Rana tigrina ssp. The Chinese edible frog , East Asian bullfrog , or Taiwanese frog Hoplobatrachus rugulosus is a species of frog in the family Dicroglossidae. Its natural habitats are freshwater marshes , intermittent freshwater marshes, arable land , pasture land, rural gardens, urban areas , ponds , aquaculture ponds, open excavations, irrigated land, seasonally flooded agricultural land, and canals and ditches. The domesticated Thai variety and wild Chinese populations of H. They are primarily insectivores.

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The bred tiger frog known as the Thailand tiger frog, is also identified as H. Our analysis of the Cyt b gene showed high genetic divergence Unexpected genetic divergence of the complete mt genome Despite this and their very similar morphology, the features of the mitochondrial genome including genetic divergence of other genes, different three-dimensional structures of ND5 proteins, and gene rearrangements indicate that H.

Using Bayesian inference, maximum likelihood, and maximum parsimony analyses, Hoplobatrachus was resolved as a sister clade to Euphlyctis , and H. We suggest that we should prevent Thailand tiger frogs bred type from escaping into wild environments lest they produce hybrids with Chinese tiger frogs wild type. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its supporting information files. Competing interests: The authors have declared that no competing interests exist. This genome typically contains 37 genes 2 rRNAs, 22 tRNAs, and 13 protein-coding genes and a long non-coding region called the control region or D-loop region [ 1 , 2 ].

The mitochondrial mt genome has several valuable characteristics, including small size, fast evolutionary rate, relatively conserved gene content and organization, maternal inheritance, as well as limited recombination [ 3 — 6 ]. Mitochondrial genomes are useful molecular markers in cryptic species identification because of the differences in compositional features, divergence of protein-coding genes, number and size of non-coding regions, and gene arrangement [ 7 — 9 ]. According to the Amphibian Species of the World 5.

Among them, species and subspecies can be found in China [ 11 ]. The genus Hoplobatrachus with five species is important among the Dicroglossini of Dicroglossinae [ 12 — 14 ].

Only one of these species, known as the Chinese tiger frog occurs naturally in wild environments in China [ 10 , 11 , 15 ]. This frog has been identified as Hoplobatrachus rugulosus [ 13 ], although some Chinese researchers insist that it should be named Hoplobatrachus chinensis , believing this to be a senior synonym of H.

A bred tiger frog introduced to China from Thailand is called the Thailand tiger frog by Chinese, and has also been identified as H. Thailand tiger frogs are bred in many farmers for local meat consumption. But more and more Thailand tiger frogs have been captured in the field after escaping from farms, which may affect the diversity of local Chinese tiger frogs.

Alam et al. They suggested that H. Pansook et al. In this study, we also found a high divergence between Chinese tiger frog and Thailand tiger frog using Cyt b gene. A cryptic species complex is a group of organisms that are typically very closely related yet their precise classification and relationships cannot be easily determined, although some can be separated by DNA sequence analyses [ 18 — 20 ].

Using the complete mt genome, Alam et al. The current work aimed to investigate further the differences between Chinese tiger frog and Thailand tiger frog, as well as to determine whether cryptic species are present in H.

Accordingly, we determined the complete mt genome sequences of Thailand tiger frog bred type BT , and then compared the differences in gene arrangement , base compositional features , and genetic divergences of mt genes between Chinese tiger frog wild type WT and Thailand tiger frog BT.

The protein structures of the ND5 genes in Hoplobatrachus were also compared. Additionally, we sequenced nuclear genes NCX1 , Rag1 , Rhod , and Tyr of wild and bred tiger frogs to examine their genetic divergence.

We also performed molecular phylogenetic analyses to discuss the relationship between Chinese tiger frog and Thailand tiger frog, and all available dicroglossids including Occidozyga martensii , based on the 11 mt protein-coding genes using five Ranidae species as out-groups. We follow the names proposed by Frost et al.

The officers of Forest Administrative Bureau of Jinhua have seized these wild frogs, which died during illegal captivity. The Thailand tiger frog samples bred tiger frogs were purchased from various farms from Jinhua, Zhejiang province and sacrificed using ether in our laboratory. The husbandry and breeding procedures of the Thailand tiger frogs in the farms were carried out under the Animal Husbandry Law of the People's Republic of China.

Information for all samples is shown in S1 Table. A sample of the Thailand tiger frog No. THW1 was used to amplify the complete mt genome; other samples were used to amplify the partial Cyt b gene.

Cyt b gene was amplified by normal PCR using primers described by Pansook et al. We amplified overlapping fragments that covered the entire mt genome of H. The long fragments were sequenced using specific primer walking of both strands. Sequences were checked and assembled using SeqMan Lasergene version 5.

The locations of the 13 protein coding genes and 2 rRNA genes were determined by comparing the homologous sequences of other anurans using Clustal W in Mega 5. The organization of the H. The complete mtDNA sequence of H. Gene name inside indicates the direction of transcription from left to right, and gene name outside indicates right to left. A total of 55 sequences of the Cyt b gene, including 19 from this study, in-group species from Pansook et al.

All 55 sequences yielded bp of Cyt b gene fragment, including variable and parsimony-informative sites. Phylogenetic relationships were constructed by neighbor joining NJ analysis in Mega 5. Statistical support was evaluated using bootstrap replicates. Using the data with 20 sequences yielded bp of Rag1 , Rhod , and Tyr genes excluding NCX1 gene as no squeence of Hoplobatrachus is reported in GenBank , including 36 variable sites.

The outgroup was combined H. Phylogenetic relationships were also constructed by neighbor joining NJ analysis in Mega 5. To confirm further the phylogenetic relationships of the Chinese tiger frog and the Thailand tiger frog among dicroglossids, 16 available complete mt genomes of anurans based on the addition of two Hoplobatrachus mt genomes, including five species of Ranidae Babina adenopleura , Pelophylax nigromaculata , Pelophylax plancyi , Odorrana ishikawae , and Odorrana tormotus [ 22 , 36 , 37 ] as out-groups, were retrieved from GenBank S2 Table.

We constructed the phylogenetic trees based on a concatenated set of 11 mt protein-coding genes excluding ND5 because of the high sequence divergence of its two copies in H. The nucleotide sequences for each of the 11 mt protein-coding genes were aligned using Clustal W in Mega 5. To select the conserved regions of the sequences, each alignment was analyzed with Gblocks 0. We concatenated the alignments of the 11 mt protein-coding genes, and recovered an alignment consisting of amino acid residues.

An alignment of nucleotides was obtained using the amino acid alignment as the backbone reference. A saturation analysis was performed for subsets with first, second, and third codon positions using DAMBE 4. The results showed that the third codon positions were saturated.

Consequently, they were excluded from the final nucleotide alignment and an alignment of nucleotides was obtained. A total of bootstrap replications were generated, each with 10 replications with random taxon order. Model selection for the nucleotide dataset was performed with Modeltest version 3.

Bayesian inference BI analysis of the nucleotide dataset was performed with MrBayes 3. Eight chains were run in parallel for 10 generations, with trees sampled every generations. Bayesian posterior probabilities were calculated according to the remaining set of trees. All Markov chain Monte Carlo runs were repeated twice to confirm consistent estimation of the posterior parameter distributions.

The complete mt genome of H. The overall base composition of the H-strand was as follows: A The nucleotide divergence of the complete mt genome was There were six reading frame overlaps in the mt genome of H. Other overlaps are shown in Table 2. Two ND5 genes with The dataset comparing the two ND5 genes in H. The codon frequency of the two ND5 genes in H.

Protein-coding genes in H. In the ND gene, H. Three protein-coding genes surprisingly showed different lengths between H. The divergence of nucleotides and amino acids in protein-coding genes using the uncorrected p -distance model between H. The divergence uncorrected p -distances of nucleotides and amino acids in protein-coding genes among Hoplobatrachus ranged from The nucleotide divergence of the two pairs of ND5 genes in H. The mt genome of H.

The non-coding regions of H. The length of control regions was similar to those of other anurans from bp to bp. Some short non-coding sequences also occurred in H. This region was 30 bp long and had the potential to fold into a stem-loop secondary structure with a stem formed by 9 paired nucleotides and a loop of 12 nucleotides.

In this study, we analyzed 12 sequences of O L in Dicroglossidae, and found that the sequence of H. Dashes — indicate Watson—Crick base pairing. Fig 4 shows the NJ phylogenetic analysis among Hoplobatrachus based on Cyt b gene. Chinese tiger frog H. Thailand tiger frog H. The divergence between two clades of H. Phylogenetic analysis was carried out for the 41 tiger frogs using the Cyt b gene.

The tree was rooted with two out-groups E. Numbers at the nodes are NJ bootstrap values. BI, ML, and MP phylogenetic analyses based on the nucleotide dataset of 11 protein-coding genes had a similar topology Fig 5 , which is consistent with Zhang et al. In this study, we recovered topological relationships among dicroglossid clades with high bootstrap and posterior probability Fig 5.

The nucleotide dataset also favored a topology that placed Hoplobatrachus as a sister clade to Euphlyctis posterior probabilities 1.


Chinese edible frog

Systems used to automatically annotate proteins with high accuracy:. Select item s and click on "Add to basket" to create your own collection here entries max. Automatic assertion inferred from database entries i. You are using a version of browser that may not display all the features of this website.



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