4. Discussion

4.1 DNA barcoding threshold of Tetraodontiformes

DNA barcoding is used to identify species at molecular level, which is independent of time, region and individual morphology (Hebert, Ratnasingham & Waard, 2003). This study shows that DNA barcoding can quickly and accurately identify the species of Tetraodontiformes, without being affected by key factors such as the integrity and development stage of the sample and the taxonomic expertise of the appraiser. Besides, for those closely related species, the COI gene sequence may have high homology, but they can still be distinguished by phylogenetic tree. For example, the sequence similarity betweenT. flavidus (Hap 100) and T. bimaculatus (Hap 91) has reached 98.7% (678 conserved sites in the 687 bp COI gene sequence). And in Takifugu , 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 between T. 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. Therefore, the genetic distance threshold of 0.02 proposed by Hebert et al. is not applicable to the species of this genus.

4.2 Quality control of sequences downloaded from databases

NCBI’s GenBank and BOLD are the two most abundant databases of DNA barcode resources. In recent years, the number of DNA barcode sequences submitted to NCBI and BOLD has increased explosively. When we use these two databases for DNA barcode sequence alignment analysis, we sometimes encounter inaccurate matching results. It has been reported that there are many errors in the sequences of NCBI database (Shen et al., 2013; Liu et al., 2020). Some studies also believe that BOLD database has conducted more rigorous review and screening on the submitted sequences, so the data in this database is more reliable (Wang et al., 2009; Macher, Macher & Leese, 2017). In fact, there are also wrong sequences in BOLD database (Lis, Lis & Ziaja, 2016).
In this study, all sequences were run BLAST in NCBI and BOLD databases. When BLAST is run on the sequence of most samples in the database, the sequences with the top similarity match are only the sequences of one species, which is consistent with the identification result. However, sometimes in the highly similar sequences of the results, in addition to the sequences of the species that are consistent with the identification results, there will also be a few sequences of the species that are inconsistent with the identification results. For instance, two published COI gene sequences of T. porphyreus (KT951818, KT951819) in the database are 100% similar to the sequences of T. alboplumbeus samples. When these two sequences were run BLAST in the database, the highly similar sequences in the result are all sequences of T. alboplumbeus , except for the two sequences of T. porphyreus , and these two sequences cannot match other sequences ofT. porphyreus in the database. Therefore, the information of the two sequences in the database is incorrect. In addition, the sequence ofT. chinensis , T. pseudommus and T. rubripes showed 99.56% similarity to the two mitochondrial genome sequences (NC_024199, KJ562276) of T. flavidus in BOLD and NCBI. After checking, it was found that the information of these two sequences was also wrong.Therefore, there are some incorrect sequences of Tetraodontiformes in the NCBI and BOLD databases.
The unreliable data in the databases will directly lead to the misidentification of species. In order to reduce the interference of the wrong sequence in the database on the analysis results, it is suggested that the relevant sequences in the database should be strictly screened when using DNA barcode technology to identify species. In particular, if the information in NCBI and BOLD databases is found to be inaccurate when the voucher specimens with complete morphological characteristics are available, the database or data submitter shall be contacted in time to correct the sequence information.

4.3 Synonym phenomenon in the order of Tetraodontiformes

For reasons such as untimely information exchange, and the morphological characteristics of fish are not only susceptible to subjective factors, but also will change significantly in different developmental stages and environmental conditions, the same species may have two or more different Latin names, which is also called synonym.
This study shows that there are a lot of synonyms in Tetraodontiformes. For example, sequences of the samples morphologically identified as P. leiuruswere identified as M. Leiurus in NCBI, and there is no information about P. leiurus can be found in NCBI. In the FishBase (https://www.fishbase.de/), P. leiurus and M. Leiurus are synonyms, and P. leiurus is the valid name for this species. What’s more, the samples which morphologically identified asD. Nigroviridis , in NCBI and BOLD databases, they were identified as T. Nigroviridis . In fact, in the FishBase D. Nigroviridis and T. nigroviridis are synonyms, and the name ofD. nigroviridis is valid. The variety of species names in the database will affect the molecular identification results of species.We suggest that the unsynchronized information in the database is also one of the reasons for the confusion of species identification of Tetraodontiformes.
In addition, in the FishBase, it is considered that both L. wheeleri from L. spadiceus are effective species. And the morphological characteristics, can distinguish Lagocephalus wheeleri (Abe, Tabeta & Kitahama, 1984) from Lagocephalus spadiceus (Richardson, 1845) The former has elliptical dorsal spinule patch, and the latter has a rhomboidal patch with a posterior extension. Some studies have suggested that these morphological features cannot be used to distinguish L. wheeleri from L. spadiceus , and that L. wheeleri may be a form of L. spadiceus , so the two species are actually the same species (Matsuura, 2010; Sakai, Sakamoto, & Yoshikawa, 2021). In this study, the results of haplotype analysis showed that they had the same haplotype. And in the Neighbor-joining tree, they clustered on the same clade, and the interspecific genetic distance was only 0.003. It was supported the view that L. wheeleri is the synonym of L. spadiceus .
Moreover, there are many reports believe that T. rubripes ,T. chinensis and T. pseudommus may be different phenotypes of the same species, T. chinensis and T. pseudommus are the synonyms of T. rubripes (Liu et al., 1999; Song et al., 2001; Cui et al., 2005; Reza et al., 2008; Park et al., 2020). However, it is considered that all three species are are valid in the FishBase, and they can be distinguished with morphological characteristics. 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. pseudommus has 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 asT. chinensis , 4 as T. pseudommus , and 27 as T. rubripes , and it was found that the samples morphologically identified as the three species 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. It was proved that the T. chinensis and T. pseudommus are the synonyms of T. rubripes , and T. rubripes is the valid name.
And 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 several dark bands on the back of T. alboplumbeus . In this study, 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. Matsuura (2017) regarded T. niphobles and T. alboplumbeus as synonyms based on the same color patterns and no differences in morphological characteristics between the type specimens. Some researchers support this view as well (Okabe et al., 2019), and these two species are also considered to be synonyms in NCBI. However, in the FishBase, it is considered that both T. alboplumbeus and T. niphobles are effective species. Zhang & He (2008) showed that 12s and cytb genes could separate T. alboplumbeus and T. niphobles , but the study did not describe the morphological characteristics of the two species. Zhou et al. (2020) Showed that the mitochondrial genome can separate T. alboplumbeusand T. niphobles effectively. However, the study did not describe the morphological characteristics of the two species as well. Therefore, whether T. alboplumbeus and T. niphobles are synonyms has not been determined.

4.4 Reconstruction of Tetraodontiformes phylogenetic relationships

Many researchers have studied the relationship between families of Tetraodontiformes based on different methods (Breder & Clark, 1947; Winterbottom, 1974; Rosen, 1984; Leis, 1984; Tyler & Sorbini, 1996; Santini & Tyler,2003; Holcrof, 2005; Alfaro, Santini & Brock, 2007; Yamanoue et al., 2007; Yamanoue et al., 2008;). In their study, there were more or less changes in the phylogenetic tree of Tetraodontiformes. Similarly, they agreed that Balistidae and Monacanthidae were closely related, and Diodontidae and Tetraodontidae were closely related as well.
In this study, the Neighbor-joining tree based on the haplotype of COI gene of Tetraodontiformes shows that the species of 6 families are splited into two clades. And Diodontidae and Tetraodontidae clustered into a clade, Monacanthidae, Balistidae, Ostraciidae and Triacanthidae clustered into a clade, as shown in Figure 1. The result of that Diodontidae and Tetraodontidae clustered in a separate clade, is consistent with the research results of others. However, unlike other studies, Balistidae and monacanthidae did not cluster into a clade in this study. The reason for this result may be that there are not enough samples collected in this study. For example, only one sample of Triacanthidae was collected. It is also possible that the COI gene sequence is not suitable for the systematic relationship study of families and higher taxonomic levels.
In the Neighbor-joining tree based on the haplotype of COI gene of Tetraodontiformes, the species of the same genus clustered together, and the same species are clustered into a clade, which is basically consistent with the results of morphological identification. Although the interspecific genetic distance of some species of Takifugu is less than 0.02(as shown in Table 2), in the Neighbor-joining tree, except for T. pseudommus , T. rubripes and T. chinensis , each species can gather into a separate clade. It indicates that the COI gene sequence is suitable for the classification and identification of genera and lower taxonomic levels.