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.