DNA Barcoding Of Tetraodontiformes In Coastal Waters Of China

The order Tetraodontiformes comprises approximately 350 species overall and are divided into 100 genera, 10 families, 6 superfamilies. Most dwell in tropical and subtropical Pacific, Indian and Atlantic Ocean, but a few species live in freshwater or enter into rivers in some season. Many species are economically-important subjects of aquaculture and marine fisheries, of which Thamnaconus modestus and T. hypargyreus are facing up resource depletion, Takifugu species are highly valuable in aquaculture, tetrodotoxin exploitation and vertebrate genome research, meanwhile poisoning and even death events by eating puffer fish have been frequently reported. Correct species identification can effectively differentiate tetrodotoxin species, and promote the exploitation of fisheries resources, ecological investigation and biodiversity conservation, however Tetraodontiforms are notable for highly modified skeletons and various morphological characters, but traditional taxonomy are mainly restricted to stripe and color patterns, body shape and other external appearances, which are easily subjected to the effects of subjective and environmental factors, thus the taxonomy of Tetraodontiformes are still controversial. By comparing a standard short DNA fragments DNA Barcoding is recommended as a method for rapid and accurate species identification and has been successfully used in animals, plants and microorganism. In present study, 652 bp segments of cytochrome oxidase subunit I(COI) gene from 110 specimenss representing 33 species, 19 genus, 8 familes were determined, combined with available data downloaded from Gen Bank, overall 210 barcodes from 56 species, 25 genus, and 8 families were evaluate the species status and phylogenetic relationships among species in Tetraodontiformes in China waters. The main results are as following:In the neighbor-joining tree, the majority of putative species clustered together, which are largely concordant with morphological taxonomy. Except for Lagocephalus, the monophyly of most genus were supported with high bootstrap values, but phylogenetic relationships above genus were poorly resoloved. The average K2 P distances within species, genus, family and order were 0.56%(0-5.41%)、7.35%(0.03%-17.09%)、16.57%(0.89%-22.74%)and 20.38%(18.59%-27.04%)respectively, of which genetic distance within 53 species were less than 2%, the intraspecies genetic distance threshold, and genetic distances within 48 species were less than 1%. The ratio of average interspecies vs intraspecies genetic distances of 56 species were 34, of which the minimum interspecies genetic distances of 51 species were larger than the maximum intraspecies genetic distances, and there were overlap between interspecies and intraspecies genetic distances, suggesting the existence of good DNA barcoding gap. As interspecies genetic distances in Lagocephalus is 16.57%, approaching those among genus, the phylogenetic position for this genus remained to be resolved. The genetic distances within species like Kentrocapros aculeatus and Rhynchostracion nasus were much higher than 2%, suggesting that the possibility of cryptic species. Thamnaconus septentrionalis might be junior synonymous to T. hypargyreu, and Takifugu poecilonotus might be junior synonymous to T. alboplumbeus. No barcoding gap were found in T.basilevskianu and T.rubripes、T.bimaculatus and T.xanthopterus in genus Takifugu, which might reflect production of evolutionary radiation and the explosive speciation. In conclusion, the 652 bp segement of mt DNA COI gene is an effective DNA barcoding marker for accurate identification of Tetraodontiformes species and preliminary evaluation of relationships among species within the same genus, it contains too few information to resolve phylogenetic relationship among genus level and above.