Molecular phylogeny and species identification of pufferfish of the genus Takifugu (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships and species identification of pufferfishes o f the genus Takifugu were examined by use of randomly amplified polymorphic DNA (RAPD) and sequencing of the amplified partial mitochondrial 16S ribos omal RNA genes. Amplifications with 200 ten-base primers under predetermine d optimal reaction conditions yielded 1962 reproducible amplified fragments ranging from 200 to 3000 bp. Genetic distances between 5 species of Takifu gu and Lagocephalus spadiceus as the outgroup were calculated from the pres ence or absence of the amplified fragments. Approximately 572 bp of the 16S ribosonial RNA gene was amplified, using universal primers, and used to de termine the genetic distance values. Topological phylogenic trees for the 5 species of Takifugu and outgroup were generated from neighbor-joining anal ysis based on the data set of RAPD analysis and sequences of mitochondrial 16S rDNA. The genetic distance between Takifugu rubripes and Takifugu pseud ommus was almost the same as that between individuals within cacti species, but much smaller than that between T. rubripes, T. pseudommus, and the oth er species. The molecular data gathered from both analysis of mitochondria and nuclear DNA strongly indicated that T. rubripes and T. pseudommus shoul d be regarded as the same species. A fragment of approximately 900 bp was a mplified from the genome of all 26 T. pseudommus individuals examined and 4 individuals of intermediate varieties between T. rubripes and T. pseudommu s. Of the 32 T. rubripes individuals, only 3 had the amplified fragment. Th ese results suggest that this fragment may be useful in distinguishing betw een T. rubripes and T. pseudommus.