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.