DNA barcoding for identification of fish species in the Taiwan Strait
DNA barcoding for identification of fish species in the Taiwan Strait
Xing Bingpeng☯ 0 1 2
Lin Heshan☯ 0 1 2
Zhang Zhilan 0 1 2
Wang Chunguang 0 1 2
Wang Yanguo 0 1 2
Wang Jianjun 0 1 2
0 Laboratory of Marine Biology and Ecology, Third Institute of Oceanography State Oceanic Administration , Xiamen, Fujian , China
1 Research Foundation of Third Institute of Oceanography, SOA (2015012) ± ZZL; 2) Key Laboratory of Marine Ecology and Environmental Science and Engineering , SOA (MESE-2017-04) ± XBP; 3) National Natural Science Foundation of China (41406216); The Marine Biological Sample Museum Upgrade And Expansion (GASI-01-02-04) ± WCG; and 4) The Marine Biological Sample
2 Editor: Sebastian D. Fugmann, Chang Gung University , TAIWAN
DNA barcoding based on a fragment of the cytochrome c oxidase subunit I (COI) gene in the mitochondrial genome is widely applied in species identification and biodiversity studies. The aim of this study was to establish a comprehensive barcoding reference database of fishes in the Taiwan Strait and evaluate the applicability of using the COI gene for the identification of fish at the species level. A total of 284 mitochondrial COI barcode sequences were obtained from 85 genera, 38 families and 12 orders of fishes. The mean length of the sequences was 655 base pairs. The average Kimura two parameter (K2P) distances within species, genera, families, orders and classes were 0.21%, 6.50%, 23.70% and 25.60%, respectively. The mean interspecific distance was 31-fold higher than the mean intraspecific distance. The K2P neighbor-joining trees based on the sequence generally clustered species in accordance with their taxonomic classifications. High efficiency of species identification was demonstrated in the present study by DNA barcoding, and we conclude that COI sequencing can be used to identify fish species.
Data Availability Statement: All DNA sequences
files are available from the Genbank database.
Accession numbers are provided in Table 1.
More than 30,000 species of fish exist worldwide, accounting for more than half of all
vertebrates. Aside from being an important component of biodiversity, fish also possess direct
economic value and are important animal protein sources for humans[
]. The classification
and identification of fish is not only the subject of taxonomy studies but also the key to fishery
investigations, the assessment of nature reserves and the identification of food and drug
]. The identification of fish species also mainly relies on morphometric and meristic
]. Fish have remarkable diversity of morphological characteristics, and most fish
go through ontogenetic metamorphism. Many morphometric characteristics change during
the stages of ontogenetic development[
]. Convergent and divergent adaptation also lead to
changes in the morphological characteristics of fish species, imposing great challenges to
morphological taxonomy, in which species identification is mainly based on morphological
characteristics, and the classification of many species has thus also been controversial[
limitations inherent in morphology-based identification systems and the declining number of
taxonomists call for a molecular approach to identify species[
Museum Upgrade And Expansion (GASI-01-02-04)
DNA sequence analysis has been used to assist species identification with the development
of molecular biology. However, the accuracy of molecular identification relies on having a
reliable and complete reference database, as inconsistent genetic marker usage could impede the
application of molecular authentication[
]. Different DNA markers have been used in
different taxonomic groups. In 2003, Hebert et al.[
] proposed DNA barcoding technology, in
which the mitochondrial cytochrome c oxidase subunit I (COI) gene sequence was used as a
barcode for species identification with the expectation of barcoding all species for the purpose
of species identification and classification. It was found that the intraspecific diversity of the
COI gene in animals was significantly lower than the interspecific diversity, using the COI
gene as a barcode was effective for classifying and identifying vertebrates and invertebrates,
and the COI gene has been widely used in various biological groups[11±14]. Compared with
the traditional morphological classification methods, the advantages of the DNA barcoding
technology are mainly as follows: 1) Some species have extremely similar external
morphological characteristics; therefore, it is difficult to distinguish them from each other merely by
morphological characteristics. DNA barcoding technology can help accurately distinguish such
species. 2) Morphological differences can vary considerably during various developmental
stages, but individuals at different developmental stages can be identified by the DNA
barcoding technology. 3) The DNA barcoding technology can allow for the discovery of cryptic
species. Cryptic species are two or more species that are morphologically similar yet genetically
distinct. Because of their morphological similarities, cryptic species are identified as the same
species in the existing system. Using DNA barcoding technology, the large molecular
evolutionary distance between these species can be revealed, thereby discovering cryptic species.
The Taiwan Strait belongs to the shallow sea of the subtropical continental shelf and is the
passage between the East China Sea and the South China Sea. The sea area of the Taiwan Strait
has a complex physical and chemical environment and is influenced by the Kuroshio
tributaries, the drifting of the South China Sea monsoon and the coastal flows in Fujian and Zhejiang
Provinces, China. The Taiwan Strait has rich fishery resources and is one of the most
important fishing grounds off the coast of China. The Taiwan Strait has been drawing much
attention due to its unique geographical location and marine environment characteristics, and
studies on its ecosystem have gained attention. As human activities have intensified,
over-fishing, habitat destruction and climate change have generated significant impacts on the
biodiversity and structure of the fish community in the Taiwan Strait. The decline in genetic
variation of a population diminishes the ability of fish to adapt to environmental changes and
decreases their chances of long-term survival[
]. To promote sustainability, better control and
management of fisheries should be implemented. The identification of fish species still stands
as one of the most basic but important issues in fisheries management. In the present study,
we examined COI diversity within and among 85 fish species, most of which were commercial
species, with the goal of testing the utility of DNA barcoding as a tool to identify fish species.
The DNA barcode records generated in this study will be available to researchers to monitor
and conserve the fish diversity in this region.
Materials and methods
All fish species were caught in the offshore area (not national parks, other protected areas, or
private areas, etc.), so no specific permissions were required for these locations/activities.
Ethical approval was not required for this study because no endangered or protected fish species
were involved. Specimen collection and maintenance were performed in strict accordance
with the recommendations of Animal Care Quality Assurance in China.
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All fish specimens were captured with a drawl net at nine locations in the Taiwan Strait (Fig 1,
Table 1). All specimens were morphologically identified by experts and taxonomists, who
mainly followed the identification keys of Liu et al. (2013)[
]. A total of 284 fish samples was
chosen for the research, and the mean number of individuals per species was 3. The voucher
specimens were fixed with 95% ethanol and deposited in the Marine Biological sample
Museum at the Third Institute of Oceanography, State Oceanic Administration. After
morphological examination, muscle tissue samples were dissected from each specimen and stored
in 95% ethanol at −20ÊC.
Total DNA was extracted from a small piece of ethanol-preserved tissue according to the
standard DNA barcoding methods for fish[
]. Approximately 655 bp were amplified from the
5' region of the COI gene employing the primers described in Ward et al.[
Fig 1. Distribution of the sampling localities for the specimens collected in this study.
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The amplification reaction was performed in a total volume of 25 μl, including 16.25 μl
ultrapure water, 2.25 μl 10× PCR buffer, 1.25 mM MgCl2, each dNTP at 0.2 mM, each primer at 2
mM, 1.25 U Taq DNA polymerase and 1 μl DNA template. The thermal cycling conditions
consisted of an initial step of 2 min at 95ÊC followed by 35 cycles of denaturing (94ÊC, 30 s), annealing
(54ÊC, 30 s) and extension (72ÊC, 1 min), with a final extension at 72ÊC for 10 min; the samples
were then held at 4ÊC. The PCR samples were screened for the existence of PCR products on a
1.0% agarose gel. Sequencing in both directions was performed by Sangon Biotech (Shanghai).
Sequences were manually edited using the SeqMan program (DNAStar software) combined
with manual proofreading; each base of the spliced sequences was ensured to be correct before
submitting them to GenBank (Table 1). Next, the sequences were aligned using ClustalW in
GenBank accession numbers
Takifug u fasciatus
Takifug u oblongus
Takifug u xanthopterus
Takifug u poecilonotus
GenBank accession numbers
MEGA 6.0 software, and parameters including the sequence length, GC content, polymorphic
loci and parsimony informative sites were calculated. The distances within species and
between species were calculated using the Kimura-2-parameter (K2P) model[
phylogenetic tree was constructed using the neighbor-joining (NJ) method. The clade credibility in the
tree that was obtained using the NJ method was tested by bootstrapping, in which 1000
repeated sampling tests were performed to obtain the support values of the clade nodes.
A total of 284 mitochondrial COI barcode sequences were obtained from 85 genera, 39
families and 11 orders of fishes (GenBank accession numbers and taxonomic data are listed in
Table 1). After editing, the consensus length of all barcode sequences was 655 bp, and no stop
codons, insertions or deletions were observed in any of the sequences. All analyzed sequences
were larger than 600 bp.
Nucleotide pair frequency analysis of the entire dataset revealed that 325 of 655 (49.62%)
sites were conserved, 330 of 655 (50.38%) sites were variable, 330 of 655 (56.27%) sites were
parsimony informative, and no singleton sites were present. The average number of identical
pairs (ii) was 519, of which 202, 216 and 101 were found at the first, second and third codon
positions, respectively. Transitional pairs (si = 76) were found to be more common than
transversional pairs (sv = 60), with a si/sv (R) ratio of 1.27 for the dataset. Both transitional and
transversional pairs were most common at the third codon position (si = 62 and sv = 56).
The overall mean nucleotide base frequencies observed for these sequences were T
(28.90%), C (28.40%), A (24.30%) and G (18.40%). The base composition analysis for the COI
sequence showed that the average T content was the highest and the average G content was the
lowest; the AT content (53.20%) was higher than the GC content (46.80%). The GC contents
at the first, second and third codon positions for the 11 sole fish were 56.70%, 43.10% and
40.60%, respectively (Table 2). Of these, the GC content at the first codon position was the
highest, which can be attributed to base usage bias among the three codon positions. The
usage frequency of only C was similar among the three codon positions, whereas the other
three bases had significantly different usage frequencies. At the first codon position, the usage
of T (18.00%) was the lowest, and the usages of the other bases were C (25.60%), A (25.60%)
and G (31.10%). At the second codon position, the content of T (42.00%) was highest, and the
contents of the other bases were C (28.40%), A (15.00%) and G (14.70). At the third codon
position, the base usage was T (27.00%), C (31.10%), A (32.20%) and G (9.50%); the G content
was the lowest, exhibiting a clear pattern of anti-G bias.
The Kimura-2-parameter model is recommended by the Consortium for the Barcode of
Life (CBOL) for calculating genetic distance [
]. In this study, the Kimura-2-parameter
model was used to calculate the genetic distances within and between species for the fish used
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in this study. As shown in Table 3, the K2P distances of the COI sequence within species
ranged from 0 to 1.83%, with an average distance of 0.21%; the largest distance of 1.83% was
found in Terapon jarbua, and the distances for all remaining species were less than 1%. The
genetic distances between species ranged from 0 to 21.70%, with an average of 6.50%, which
was 31 times the average genetic distance within species. The genetic distance between genera
was 7.70±30.50%, with an average of 23.70%, and the genetic distance between families was
17.60±31.80%, with an average of 25.60%. Only genetic distances within species were less than
2%, and the mean genetic distances between species, between genera and between families
were all greater than 5%, which were much higher than the distances within species. The data
also show that the genetic distance (K2P) was larger at higher taxonomic levels, and the
increases in genetic distances (K2P) above the species level were smaller and less pronounced
at higher taxonomic levels.
The NJ tree, including 284 species with all haplotypes and 71 species from NCBI (Table 1),
is provided in Fig 2. Most of the specimens of the same species were clustered together, which
reflected the prior taxonomic assignment based on morphology. No taxonomic deviation was
detected at the species level, indicating that the majority of the examined species could be
authenticated by the barcode approach.
DNA barcoding technology allows for species identification by taking advantage of a DNA
sequence fragment that is shared by organisms that have significant interspecies differences.
This technology breaks through the over-reliance on the personal abilities and experiences of
Fig 2. Neighbor-joining tree based on COI sequences using K2P distances.
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taxonomists in traditional morphological classification and enables the informatization and
standardization of species identification. The mitochondrial COI gene, which exhibits high
levels of conservation within species and modest levels of genetic variability between different
species, is usually utilized as a species barcode, and its high efficiency in species identification
has been reported in Japanese marine fishes [
], Indian freshwater fishes[
] and Mediterranean fishes[
]. In this study, we successfully amplified the COI
barcode sequences for 89 marine fish species. The primer pairs used in this study could amplify
the target region without any deletions or insertions, indicating that DNA barcoding could be
used as a global standard for identifying fish species. All barcode sequences were a consensus
length of 655 bp, no stop codons were detected, and the sequences were free of nuclear
mitochondrial pseudogenes (NUMTs). Vertebrate NUMTs are typically smaller than 600 bp [
]. The presence of NUMTs can cause misestimation of the biodiversity. The number of COI
genes is greater than that of pseudogenes, so conserved primers should preferentially amplify
mitochondrial DNA over NUMTs, and there was no evidence of NUMTs in the fish. A
nucleotide pair frequency analysis resulted in 325 conserved sites, 330 variable sites, 330 bp
parsimony informative sites and no singleton sites. There were more transitional pairs (si = 76)
than transversional pairs (sv = 60). The observed nucleotide pair frequencies were similar to
those reported in studies of fishes in Turkey. Both transitional and transversional pairs were
highest at the third codon position (62 and 56 for si and sv, respectively), and synonymous
mutations mostly occurred at the third position. The amount of variation observed in
mitochondrial DNA can lead to demographic changes in fish populations.
The base composition analysis of the COI sequence revealed AT content (53.20%) that was
higher than GC content (46.80%), which is a result similar to the results found in Australian
], Canadian [
] and Cuban fish species[
]. The GC contents in the first, second and third
codon positions for the 11 sole fish were 56.70%, 43.10% and 40.60%, respectively. Of these,
the GC content of the first codon position was significantly higher than those of the other two
positions, which can be attributed to base usage bias at the three codon positions. At the first
codon position, the usage of T (18.00%) was the lowest, and the usage of the other bases was C
(25.60%), A (25.60%) and G (31.10%). At the second codon position, the usage of T (42.00%)
was the highest, and the usage of the other bases was C (28.40%), A (15.00%) and G (14.70%).
At the third codon position, the base usage was T (27.00%), C (31.10%) and A (32.20%), each
of which was higher than G (9.50%). The third codon position had a clear pattern of anti-G
bias, and similar patterns have also been observed in Ophichthyidae and Soleidae [
species evolution, the codon positions of mitochondrial genes are subjected to varying degrees
of base-mutation selection pressure, and base usage bias may be caused by base-mutation
pressure in codon positions.
The efficiency of species identification through DNA barcoding depends on both
interspecific divergence and intraspecific divergence. Barcode analysis attempts to identify the
boundaries to delineate species, which corresponds to the divergence between the nearest neighbors
within a group[
]. However, there is still no universal standard threshold defined for
interspecies demarcation. The difference between minimum congeneric and maximum
conspecific divergence was recently used to define the barcoding gap, and this difference was
more efficient than the mean of intra- and interspecific sequence variability[
In this study, the average intraspecific K2P distance was 0.21%, compared with 6.50% for
species within genera. The mean interspecific distance was found to be 31-fold higher than the
mean intraspecific distance, which was similar to the 25-fold difference observed in Australian
marine fishes [
] and the 26.2-fold difference observed in Canadian mesopelagic and upper
bathypelagic marine fishes[
]; this result corresponds to the DNA barcoding principle that
interspecific divergence sufficiently outscores intraspecific divergence. In addition, the
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difference was greater than the 13.9-fold difference observed in the marine fishes commonly
encountered in the Canadian Atlantic [
]. The amount of variation in mitochondrial DNA
observed in this study can lead to demographic changes in fish populations. The mean K2P
distance increased gently within the higher taxonomic ranks of families and species classes,
with values of 23.70% and 25.60%, respectively. The rate of increase declines in the higher
taxonomic categories due to substitutional saturation.
The entire NJ tree derived from the study is shown in Fig 2. Most species were clustered
into monophyletic units in the NJ tree, indicating that DNA barcoding has high efficiency in
species identification. Morphological misidentification can change the outcome of the NJ tree.
We detected deep divergence of 5.99% among individuals of Muraenesox cinereus (MG220575,
KX215195, KX215196) at first. Three sequences obtained from M. cinereus formed two
different clusters. The sequence MG220575 clustered away from the rest and clustered with
Uroconger lepturus with a K2P distance of zero. Without this single sequence, the conspecific
divergence of M. cinereus was zero. We checked the preserved specimens and discovered that
the single specimen was a larva of U. lepturus, which was originally classified as M. cinereus.
Morphological misidentifications of voucher specimens, DNA contamination and incomplete
knowledge of the taxonomic literature can contribute to ambiguous barcoding results [
On the other hand, the reference library of barcodes and species identification requires a large
number of specimens, including eggs, larvae and adults, and many morphometric
characteristics change during distinct developmental stages. Therefore, occasional instances of
misidentification are inevitable, and this example reflects that DNA barcoding can detect cases of
morphological misidentification. The combination of morphological and molecular
characteristics is a necessary condition for establishing a molecular database. A successful reference
barcode library can be used to better characterize and broadly identify species.
Two other cases can be found in the NJ tree: sequences that have the same species name
but do not exhibit cohesive clustering by conspecies in which detect deep divergence can be
detected among them, and sequences that have different species names and form a cohesive
cluster. The sequences of Thryssa kammalensis clustered separately in the NJ tree; one sequence
showed a genetic divergence of 3.30% with others and 4.30% with Thryssa dussumieri, but it
clustered with Thryssa hamiltonii with a K2P distance of zero. We rechecked the identification
history of the preserved samples and found some intermediate morphological characteristics in
them. The main factors responsible for this case may be introgressive hybridization.
Mitochondrial genes are maternally inherited, and the hybrid would have only maternal species DNA.
When species with a close phylogenetic relationship mate, the subsequent generation can have
the morphological characteristics of either parent species. Introgressive hybridization would
lead to phylogenetic paraphyly. The species of Takifugu, which formed a cohesive cluster,
exhibited fairly low interspecies divergence with a value of zero and could not be discriminated by
DNA barcoding. This failure was due to recent and rapid speciation, and the specimens of these
species were genetically similar at the DNA barcode region. The factors of erroneous taxonomy,
low sister species divergence, introgressive hybridization and the scarcity of specimens were
also theoretically associated with the failure of DNA barcoding. A more rapidly evolving DNA
fragment, such as the mitochondrial control region, should be used in such specimens, and
nuclear genes should also be sequenced to establish whether hybridization has occurred.
Fish diversity in the Taiwan Strait is highly threatened by overexploitation, and some
scientists predict that all fisheries will have collapsed by 2048[
]. With the significant decline in
biodiversity, species extinction enhances the need for the conservation of marine biodiversity
]. Our results reveal that DNA barcoding was successful in identifying the vast majority
of fish species. Identification supported by DNA barcoding could be used to evaluate fish
biodiversity, monitor fish conservation and manage fisheries [
]. This technique will provide
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direction for future studies of fish species that need to be barcoded. Once a fish DNA barcode
database has been established, the scientific and practical benefits of fish barcoding are diverse.
DNA barcoding can discriminate all fish species and identify the eggs, larvae and carcass
fragments of these species. The results will provide more information on fish diversity to the
fisheries managers and ecologists who craft the policies for the conservation and sustainable use of
Conceptualization: Xing Bingpeng, Lin Heshan, Wang Jianjun.
Data curation: Xing Bingpeng, Lin Heshan, Zhang Zhilan.
Formal analysis: Xing Bingpeng, Lin Heshan, Wang Chunguang, Wang Yanguo.
Funding acquisition: Wang Chunguang, Wang Jianjun.
Investigation: Xing Bingpeng, Lin Heshan, Zhang Zhilan, Wang Chunguang, Wang Yanguo,
Methodology: Xing Bingpeng, Lin Heshan, Wang Jianjun.
Project administration: Xing Bingpeng, Lin Heshan, Wang Jianjun.
Resources: Xing Bingpeng, Wang Chunguang, Wang Jianjun.
Software: Zhang Zhilan, Wang Yanguo.
Supervision: Wang Yanguo.
Validation: Zhang Zhilan.
Writing ± original draft: Xing Bingpeng, Lin Heshan.
Writing ± review & editing: Xing Bingpeng, Lin Heshan, Wang Jianjun.
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