3. Result

3.1 Morphological identification

According to the morphological characteristics, 616 samples were identified as 50 species, belonging to 23 genera, 6 families and 4 Suborder (Table 1). Among them, 612 samples can be identified to the level of species, 4 samples can only be identified to the level of genus because the sample is juvenile fish and the morphological characteristics are not obvious.
Among the 23 genera of Tetraodontiformes collected in this study, the morphology of the related species of Takifugu is very similar and difficult to distinguish. For example, the morphologic features ofT. alboplumbeus and T. niphobles are so similar that they are difficult to distinguish. It is generally believed that T. niphobles have obvious chest spots, while the chest spots of T. alboplumbeus are not obvious, and there are 4-6 dark bands on the back of T. alboplumbeus . Of the 616 specimens collected in this study, 20 specimens were identified as the T. niphobles , and 11 specimens had the characteristics of the T. alboplumbeus , but they were different from the T. alboplumbeus , because they had only two dark bands. Furthermore, it is also difficult to distinguishT. rubripes , T. pseudommus and T. chinensis , some study suggest T. rubripes has irregular round black spots and white patterns in front of the caudal fin on the sides of the body,T. chinensis has no such black marks while T. pseudommushas white spots scattered on a black background on the dorsal and lateral sides of the body (Baek et al., 2018; Reza et al., 2008). In this study, 8 samples were identified as T. chinensis , 4 asT. pseudommus , and 27 as T. rubripes .
Additionally, the morphological characteristics can distinguishLagocephalus wheeleri (Abe, Tabeta & Kitahama, 1984) fromLagocephalus spadiceus (Richardson, 1845) The former has elliptical dorsal spinule patch, and the latter has a rhomboidal patch with a posterior extension. In this study, 82 specimens were identified as L. wheeleri ,10 were identified as L. spadiceus , and 4 were the intermediate individuals of the two species, that is they have discontinuous patch.

3.2 DNA Barcoding of the Tetraodontiformes

3.2.1 Characteristics of DNA Barcode Sequences of Tetraodontiformes

The results showed that among the 616 COI gene sequences of Tetraodontiformes with a length of 687 bp, there were 370 conserved sites, 62 transition sites (si), 43 transversional site (sv), and the si/sv ratio was 1.4. The results of DNA base content analysis showed that the average DNA base composition of 687 bp COI gene of Tetraodontiformes was: T: 27.7%, C: 29.9%, a: 23.8%, G: 18.6%, and the content of A+T was 51.5%, and the content of C+G was 48.5%. Moreover, the average G+C content of the first base of codon was the highest reaching 56.9%. Additionally, it shows that the sequence similarity of COI gene among the species of Takifugu is very high, and the number of conservative sites was 561 in 236 sequences ofTakifugu with a length of 687bp.

3.2.2 Haplotype analysis of DNA barcodes in Tetraodontiformes

Haplotype analysis showed that 616 COI gene sequences of Tetraodontiformes were divided into 171 haplotypes. Among them, 253 COI gene sequences of Takifugu were divided into 65 haplotypes, and Hap 99 is the same hyplotype of T. pseudommus, T. rubripesand T. chinensis , Hap 136 is the same hyplotype ofT.rubripes and T. pseudommus and Hap 88 is same hyplotype of T. alboplumbeus and T. niphobles . 121 COI gene sequences of Lagocephalus were divided into 36 hapltypes, and Hap 50 and Hap 53 is the same hyplotype of L. wheeleri and L. Spadiceus .

3.2.3 Genetic distance of Tetraodontiformes

The intraspecific genetic distance ranged from 0 to 0.009, and the average intraspecific genetic distance was 0.003. The interspecific genetic distance ranged from 0.001 to 0.298, the average interspecific genetic distance was 0.2, and the ratio of the average interspecific genetic distance to the average intraspecific genetic distance was 70.63. The average genetic distance within genus was 0.016. The genetic distance between genera ranged from 0.12 to 0.298, and the average genetic distance between genera was 0.223. The average genetic distance within the family was 0.106. The genetic distance between families ranged from 0.192 to 0.247, and the average genetic distance between families was 0.229.
The results showed that the interspecific genetic distance betweenT. pseudommus, T. rubripes, and T. chinensis was very small, and the range of interspecific genetic distance between them was 0.001 to 0.002, the average interspecific genetic distance was 0.002, and the interspecific genetic distance betweenT. alboplumbeus and T. niphobles is 0.002. Additionally, the interspecific genetic distance between L. wheeleri and L. Spadiceus is also very small, only 0.003.
Moreover, the results showed that the species of Takifugu have close genetic relationship. If T. rubripes , T. pseudommusand T. chinensis are taken as one species, alboplumbeusand T. niphobles are considered as one species, there are 14 species of this genus in this study. And the average interspecific genetic distance is 6.21 times of the average intraspecific genetic distance, which does not reach the DNA barcode threshold of more than 10 times proposed by Hebert. Among them, the genetic distance betweenT. oblongus and T. stictonotus is the largest, 0.045; The interspecific genetic distance of several species in the genera is less than 0.02 (as shown in Table 2), the genetic distance between T. bimaculatus and T. flavidus is the smallest, only 0.013.

3.2.4 Phylogenetic relationships of Tetraodontiformes

As shown in Figure 1, in the Neighbor-joining tree based on the haplotype of COI gene of Tetraodontiformes, the species of 6 families collected in this study are divided into two clades, Diodontidae and Tetraodontidae clustered into a clade, Monacanthidae, Balistidae, Ostraciidae and Triacanthidae clustered into a clade, as shown in Figure 1. Moreover, the results showed that Ostraciidae and Triacanthidae clustered in separate clades, as did Diodontidae and Tetraodontidae.
In the Neighbor-joining tree, T. rubripes , T. pseudommusand T. chinensis clustered into the same clade, and T. alboplumbeus and T. niphobles clustered into the same clade, as did L. wheeleri and L. Spadiceus . If taken the species clustered into the same clade as one species. In the Neighbor-joining tree, each species can gather into a separate clade except for the species that may be synonyms, although the interspecific genetic distance of some species of Takifugu is less than 0.02(as shown in Table 2).

3.2.5 DNA barcode classification and identification of Tetraodontiformes

The DNA barcoding identification result shows that 616 samples were identified as 46 species, belonging to 23 genera, 6 families and 4 Suborder (Table 1). Among them, DNA barcoding analysis showed that the specimens identified as T. rubripes , T. pseudommus , and T. chinensis had the same haplotype, the range of interspecific genetic distance between them was 0.001 to 0.002, and the average interspecific genetic distance was 0.002. In additional, in the NJ tree, they clustered into one clade. According to the DNA barcode identification results, T. rubripes , T. pseudommus and T. chinensis should be the synonyms. According to the principle that the first published species name is the valid name, for the three species,T. rubripes is the valid species name. Furthermore, the same results were found in L. wheeleri and L. Spadiceus . It showed that the specimens identified as L. wheeleri and L. Spadiceus had the same haplotype, the interspecific genetic distance between them was only 0.003, and they clustered into one clade in NJ tree. The results showed that L. wheeleri and L. Spadiceusshould be synonyms, and L. Spadiceus is valid.
For T. alboplumbeus and T. niphobles , the DNA barcode identification results also showed that the two species may be synonyms. However, since the 11 specimens identified as T. alboplumbeus do not completely have the morphological characteristics of T. alboplumbeus , it is not certain that these specimens are hybrids ofT. alboplumbeus and T. niphobles . Or that T. alboplumbeus and T. niphobles are the same species, but they will undergo a morphological transformation process.