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Comparisons of mitochondrial DNA and ribosomal DNA to elucidate the phylogeny and speciation of amphibians
(a) Comparisons of mitochondrial DNA to elucidate phylogeny and speciation
The nucleotide sequences of the mtDNA genes were investigated using Japanese pond frogs to clarify the phylogenetic relationships at the nucleotide sequence level. Based on a conventional mtDNA clock calibration of 2~4% nucleotide sequence divergence per million years, Rana nigromaculata and Rana porosa are roughly estimated to have diverged about three to five million years ago, and Rana porosa porosa and the two local races of Rana porosa brevipoda are estimated to have diverged about one to two million years ago (Fig. 1). These results imply that the rate of sequence change for the cytochrome b gene is about two or three times greater than that for the 12S rRNA gene at the specific level of the Japanese pond frog. The cytochrome b gene sequences are apparently adequate to resolve phylogenetic relationships among Japanese pond frogs, whereas the 12S rRNA gene sequences are not informative at racial or subspecific level and should be used only at taxonomic levels higher than species (Fig. 2).
(b) Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the Japanese pond frog Rana nigromaculata
The complete nucleotide sequence of the mitochondrial genome of the Japanese pond frog Rana nigromaculata was determined. The length of the sequence was 17,804 bp (Fig. 3). A comparison of gene organization between two amphibian species, Rana and Xenopus, provided evidence that the gene arrangement of Rana differs by four tRNA gene positions from that of Xenopus, a species with a gene arrangement common among vertebrates (Fig. 4). Evolutionary relationships were estimated from the concatenated sequences of the 12 proteins encoded in the H-strand of mtDNA among 37 vertebrate species. A quartet puzzling tree showed, firstly, that three amphibian species form a monophyletic clade, and secondly, that the caecilian is a sister group of the monophyletic Anura (Fig. 5).
(c) Natural hybridization and introgression in a hybrid zone
The pond frogs of the Sengokuhara population from central Japan were analyzed for nuclear and mitochondrial DNA to elucidate the extent of natural hybridization and gene introgression in this population. As the outcome of long-term hybridization, there was some likelihood of introgression of genetic material between Rana nigromaculata and Rana porosa porosa through female hybrids and backcrosses (Fig. 6).
(d) Sequencing and analysis of the ribosomal DNA of the Japanese pond frog Rana nigromaculata
The nucleotide sequence of the EcoR I fragment of the rDNA of Rana nigromaculata was determined by the use of the deletion mutant and primer extension methods. The results showed that the EcoR I fragment was 4,806 bp long and included the ITS 1, 5.8S rRNA, ITS 2, the 3'-terminus of 18S rRNA, and the 5'-terminus of 28S rRNA (Fig. 7). The pylogenetic analysis was carried out using the nucleotide sequences of the 5.8S rDNA gene of various taxa (Fig. 8).
The nucleotide sequences of the mtDNA genes were investigated using Japanese pond frogs to clarify the phylogenetic relationships at the nucleotide sequence level. Based on a conventional mtDNA clock calibration of 2~4% nucleotide sequence divergence per million years, Rana nigromaculata and Rana porosa are roughly estimated to have diverged about three to five million years ago, and Rana porosa porosa and the two local races of Rana porosa brevipoda are estimated to have diverged about one to two million years ago (Fig. 1). These results imply that the rate of sequence change for the cytochrome b gene is about two or three times greater than that for the 12S rRNA gene at the specific level of the Japanese pond frog. The cytochrome b gene sequences are apparently adequate to resolve phylogenetic relationships among Japanese pond frogs, whereas the 12S rRNA gene sequences are not informative at racial or subspecific level and should be used only at taxonomic levels higher than species (Fig. 2).
(b) Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the Japanese pond frog Rana nigromaculata
The complete nucleotide sequence of the mitochondrial genome of the Japanese pond frog Rana nigromaculata was determined. The length of the sequence was 17,804 bp (Fig. 3). A comparison of gene organization between two amphibian species, Rana and Xenopus, provided evidence that the gene arrangement of Rana differs by four tRNA gene positions from that of Xenopus, a species with a gene arrangement common among vertebrates (Fig. 4). Evolutionary relationships were estimated from the concatenated sequences of the 12 proteins encoded in the H-strand of mtDNA among 37 vertebrate species. A quartet puzzling tree showed, firstly, that three amphibian species form a monophyletic clade, and secondly, that the caecilian is a sister group of the monophyletic Anura (Fig. 5).
(c) Natural hybridization and introgression in a hybrid zone
The pond frogs of the Sengokuhara population from central Japan were analyzed for nuclear and mitochondrial DNA to elucidate the extent of natural hybridization and gene introgression in this population. As the outcome of long-term hybridization, there was some likelihood of introgression of genetic material between Rana nigromaculata and Rana porosa porosa through female hybrids and backcrosses (Fig. 6).
(d) Sequencing and analysis of the ribosomal DNA of the Japanese pond frog Rana nigromaculata
The nucleotide sequence of the EcoR I fragment of the rDNA of Rana nigromaculata was determined by the use of the deletion mutant and primer extension methods. The results showed that the EcoR I fragment was 4,806 bp long and included the ITS 1, 5.8S rRNA, ITS 2, the 3'-terminus of 18S rRNA, and the 5'-terminus of 28S rRNA (Fig. 7). The pylogenetic analysis was carried out using the nucleotide sequences of the 5.8S rDNA gene of various taxa (Fig. 8).