China

Abstract In this study, the complete nucleotide sequence (18,321 bp) of the mitochondrial (mt) genome of the round-tongued ﬂoat- ing frog, Occidozyga martensii was determined. Although, the base composition and codon usage of O. martensii conformed to the typical vertebrate patterns, this mt genome contained 23 tRNAs (a tandem duplication of tRNA-Met gene). The LTPF tRNA-gene cluster, and the derived position of the ND5 gene downstream of the control region, were present in this mitogenome. Moreover, we found that in the WANCY tRNA-gene cluster, the tRNA-Asn gene was located between the tRNA- Tyr and COI genes instead of between the tRNA-Ala and tRNA-Cys genes, which is a novel mtDNA gene rearrangement in vertebrates. Based on the concatenated nucleotide sequences of the 13 protein-coding genes, phylogenetic analysis (BI, ML, MP) was performed to further clarify the phylogenetic relations of this species within anurans.

[Li E., Li X., Wu X., Feng G., Zhang M., Shi H., Wang L. and Jiang J. 2014 Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of Occidozyga martensii. J. Genet. 93, 631–641 ]

Introduction archaeobatrachians are identical to that of vertebrates (Roe et al. 1985;SanMauroet al. 2004a; Pabijan et al. 2008); The mitochondrial DNA (mtDNA) of vertebrates is a closed however, further gene rearrangements have been reported circular double-stranded molecule, typically containing 37 in the neobatrachians, Pelophylax nigromaculata (Sumida genes for two rRNAs, 13 protein-coding genes and 22 et al. 2001), Buergeria buergeri (Sano et al. 2004), Fejer- tRNAs with a long noncoding region called the D-loop varya limnocharis (Liu et al. 2005), Rhacophorus schlegelii that includes signals required for regulating and initiating (Sano et al. 2005), Polypedates megacephalus (Zhang mtDNA replication and transcription (Wolstenholme 1992). et al. 2005b), Mantella madagascariensis (Kurabayashi The content is highly conserved in vertebrates, with only et al. 2006), Amolops tormotus (Su et al. 2007), Hyla japon- a few exceptions (Liu et al. 2005). Comparison of many ica, Bufo japonicus, Microhyla okinavensis (Igawa et al. mitochondrial complex features: modes of replication and 2008), Fejervarya cancrivora (Ren et al. 2009), Limnonectes transcription; RNA processing; genetic code variations; pro- bannaensis (Zhang et al. 2009), Paa spinosa (Zhou et al. tein, tRNA and rRNA secondary structures and the rela- 2009)andHoplobatrachus rugulosus (Yu et al. 2012). Most tive arrangement of genes are helpful for modelling the of these rearrangements involve tRNA genes, ND5 gene and genome evolution and phylogenetic inference (Garesse et al. the D-loop region. 1997; Boore 1999). Among these mt genomic features, gene The taxonomy of Ranidae is extremely controversial and arrangements have attracted the attention of evolutionary has received much attention. Lately, a series of studies, inclu- biologists as novel phylogenetic markers (Kumazawa and ding several using DNA sequences, have proposed many Nishida 1995; Quinn and Mindell 1996; Macey et al. 1997; updates (Dubois 1992, 2005; Inger 1996;Frostet al. 2006; Kurabayashi and Ueshima 2000;Sanoet al. 2004, 2005;Ren Che et al. 2007;Frost2010). To clarify these controversies, it et al. 2009). Among anurans, the mtDNA arrangements in is apparent that intensive sampling is needed, in particular for the Chinese species. Chinese ranids are classiﬁed into ∗ For correspondence. E-mail: [email protected]. three subfamilies: Raninae, Amolopinae and Occidozyginae Keywords. mitochondrial genome; novel gene rearrangement; phylogenetic relationships; Occidozyga martensii.

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(Fei et al. 1990). However, the subfamily Amolopinae was Gel Extract Purification Kits (V-gene) (Hangzhou, China) shown to be paraphyletic by the results of molecular system- for sequencing on an automated DNA sequencer (Applied atics (Roelants et al. 2004;Wienset al. 2009), while Occi- Biosystems, Shanghai, China) from both strands with the dozyginae was considered monophyletic and is still treated primer walking strategy. as a subfamily of the family Ranidae or Dicroglossidae (Frost 2010). Apparently, only morphology will not be able Sequence analyses to resolve these controversies on account of the high morphological homoplasy existing in amphibians, but molecular Nucleotide sequences were checked and assembled using the data could provide the additional vital information. Com- program Seqman (DNASTAR) and ClustalX (Jeanmougin plete mtDNA sequences have been published for relatively et al. 1998). The locations of 13 protein-coding genes were few species in family Ranidae until now. However, addi- determined using software SEQUIN (ver. 5.35) (http://www. tional complete mtDNA sequences will be useful to elucidate ncbi.nlm.nih.gov/projects/Sequin/download/seq_win_download. relationships within Ranidae and among Anurans. html) and two rRNA genes were identified by comparison To further understand the various aspects of mitochondrial with the homologous sequences of other amphibian mtDNA. gene rearrangement, the complete mtDNA sequence of the The 22 tRNA genes were identified using the software tRNA round-tongued floating frog O. martensii was determined. Scan-SE 1.21 (http://lowelab.ucsc.edu/tRNAscan-SE/)and The origin of the features of this mtDNA was discussed. their cloverleaf secondary structure and anticodon sequences Based on the data set, phylogenetic analyses were performed were identified using DNASIS (ver. 2.5, Hitachi Software to confirm the relationship among 40 available anurans. Engineering, Yokohama, Japan). The complete mtDNA sequence of O. martensii reported in this paper was deposited in GenBank under the accession number GU177877. Materials and methods

Sample and DNA extraction Molecular phylogenetic analyses The round-tongued floating frog (O. martensii) (two indi- In an attempt to further confirm the phylogenetic rela- viduals) was collected from Hainan province in China, and tions within Neobatrachia and the phylogenetic position of ◦ immediately preserved in 95% ethanol at −20 C until use. O. martensii among the ranoid group, the complete Total genomic DNA was extracted from the muscle using mtDNA sequences of 39 anurans and three Caudata species proteinase K digestion and phenol extraction (Sambrook and (Lyciasalamandra atifi, Paramesotriton hongkongensis and Russell 2001). The frog sample (sample no. AM07357) is Ranodon sibiricus) were retrieved from GenBank database preserved in the Animal Conservation Biology Laboratory, (table 1). The latter three species were used as outgroups. College of Life Sciences, Anhui Normal University, China. We mainly followed the taxonomical schemes proposed by Frost (2010). Phylogenetic analyses were carried out on 13 mitochondrial protein-coding genes. The nucleotide and PCR and sequencing the deduced amino acid sequences of the 13 mitochon- The entire mt genome of O. martensii was amplified by normal drial protein genes were aligned using the Clustal W in or long-and-accurate (LA) PCR. According to the conserved Mega 5.2 (Tamura et al. 2011). To fill the gaps and poorly sequences of mtDNA of other frogs, 16 pairs of overlapping aligned positions, the amino acid alignment was analysed amplification primers were designed (sequences are available using GBlocks ver. 0.91b (Castresana 2000) with default set- upon request) which were used to amplify and sequence the tings. The alignments of 13 proteins were then concatenated complete mtDNA sequence of O. martensii by using Oligo to form a single alignment consisting of 3260 amino acid 6.0 (Rychlik and Rychlik 2000). Each PCR mixture (30 µL) residues. Similar positions were eliminated from the corre- included 100 ng template DNA, 3 µL10× reaction buffer, 2 µL sponding nucleotides alignment to obtain an alignment of dNTPs (2.5 mM), 2 µLMgCl2 (25 mM), 1 µL of each 9780 nucleotides. According to the saturation analysis using primer (10 µM), 1 U Taq DNA polymerase (Promega, Beijing, DAMBE5 (Xia 2013), the third codon positions of dataset China). The PCR reactions were carried out in a MJ Model were saturated, so they were excluded and a final dataset of PTC-200 thermal cycler (MJ Reasearch, Watertown, USA) 6520 nucleotides was obtained. with heated lid under the following conditions: an initial The dataset were subjected to three different meth- denaturation at 95◦C for 5 min, 30 cycles of denaturation at ods of phylogenetic analyses: the Bayesian inference 94◦C for 50 s, annealing at 50–58◦C for 1 min and extension (BI), maximum likelihood (ML) and maximum parsi- at 72◦C for 55 s and a final extension at 72◦Cfor7min. mony (MP). MP analyses were carried out with PAUP* The LA-PCR mixtures and reactions were prepared with a ver. 4.0b10 (Swofford 2002). Heuristic MP searches TaKaRa LA TaqTM Kit according to the manufacturer’s were executed in 1000 replicates of randomly added instructions (TaKaRa, Dalian, China). The PCR products sequences with all characters unordered and equally were separated by electrophoresis in 1% agarose gels, then weighted, using tree-bisection-reconnection (TBR) branch they were purified from excised pieces of gel using DNA swapping. ML analyses were performed using PhyML 3.0

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Table 1. Species classiﬁcation and sequence accession numbers of amphibian mt genomes used in phylogenetic analyses.

Taxon Species Family GenBank no.

Archaeobatrachia Bombina fortinuptialis Bombinatoridae AY458591 B. orientalis Bombinatoridae AY585338 B. variegata Bombinatoridae AY971143 B. maxima Bombinatoridae EU789363 Ascaphus truei Leiopelmatidae AJ871087 Leiopelma archeyi Leiopelmatidae HM142901 Alytes obstetricans Discoglossidae AY585337 Discoglossus galganoi Discoglossidae AY585339 Leptolalax oshanensis Megophryidae KC460337 Megophrys shapingensis Megophryidae JX458090 Pelobates cultripes Pelobatidae AJ871086 Xenopus laevis Pipidae NC_001573 X. tropicalis Pipidae NC_006839

Neobatrachia Bufo japonicus Bufonidae AB303363 B. gargarizans Bufonidae DQ275350 B. melanostictus Bufonidae AJ584640 Fejervarya limnocharis Dicroglossidae AY158705 F. cancrivora Dicroglossidae EU652694 Limnonectes fujianensis Dicroglossidae AY974191 L. bannaensis Dicroglossidae AY899242 Hoplobatrachus rugulosus Dicroglossidae HM104684 H. tigerinus Dicroglossidae AP011543 Euphlyctis hexadactylus Dicroglossidae AP011544 Paa spinosa Dicroglossidae FJ432700 Occidozyga martensii Dicroglossidae GU177877* Hyla japonica Hylidae AB303949 H. chinensis Hylidae AY458593 Mantella madagascariensis Mantellidae AB212225 Microhyla ornata Microhylidae DQ512876 M. heymonsi Microhylidae AY458596 Kaloula pulchra Microhylidae AY458595 K. borealis Microhylidae JQ692869 Microhyla okinavensis Microhylidae AB303950 Pelophylax nigromaculatus Ranidae AB043889 P. plancyi Ranidae EF196679 Amolops tormotus Ranidae DQ835616 Odorrana ishikawae Ranidae AB511282 Babina adenopleura Ranidae NC_018771 Buergeria buergeri Rhacophoridae AB127977 Rhacophorus schlegelii Rhacophoridae AB202078

Caudata Ranodon sibiricus Hynobiidae NC_004021 Lyciasalamandra atiﬁ Salamandridae AF154053 Paramesotriton hongkongensis Salamandridae NC_006407

*This study.

(Guindon et al. 2010) based on the best-ﬁtting model of resultant ML and BI trees were calculated using nonparamet- evolution selected by Modeltest 3.7 (Posada and Cran- ric bootstrap probabilities (BP) with 500 pseudo-replications dall 1998). A GTR+I+G model was selected by the and Bayesian posterior probability (BPP), respectively. Akaike information criterion (AIC) (−lnL = 83,011.9297, AIC = 166,043.8594). BI analyses were conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) under the model GTR+I+G chosen by Modeltest 3.7, and Markov chains Results were run simultaneously for 1,000,000 generations sampled Genome content one out of every 100 generations; the ﬁrst 2500 trees were discarded as burn-in samples and the remaining trees were The complete mtDNA nucleotide sequence of O. marten- used to generate Bayesian consensus trees. Robustness of the sii is 18,321 bp in length containing 13 protein-coding

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Ribosomal and transfer RNA genes In O. martensii mitochondrial genome, the lengths of 12S and 16S rRNA genes are 939 and 1581 bp, respectively, which is similar to those of P. nigromaculatus. Twenty-three tRNA genes (including an extra copy of tRNA-Met gene) are identiﬁed in the mitochondrial genome of O. martensii, which are interspersed in the genome and range in size from 66 to 73 bp. All tRNAs except tRNA-Ser (AGY) can be folded into the canonical cloverleaf secondary structures. The tRNA-Ser (AGY) is similar to the metazoan homologues with the absence of dihydrouridine (D) arm. Two tRNA-Met genes hold the same anticodon, CAT, which is common in most metazoan mt tRNA-Met gene (ﬁgure 2b).

Protein-encoding genes All the 13 protein-encoding genes found in other ani- mals are identified in the mtDNA of O. martensii by software SEQUIN and compared with the homologues of other amphibians, all are encoded by heavy strand except for ND6 gene. Most protein-encoding genes begin with ATG or ATA as start codon except ND1, ND2 with ATT and COI with Figure 1. Gene organization of the O. martensii mt genome. GTG. The ND2, ATPase8, ND3, ND4L and Cytb end with Genes encoded by the light strand are underlined. Each tRNA gene is represented by the standard one-letter amino acid code. the usual TAR (TAA and TAG) as stop codon, COI, ND4 OH and OL represent the replication origins of heavy-strands and and ND6 end with AGR (AGG and AGA), and the remain- light-strands, respectively. Genes are abbreviated as in table 2, ing five genes have an incomplete stop codon of T (table 2), except for A6 and A8, which denote ATPase6 and ATPase8, respec- which can be completed (TAA) by polyadenylation after tively. M’ shows the extra copy of tRNA-Met. transcription (Boore 2001). genes, two rRNA genes, 23 tRNA genes (including an addi- Noncoding regions tional copy of tRNA-Met) and noncoding regions (including The noncoding regions in O. martensii mtDNA include the the D-loop region) (figure 1;table2). As found in other control region (D-loop) and a few intergenic spacers. Due vertebrates, most of these genes are coded on the heavy to the translocation of ND5,theO. martensii D-loop is strand except for ND6 and eight tRNA genes. The base positioned between Cytb and ND5 and the position coincides composition of the light strand is as follows: A (29.5%), with those of the seven ranoids, F. cancrivora, F. limnocharis T (32.8%), G (14.2%) and C (23.5%). The low G+Cand (Dicroglossidae), B. buergeri, R. schlegelii (Rhacophori- high A+T contents are similar to those observed in other dae), H. rugulosus, H. tigerinus (Dicroglossidae) and M. vertebrates. madagascariensis (Mantellidae). The latter four species pos- sess two copied D-loops. Compared with the homologues of other frogs, the D-loop region of O. martensii contains Gene arrangement several components characteristic of those in vertebrates: a As shown in figure 1, the mtDNA gene arrangement of termination-associated sequence (TAS), an origin of heavy- O. martensii differs from that of typical vertebrates. The strand replication (OH) and three conserved sequence blocks ND5 gene, accompanied by the tRNA-gene cluster of tRNA- (CSB-1, CSB-2 and CSB-3). The CSB-1 motif of O. marten- Leu(CUN)/tRNA-Thr/tRNA-Pro/tRNA-Phe (LTPF cluster), sii is not reduced to a truncated pentamotif (5-GACAT- are located between the D-loop and 12S rRNA gene. The 3) as in fishes. In addition, a pyrimidine-rich (PP) stretch ND5 gene and LTPF cluster upstream of the 12S rRNA gene is identified between CSB-1 and CSB-2 (figure 3), which are also identified in the bell-ring frog B. buergeri (Sano may be involved in regulatory aspects of OH (San Mauro et al. 2004) with the same order. Further, in tRNA-gene et al. 2004b). There are several small noncoding sequences in cluster of tRNA-Trp/tRNA-Ala/tRNA-Asn/tRNA-Cys/tRNA- O. martensii mtDNA. A 22-base noncoding sequence exists Tyr (WANCY cluster), the tRNA-Asn gene is located between between ND5 and tRNA-Leu genes, a 38-base noncoding tRNA-Tyr and COI genes instead of between tRNA-Ala and sequence is between tRNA-Ser and ND6 genes and 19-base tRNA-Cys genes. The translocation of the tRNA-Asn gene has noncoding spacer exists between ND4L and ND4 genes. In not yet been reported in other vertebrate sequences. addition, there is a 21-base noncoding sequence interspaced

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Table 2. Location of features in the mtDNA of O. martensii.

Length Position Spacer (+); Codon Feature (bp) From To overlap (−) Strand Start Stop

D-loop 2766 1 2766 H ND5 1807 2767 4573 +22 H ATG T tRNA-Leu(CUN) 71 4596 4666 +3H tRNA-Thr 71 4670 4740 H tRNA-Pro 69 4741 4809 −1L tRNA-Phe 70 4809 4878 H 12S rRNA 939 4879 5817 H tRNA-Val 68 5818 5885 H 16S rRNA 1581 5886 7466 H tRNA-Leu(UUR) 71 7467 7537 H ND1 961 7538 8498 H ATT T tRNA-Ile 71 8499 8569 +1H tRNA-Gln 72 8571 8642 +6L tRNA-Met 69 8649 8717 +12 H tRNA-Met 69 8730 8798 +10 H ND2 1027 8809 9835 H ATT TAG tRNA-Trp 69 9836 9904 H tRNA-Ala 70 9905 9974 +21 L OL 33 9996 10028 −9H tRNA-Cys 71 10020 10090 L tRNA-Tyr 67 10091 10157 L tRNA-Asn 73 10158 10230 +3L COI 1548 10234 11781 −5 H GTG AGA tRNA-Ser(UCN) 70 11777 11846 L tRNA-Asp 69 11847 11915 H COII 688 11916 12603 H ATG T tRNA-Lys 69 12604 12672 +1H ATPase8 162 12674 12835 −7HATGTAA ATPase6 682 12829 13510 H ATG T COIII 784 13511 14294 H ATG T tRNA-Gly 69 14295 14363 −3H ND3 345 14361 14705 H ATA TAA tRNA-Arg 68 14706 14773 +1H ND4L 279 14775 15053 +19 H ATG TAA ND4 1368 15073 16440 +5 H ATG AGG tRNA-His 69 16446 16514 H tRNA-Ser(AGY) 66 16515 16580 +38 H ND6 492 16619 17110 +2 L ATG AGA tRNA-Glu 69 17113 17181 +3L Cytb 1137 17185 18321 H ATG TAA

L, gene encoding by the light-strand; H, gene encoding by the heavy-strand.

between tRNA-Ala gene and origin of light-strand replication Phylogenetic analyses ) (OL (table 2). The three phylogenetic analyses of BI, ML and MP, which Although O is located within the WANCY cluster, the L are based on the nucleotide dataset of 13 mt protein-coding OL exists between tRNA-Ala and tRNA-Cys genes instead genes, arrived at similar topologies (only the BI and ML trees of between tRNA-Asn and tRNA-Cys genes (figure 1). This are shown in figure 4). In the reconstructed trees, archaeo- region takes up 33 nucleotides in length and has the poten- batrachians and neobatrachians form monophyletic groups, tial to fold into a stem-loop secondary structure with 11 respectively. Within the archaeobatrachian clade, Pipidae paired nucleotides in the stem and 11 nucleotides in the loop acts as a sister clade to (Bombinatoridae + Discoglossidae). (figure 2a). The 5 -GCCGG-3 conserved motif characteristic The neobatrachian clade is recognized to include two sub- of vertebrate OL is also found at the base of the stem within groups corresponding to the Hylidae + Bufonidae clade (the the tRNA-Cys gene in O. martensii. superfamily Hyloidea, BPP = 1.00 and BP = 100%) and

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Figure 2. (a) Putative secondary structures for the light-strand origin of mtDNA replication of O. martensii and X. laevis (Roe et al. 1985). The 5-GCCGG-3 motif that is predicted to be involved with the transition of RNA to DNA synthesis is indicated by a box. Lines show the nucleotides partially shared with ﬂanking tRNAs. (b) Putative secondary structures for the tRNA-Met in O. martensii and P. nigromaculatus. Copy 1, upstream copy of tRNA-Met; copy 2, downstream copy of tRNA-Met.

Ranidae + Dicroglossidae + Microhylidae + Mantellidae + genes in O. martensii mt genome show high sequence Rhacophoridae clade (the superfamily Ranoidea, BPP = 1.00 similar with other frogs (72.6–81.4%). These sequences and BP = 100%). In addition, within the monophyly of could be folded into typical cloverleaf structures and hold Dicroglossidae, O. martensii stands at the base of the the same anticodon, CAT, which is common in most meta- dicroglossid tree, as a sister to all the remaining groups of the zoan mt tRNA-Met genes. Although, two tRNA-Met genes family. have been reported in the complete mtDNA of F. cancrivora (Ren et al. 2009), F. limnocharis (Liu et al. 2005), L. Discussion bannaensis (Zhang et al. 2009), L. fujianensis (unpub- lished data), H. rugulosus (Yu et al. 2012), H. tigerinus, E. Two tandem tRNA-Met genes and gene rearrangement hexadactylus (Alam et al. 2010), P. spinosa (Zhou et al. In general, most metazoan mtDNAs have only one copy of 2009)andM. madagascariensis (Kurabayashi et al. 2006), tRNA-Met gene, but we found that two tandem tRNA-Met the position of the extra tRNA-Met gene differs between Dicroglossidae and Mantellidae. Based on these differences, Kurabayashi et al.(2006) suggested that two tRNA-Met genes in each lineage are of different origin. The tandem duplication of tRNA-Met gene can be regarded as a synapo- morphic character of Dicroglossidae. Macey et al.(1998) ﬁrst reported a tandem duplication of the mitochondrial tRNA-Thr and tRNA-Pro genes in the amphisbaenian reptile Figure 3. Structure of the O. martensii mtDNA control region. Bipes biporus. They suggested that pseudogene formation in TAS, termination-associated sequence; CSB, conserved sequence tandemly duplicated sequence might be an intermediate step block; PP, pyrimidine-rich region. in mtDNA rearrangement by tandem duplication/deletion

636 Journal of Genetics, Vol. 93, No. 3, December 2014 Mitochondrial genome of round-tongued ﬂoating frog tein-coding genes. the inferred lineages in which gene lyses. Arrows indicate m ML (500 replicates) ana obabilities fro probability from BI and bootstrap pr Bayesian inference (BI) (a) and maximum likelihood (ML) (b) trees of the relationships among 40 anurans based on the nucleotide dataset of the 13 mt pro Numbers above the branches represent posterior Figure 4. rearrangement events occurred.

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Figure 5. Comparison of gene arrangements in the mtDNA genomes of O. martensii and typical vertebrates. Arrows indicate the rearranged homologous genes. model. Our finding of two tandem tRNA-Met genes in O. is not seen in other vertebrate sequences investigated. martensii supports their opinion. OL was not located between tRNA-Asn and tRNA-Cys The ND5 gene accompanied with the LTPF cluster and genes but was located between tRNA-Ala and tRNA-Cys located between the D-loop and 12S rRNA gene have been genes. reported in B. buergeri mt genome. Gene rearrangement The ‘GCCGG’ motif involved in the transition from RNA of mt genomes is generally believed to occur through to DNA synthesis (Hixson et al. 1986) was also found at the tandem duplication of gene regions as a result of slipped- base of the stem within the tRNA-Cys gene in O. marten- strand mispairing followed by multiple deletions of redun- sii. However, the 5 end of OL overlaps by nine bases with dant genes (Moritz and Brown 1987; Boore and Brown the tRNA-Cys gene longer than the corresponding space 1998). According to the gene rearrangement mechanism, (3–4 bp) in other frogs (figure 2a). The rearrangement of Sano et al.(2004) presumed that minimal two duplication– OL and the five tRNA genes surrounding it were reported in deletion events through an intermediate arrangement would Marsupials (Pääbo et al. 1991). In addition, there was a be needed to generate this gene order from that of typical 21-base noncoding sequence interspaced between tRNA- vertebrates. However, according to the parsimony principle, Ala gene and OL. This short noncoding sequence showed they suggested that there might be two rearrangement path- moderate similarity to the relocated tRNA-Asn gene ways. In these hypothetical pathways, the intermediate and it is speculated to represent remnants of deletion arrangement (i.e. D-loop, tRNA-Leu, tRNA-Thr, tRNA-Pro, events. tRNA-Phe and 12S rRNA) of one pathway is identical to that found in ranid frogs, so they suggested that the pathway occurred during anuran evolution. In anurans, although the members and arrangement of the LTPF cluster are slightly different in some taxa, all neobatrachian families reported previously (Bufonidae, Ranidae, Rhacophoridae, Dicroglossidae, Hylidae, Microhylidae and Mantellidae) share this arrangement. Based on this observation, some researchers suggested that the LTPF cluster have occurred in a common ancestral lineage of neobatrachian anurans (Zhang et al. 2005b;Renet al. 2009). The presence of the same tRNA-gene cluster in O. martensii also supports this conclusion. The mtDNA genes arrangement of O. martensii dif- fered from the gene arrangements so far obtained in all other vertebrates reported in the following respects: the tRNA-Asn gene is located between tRNA-Tyr and Figure 6. Putative mechanism of gene rearrangements of the COI genes instead of between tRNA-Ala and tRNA-Cys WANCY cluster in O. martensii according to a model of tandem genes (figure 5). The translocation of the tRNA-Asn gene duplication of gene regions and subsequent gene deletions.

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Possible mechanisms for gene rearrangement to (Mantellidae+Rhacophoridae) with high BPP (1.00) in BI and low BP (77%) in ML, which was consistent with the In general, the observed pattern is largely consistent with the result of MP analysis by Ren et al.(2009). tandem duplication/deletion model, the most general expla- The taxonomy about Chinese ranids has not reached a nation for mt gene rearrangement. According to this model, complete consensus (Che et al. 2007). Fei et al.(1990) the rearrangement of the WANCY cluster in O. martensii are classified Chinese ranids into three subfamilies: Raninae, postulated to have occurred by the tandem duplication of a Amolopinae and Occidozyginae, however, among which gene region from the tRNA-Asn to the tRNA-Tyr gene, fol- Amolopinae and Occidozyginae were only identified as lowed by multiple deletions of redundant genes (figure 6). genus Amolops (sensu lato) and genera Occidozyga, Exploration of this model may also elucidate the formation Phrynoglossus to be respectively included in Raninae and of a tandem duplication of tRNA-Met gene, while the dupli- Dicroglossinae by Dubois (1992, 2005). Later, Chinese cated gene does not come through subsequent deletion. Some ranids were divided into four subfamilies: Raninae, multiple deletions of redundant genes seemed to be incom- Dicroglossinae, Amolopinae and Occidozyginae (Fei et al. plete in O. martensii. This might be responsible for the non- 2005). Frost (2006, 2010) made some major adjustments to coding sequence occurring around the genes involved in the Anura taxonomy and elevated Dicroglossinae from Ranidae rearrangements. As these noncoding sequences were located to family status (included two subfamilies of Dicroglossi- at the original position of the rearranged genes, it was likely nae and Occidozyginae), and proposed two families (Ranidae to be a degenerating vestige of duplicate genes (Liu et al. and Dicroglossidae) for Chinese ranids. This viewpoint was 2005). well corroborated by several studies based on molecular data (Jiang and Zhou 2005;Cheet al. 2007;Wienset al. 2009). Our results are also consistent with the latest taxo- Analyses of the phylogenetic trees nomic systems, Dicroglossidae is a sister clade to (Ranidae The evolutionary relationships of anuran taxa indicated + (Mantellidae + Rhacophoridae)), and the monophyly of in the phylogenetic topologies based on the concatenated Ranidae and Dicroglossidae is well supported (BP = 100% sequences of 13 protein-coding genes (figure 4) largely sup- and BPP = 1.00), which is in accordance with the analysis port the previous classifications (Hedges and Maxson 1993; of mt genome rearrangement (Dicroglossidae retains tandem Duellman and Trueb 1994; Meyer and Zardoya 2003; Zhang duplication of tRNA-Met, but Ranidae has only one copy). et al. 2005b;Igawaet al. 2008;Renet al. 2009). In the trees, O. martensii occupies the basal phylo- Although, the monophyly of the order Anura is widely genetic position among the dicroglossids studied here. This accepted, the monophyly of Archaeobatrachia has been a indicates that Occidozyginae represents the ancient lineage controversy. Early mitochondrial rRNA gene analyses sup- in Dicroglossidae, which is consistent with the opinion ported the monophyly of Archaeobatrachia (Hedges and of Roelants et al. (2004). All trees support the divi- Maxson 1993;Hayet al. 1995). However, some studies sion of the Dicroglossidae into four clades: Occidozygini, based on the morphological data concluded the archaeo- Dicroglossini Paini and Limnonectini. Limnonectini clusters batrachians as a paraphyletic group (Ford and Cannatella 1993; with Paini, Dicroglossini as a sister taxon to Paini + Lim- Duellman and Trueb 1994; Pugener et al. 2003). Inspite of nonectini and Occidozygini as a basal clade to Dicroglos- the low support values (BP = 50%; BPP = 0.89), all phylo- sidae are all well supported. This phylogenetic relationship genetic trees of our study support the monophyly of Archaeo- in family Dicroglossidae are compatible with the viewpoint batrachia, which is consistent with the recent studies using of four groups for Dicroglossidae recommended by Dubois complete mt sequences (Kurabayashi et al. 2006;Renet al. (2005). 2009; Zhou et al. 2009). However, Archaeobatrachia is not generally regarded as a natural group, and paraphyletic rela- Acknowledgements tionships of the taxon were supported by recent studies using nuclear and partial mtDNA data with a degree of taxon sam- This work was sponsored by the special fund for Excellent Creative Research Team of Animal Biology in Anhui Normal University and pling (Hoegg et al. 2004; Roelants and Bossuyt 2005; Irisarri Key Laboratory of Biotic Environment and Ecological safety in et al. 2010; Zhang et al. 2013). Thus, some authors consid- Anhui Province, China. ered that an archaeobatrachian paraphyly could be recovered by adding a novel archaeobatrachian mt genome data (Zhang et al. 2005a). References The relationships among three ranoid families (Ranidae, Mantellidae and Rhacophoridae) have been a subject of Alam M. S., Kurabayashi A., Hayashi Y., Sano N., Khan M. M. R., debate for the last few decades. Several studies based on Fujii T. et al. 2010 Complete mitochondrial genomes and novel morphological, molecular or combined data suggested that gene rearrangements in two dicroglossid frogs, Hoplobatrachus tigerinus and Euphlyctis hexadactylus, from Bangladesh. Genes Ranidae was paraphyletic with respect to Mantellidae and Genet. Syst. 85, 219–232. Rhacophoridae) (Larson 2005;Frostet al. 2006;Igawaet al. Boore J. L. 1999 Animal mitochondrial genomes. Nucleic Acids 2008). In our results, Ranidae was found to be a sister clade Res. 27, 1767–1780.

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Received 27 February 2014, in revised form 17 April 2014; accepted 23 April 2014 Unedited version published online: 25 June 2014 Final version published online: 30 September 2014

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