Molecular Phylogeny Of Chinese Genus Limnonectes And Complete Mitochondrial Genomes Of L. Kuhlii And L. Fragilis

The present thesis includes two parts : Part One:Reviews Three species of Chinese group of Limnonectes were simply introduced,and the systematic studied on Limnonectes. Particularly, it was summarized at morphological, chromosomal and nuclear acid levels. An interesting aspect of the evolution of Limnonectes is the shift in chromosome number that has taken place.Limnonectes ( including R. kuhlii from Taiwan) was further partitioned into four species groups by Evans and Emerson (2000,2003). Part Two: Studies 1. Partial sequences of the mitochondrial 12S rRNA and 16S rRNA gene were determined for 8 populations of three species of Chinese Limnonectes, and aligned with the published sequences of Limnonectes from other parts of the world. When Nanorana parker, Paa boulengeri, Fejervarya limnocharis and Hoplobatrachus rugulosus was used as outgroup taxa (Accession Nos. AY158705, AY313685, AF206111, AF206491, AY322311). We reconstructed phylogenetic trees using Clustal X 1.8,Mega 2.1 and PHYLIP 3.5c software, and using the NJ,MP and ML methods, respectively. Our analyses suggest that these fanged frogs from China are another monophyletic group in addition to the four monophyletic groups identified by previous studies. The Chinese Limnonectes were grouped into three clades : Limnonectes fragilis from Hainan Province, Limnonectes kuhlii from Yunnan Province,and Limnonectes fujianensis from Fujian Province + one from Taiwan Province. 2. The mitochondrial DNA as a model system is being used as a powerful tool in phylogeny analysis.At least 350 chordata mt genomes were sequenced so far.They always show a great invariability in gene contents(consist of 22 tRNA ,2 rRNA, 13 protein-coding genes and a control region ) and genome size,But the variability of the sequences of mt DNA and a great variety of the gene rearrangement often happened .So,the analysis of them plays an important role in resolving the debates of vertebrate phylogeny. We obtained the complete mitochondrial genome of L. kuhlii and L. fragilis by DNA sequencing based on the PCR fragments of 20 primers we desigened.The complete mt DNA of L. kuhlii is 16 867 base pairs (bp) in length,and that of L. fragilis is 16 640 bp. L. kuhlii contains genes for 19 transfer RNAs, 2 ribosomal RNAs ,13 protein-coding genes and a D-loop region, and L. fragilis contains genes for 17 transfer RNAs, 2 ribosomal RNAs ,13 protein-coding genes and a D-loop region. They lack 4-6 tRNAs and the corresponding sequences have become a pseudogene or an aberrance of codons or noncoding sequences, Besides an extra copy of the tRNA-Met gene, There are an additional noncoding region of 60-80 bp between tRNA-Ser(AGY) and ND5, between ND5 and ND6 in the complete mitochondrial genome of L. kuhlii and L. fragilis .This thing is hardly observed in other vertebrate mitochondrial genomes. 3. In order to insure validity of our results,we validated them again by 5 primers desiged renewedly for further PCR amplifications.