| Tetraodontiformes has a long edible history in China and Japan,and it is called a precious aquatic product for its high protein content and extremely delicious taste.many of them contain toxic tetrodotxin(TTX)in the ovary,liver,kidney,eyes and blood.If they are not handled properly or eaten by mistake,people will be poisoned or even die.In traditional taxonomy,the main distinguishing basis for the classification and identification of Tetraodontiformes is the morphological characteristics of stripes,spots,body color and so on.However,it is difficult to accurately identify species only based on morphological characteristics,and the phenotypic plasticity of species may also lead to misidentifications.Moreover,most of the commodities of the puffer fish in the market have been processed and lost their original morphological characteristics.DNA barcoding is used to identify species at molecular level,which is not affected by time,region and individual morphology In this study,616 samples of Tetraodontiformes collected from coastal China were identified by DNA barcoding technology and morphological methods.At the same time,154 COI gene sequences of Tetraodontiformes were screened from NCBI.Based on the COI gene sequences of 324 fishes belonging to 10 families,55 genera and 129 species of Pufferforma,a DNA barcoding classification system for pufferforma fishes was established.The main research results are as follows:(1)According to the morphological characteristics,50 species were preliminarily identified,belonging to 23 genera,6 families and 4 suborder.Among them,DNA barcoding analysis showed that the specimens identified as Takifugu rubripes(Temminck & Schlegel,1850),Takifugu pseudommus(Chu,1935)and Takifugu chinensis(Abe,1949)had the same haplotype,and the interspecific genetic distance between them was only 0.003 And they clustered into the same clade in NJ tree.In the Fish Base and the Worms,these three species are all valid.Nevertheless,this study showed that three species are synonyms,and T.rubripes is valid.The same situation also happens in Lagocephalus wheeleri(Abe,Tabeta & Kitahama,1984)and Lagocephalus Spadiceus(Richardson,1845).Our study showed that the two species are synonyms and L.Spadiceus is valid.(2)The results of this study indicate that the species of Takifugu have close genetic relationship.It showed that the average intraspecific genetic distance of 14 species of Takifugu was 0.005,and the average interspecific genetic distance was only 0.036.And the average interspecific genetic distance is 7.20 times of the average intraspecific genetic distance,which does not reach the DNA barcode threshold of more than 10times proposed by Hebert.Therefore,the Genetic distance threshold of 0.02 proposed by Hebert et al.is not applicable to the species of this genus.(3)In addition,based on mitochondrial COI gene haplotypes,the phylogenetic tree of Tetraodontiformes was constructed.The results showed that 10 families are splited into two clades,Aracanidae and Triacanthodidae was clusted into one clade,the other 8families gather into one clade.Among them,Molidae is mixed into Monacanthidae,Triodontidae and Diodontidae are the sister groups of each other,and Tetraodontidae and Monacanthidae are the sister groups of each other.In Tetraodontidae,Takifugu and Torquigener gather into one clade,Tetraodon and Chelonodon are sister groups.In Takifugu,the sister group formed by T.snyderi and T.stictonotus is located at the base of Takifugu;T.ocellatus and T.poecilonotus gather into a monogenic sister group,T.pardalis and T.alboplumbeus show a relatively close kinship,T.bimaculatus and T.flavidus gather into a monogenic sister group.The results of this study showed that in the phylogenetic tree of Tetraodontiformes based on 324 COI genes,family can gather in their respective branches according to family,except for Molidae,which is mixed in Monacanthidae;Except for 7 species including B.viridescens in Balistoides,B.punctatus in Balistes,genera can be clustered according to families;Even if there are interspecific genetic distances in Takifugu less than 0.02,in the NJ tree,Takifugu can gather into single clade of species.Therefore,it indicates that the COI gene sequence is suitable for the classification and identification of genera and lower taxonomic levels of Tetraodontiformes.(4)Finally,this study established a rapid detection method for common puffer fish in the market by designing specific primers for common puffer fish in the market and using common PCR technology to identify the species of samples according to the length and presence of electrophoresis bands.The results showed that the common puffer fish specific primers designed in this study were universal to target species and specific to related species,and could be used for rapid identification of common puffer fish in the market.In conclusion,this study clarified the synonyms of several species,provided the molecular basis for the confirmation of effective species names,enriched the DNA barcode database of Tetraodontiformes and improved the DNA barcoding classification system of the order.And it provided a scientific basis for the study of classification,identification conservation,evolutionary of this order. |
