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Article

Validity of Pampus liuorum Liu & Li, 2013, Revealed by the DNA Barcoding of Pampus Fishes (Perciformes, Stromateidae)

by
Jiehong Wei
1,5,†,
Renxie Wu
2,†,
Yongshuang Xiao
1,
Haoran Zhang
2,
Laith A. Jawad
3,
Yajun Wang
4,
Jing Liu
1,* and
Mustafa A. Al-Mukhtar
6
1
Laboratory of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2
College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China
3
School of Environmental and Animal Sciences, Unitec Institute of Technology, 139 Carrington Road, Mt Albert, Auckland 1025, New Zealand
4
College of Marine Sciences, Ningbo University, Ningbo 315211, China
5
University of Chinese Academy of Sciences, Beijing 100049, China
6
Marine Science Centre, University of Basrah, Basrah 61004, Iraq
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this paper.
Diversity 2021, 13(12), 618; https://doi.org/10.3390/d13120618
Submission received: 28 September 2021 / Revised: 21 November 2021 / Accepted: 23 November 2021 / Published: 25 November 2021
(This article belongs to the Special Issue Aquatic Organisms Research with DNA Barcodes)
Browse Figures
Versions Notes

Abstract

:
The genus Pampus contains seven valid species, which are commercially important fishery species in the Indo-Pacific area. Due to their highly similar external morphologies, Pampus liuorum has been proposed as a synonym of Pampus cinereus. In this study, partial sequences of COI (582 bp) and Cytb (1077 bp) were presented as potential DNA barcodes of six valid Pampus species and the controversial species P. liuorum. A species delimitation of the seven Pampus species was performed to verify their validities. Explicit COI barcoding gaps were found in all assessed species, except for P. liuorum and P. cinereus, which resulted from their smaller interspecific K2P distance (0.0034–0.0069). A Cytb barcoding gap (0.0200) of the two species was revealed, with a K2P distance ranging from 0.0237 to 0.0277. The longer Cytb fragment is thus a more suitable DNA barcode for the genus Pampus. In the genetic tree, using concatenated Cytb and COI sequences, the seven species reciprocally formed well-supported clades. Species delimitations with ABGD, GMYC, and bPTP models identified seven operational taxonomic units, which were congruent with the seven morphological species. Therefore, all of the seven analyzed species, including P. liuorum, should be kept as valid species.

1. Introduction

Pomfrets, species of genus Pampus Bonaparte, 1834, family Stromateidae Rafinesque, 1810, are pelagic marine fishes widely distributed along the coast of the Indo-West Pacific region. Seven valid species of genus Pampus have been recognized, namely, Pampus argenteus (Euphrasen, 1788), P. candidus (Cuvier, 1829), Pampus chinensis (Euphrasen, 1788), Pampus cinereus (Bloch, 1795), Pampus minor Liu & Li, 1998, Pampus nozawae (Ishikawa, 1904), and Pampus punctatissimus (Temminck & Schlegel, 1845) [1,2,3,4,5,6,7,8,9]. They contribute high commercial values to fisheries of the countries along the coast of the Indo-West Pacific region. In 2016, fishery harvests of pomfret in China reached over three million tons [10].
The taxonomy of the genus Pampus has long been confused by their highly similar external morphologies. Pampus argenteus might be the most confusing name in the genus Pampus. Its holotype is not available in its original description, while the vague original morphological description was found to be applicable to multiple known pomfret species [3]. Twelve available names were assigned as junior synonyms of P. argenteus, including P. minor, P. cinereus, P. candidus, and P. punctatissimus, which have been recognized or resurrected as valid species [1,4,5,9]. Liu et al. [11] presented a morphological comparison of P. argenteus, P. cinereus, P. chinensis, P. minor, and P. punctatissimus, which indicated that the five species differed from each other in numerous external and skeletal characters, e.g., skull, gill rakers, and sensory canal systems on the head and lateral lines. Liu et al. [3], based on the original description and type locality of P. argenteus, redescribed the species and designated its neotype, which set up a reference for verifying validities of its junior synonyms. Simultaneously, the neotype of P. cinereus was assigned and described by Liu et al. [6] as a substitution of its lost holotype. Liu and Li [2] described a novel species, Pampus liuorum Liu & Li, 2013, based on its distinct morphology compared with six known pomfret species. However, the phylogenetic tree by Yin et al. [7], inferred from numerous nuclear gene loci, indicated that the specimens of P. cinereus and P. liuorum formed a mixing clade, refusing monophyly of the two species. Pampus liuorum is thus suspected to be a junior synonym of P. cinereus [7], and its monophyly and exclusiveness await further verification. Li et al. [12] proposed the resurrection of P. echinogaster from P. argenteus because of their distinct cytochrome c oxidase subunit I (COI) gene sequences. However, a morphological comparison indicated that P. echinogaster sensu Li et al. [12] is similar to the neotype of P. argenteus designated in Liu et al. [3], and thus could be a misidentification. Pampus nozawae used to be considered as a junior synonym of P. cinereus [6]. Its validity was recently proposed based on its distinct axial skeletal morphology comparing to its congeners [8], although a redescription and neotype designation of this species are currently unavailable. Therefore, the validities of P. nozawae and P. echinogaster are still uncertain. Radhakrishnan et al. [9] resurrected P. candidus based on its distinct morphological and genetic characteristics compared to P. argenteus, P. cinereus, and P. liuorum.
DNA barcoding, the idea of using short segments of genes to enable the precise identification of species, was proposed as an alternative way to clarify the species and genetic diversity of the genus Pampus [3,13,14]. Guo et al. [13] carried out preliminarily explorations on the genetic diversity of the genus Pampus using partial sequences of 16S ribosomal RNA (16S rRNA) and COI genes, and confirmed that P. minor was genetically distinct from its congeners. Cui et al. [14], using mitogenomic data, identified five species among specimens collected from the coast of China, i.e., P. minor, P. punctatissimus, P. chinensis, P. cinereus, and Pampus sp. (possibly P. argenteus or P. echinogaster). Li et al. [15] reported a new species, Pampus sp. nov., claiming its mitogenome to be different from its congeners. Radhakrishnan et al. [16] reported two new species, Pampus sp1. and Pampus sp2., from the Indian Ocean. Li et al. [17] presented an integrative comparison of morphological and genetic differences in seven Pampus species from the Indo-Pacific region. Neighbor-joining trees inferred from COI sequences suggested that Pampus sp1. and Pampus sp2. sensu (Radhakrishnan et al. [16]) are identical to P. argenteus and Pampus sp. nov. sensu (Li et al. [15]), respectively [17]. Despite the huge efforts, the misidentifications and mislabelings of the pomfret species frequently occur, especially on NCBI (National Center for Biotechnology Information) GenBank, which could hinder the application of DNA barcoding for Pampus species identification [7,17].
To establish reliable references for pomfret species identification, partial COI and cytochrome b gene (Cytb) sequences of seven pomfret species are presented in this study as potential DNA barcodes. To verify the validity of the pomfret species, we performed phylogenetic inference and species delimitation with well-identified Pampus specimens collected from the Indo-Pacific region, including type specimens of P. argenteus and P. liuorum deposited in the Museum of Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

2. Materials and Methods

2.1. Sampling and Species Identification

In this study, seven pomfret species (74 specimens) were assessed (Figure 1): Pampus argenteus, P. candidus, P. chinensis, P. cinereus, P. minor, P. liuorum, and P. punctatissimus. Due to a lack of specimens, Pampus nozawae was not included in this study. Six of the assessed Pampus species, including a total of seventy specimens, were collected from nine localities along the coast of China from August 2009 to January 2014 using commercial fishing trawl boats or gillnet fishing. Two paratypes of P. liuorum (i.e., IOCAS20120541 and 0542) were derived from Liu and Li [2], where the species was first described. Three specimens of P. argenteus (i.e., IOCAS120413, 0423, 0435) were derived from Liu et al. [6], where P. argenteus was redescribed. All specimens were carefully identified based on the type of specimen and our previous work on Pampus taxonomy [2,3,4,5,6]. Four specimens of P. candidus were collected from coastal Iraq in the northern Indian Ocean and identified based on morphological descriptions and the Cytb sequences of Radhakrishnan et al. [9]. Muscle tissues of the specimens were taken and preserved in 95% ethanol for further experiments. All voucher specimens of the barcodes were deposited at the Museum of Marine Biology, IOCAS, Qingdao, China. Sequences of Peprilus medius (COI, AB205449; Cytb, AB205471) from Doiuhi and Nakabo [18] were obtained from NCBI GenBank and selected as an outgroup for molecular analyses.

2.2. DNA Extraction, Amplification and Sequencing

Total genomic DNA was extracted from muscle tissues, following the protocol of Sambrook et al. [19]. The COI barcode sequence was amplified by two pairs of fish-specific primers (FishF1 and FishR1; FishF2 and FishR2) [20]. Based on mitochondrial genome sequences of Pampus in Cui et al. [14], a new primer (Thr20-Pam) was designed, and three primers reported by Doiuhi and Nakabo [18] were modified to form three pairs of primers for Cytb sequence amplification of the genus Pampus. The primer names and sequences are as follows: one forward primer: L14724-Pam (5′-GACTTGAAAAACCATCGTTG-3′); three reverse primers: Thr20-Pam (5′-GTTTACAAGACCGGCGCTCT-3′), H15915-Pam (5′-TTCCGACGTCCGGTTTACAAGAC-3′), and H15973-Strdei (5′-TTGGGAGYYRGTGGTAGGAGTT-3′). Polymerase chain reactions (PCR) were performed in a 50 µL volume with 50 ng template DNA, 5 µL of 10 × reaction buffer, 1.5 mM MgCl2, 200 µM dNTP mixture, 0.2 µM of each primer, and 2.5 U Taq DNA polymerase (Transgen Biotech Co., Ltd., Beijing, China). PCR cycles were conducted on a VeritiTM 96-Well Thermal Cycler (Applied Biosystems, USA) under the following protocol: initial denaturation for 4 min at 94 °C, followed by 35 cycles of 45 s at 94 °C, 45 s at 50–52 °C, 45 s at 72 °C, and a final 10 min extension at 72 °C. PCR amplification without the addition of the template DNA was used as a negative control reaction to ensure no cross-contamination during the experiments. PCR products were separated on 1.2% agarose gel, and then sent to Sangon Biotech Co., Ltd. (Shanghai, China) for bidirectional DNA sequencing with the corresponding forward and reverse primers in PCR reactions, using the ABI Prism 3730 automatic sequencer (Applied Biosystems, Foster City, CA, USA).

2.3. Phylogenetic Inference and Barcoding Gaps

The raw sequences were first assembled in EditSeq V7.1.0 (Lasergene, DNASTAR, Madison, WI, USA), and only high-quality bases with clear signals were retained for analyses. Sequence alignment was carried out in MegAlign V7.1.0 (Lasergene, DNASTAR) using the ClustalW algorithm with default settings. The sequences were trimmed to obtain uniform lengths for subsequent analyses. The COI and Cytb sequences were deposited in NCBI GenBank. Sampling information, specimen photos (whenever available), and corresponding COI and Cytb sequences of the specimens were also archived on the Barcoding of Life Database (BOLD) under a public project coded by IOCAS (https://www.boldsystems.org/index.php/Public_SearchTerms?query=IOCAS, access on 1 November 2021). Sampling information, voucher specimen numbers (Museum ID of BOLD), and NCBI GenBank accession numbers of COI and Cytb of the specimens are summarized in Table 1. Sequence variation indices of COI and Cytb sequences among Pampus species, including base composition and number of polymorphic sites, parsimony informative sites, and indels, were calculated using DnaSP v6 [21]. COI and Cytb sequences of the specimens were concatenated to form another dataset for tree inferences and species delimitations.
Due to more genetic distance references for Kimura’s two-parameter model (K2P) [22], we calculated pairwise K2P distances to estimate barcoding gaps of each species. K2P distances among and within the identified Pampus species, namely, interspecific and intraspecific K2P distances, were calculated in MEGA7 using the COI and Cytb datasets [23]. Interspecific and intraspecific K2P distances of each species were visualized using boxplots in OriginPro 2020 (OriginLab ©, Northampton, MA, USA). The barcoding gap for each species was then calculated as the difference between the minimum interspecific distance and the maximum intraspecific distance [24,25].
Three datasets were used for phylogenetic inference, i.e., the COI dataset, the Cytb dataset, and concatenated datasets of the two genes. Specially, COI and Cytb sequences were treated as two partitions in the concatenated dataset. Best-fit models available in IQtrees v 1.6.12 [26] and MrBayes v 3.2 [27] were selected in jModelTest 2 [28] using the Akaike information criterion [29]. The best fit models for COI and Cytb were HKY + G + I [30] and GTR + G [31]. Maximum likelihood trees were inferred in IQtrees v1.6.12 [26], with 1000 bootstrap replicates to estimate the bootstrap values (BSs) of nodes. For BI trees, two independent Markov chain Monte Carlo (MCMC) runs were performed in MrBayes v3.2, with four chains for 500,000 generations, sampling every 100 generations and discarding the first 25% of samples as burn-ins [27]. Sufficient convergence of the runs was evaluated with summary statistics in MrBayes v3.2 (effective sampling size > 200, potential scale reduction factors≈1). All phylogenetic trees were rooted by the outgroup Peprilus medius.

2.4. Species Delimitation

Species delimitation was performed with the concatenated dataset of COI and Cytb using a distance-based method, i.e., automatic barcode gap discovery (ABGD) [32], and two tree-based methods, i.e., the single threshold Bayesian Poisson tree processes (bPTP) model and the generalized mixed Yule-coalescent (GMYC) model [33,34,35]. The ABGD attempts to identify the barcoding gap as the first significant gap in pairwise distances among a given sequence dataset and uses the detected gap to partition the data [32]. The ABGD was performed on an online ABGD interface of Muséum National d’Histoire Naturelle, France (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html, access on 1 November 2021), scanning a range of prior intraspecific divergence values from 0.1% to 10% with 50 search steps and default settings, although applying K2P distances [22] instead of Jukes-Cantor distances [36].
The single-threshold GMYC identifies speciation events by detecting apparent branching rate increases at the transition of interspecific diversification to population-level coalescence. The GMYC model requires inputs of ultrametric trees; therefore, the ultrametric tree of the concatenated dataset was generated using BEAST2 v 2.5.1 [37], applying prior best-fit models of the two genes, the lognormal relax clock model, and constant population size coalescent tree. Specially, the root node height was constrained to an arbitrary age of 1. Two parallel MCMC runs were performed for 50,000,000 generations, with sampling trees and parameters every 1000 generations. Logfiles were combined in LogCombiner v. 2.5.1 and subsequently analyzed with Tracer v. 1.7 of the BEAST2 package. Sufficient convergence of the two runs was checked by the convergence of parameter values, and ESS should be greater than 200. Trees were summarized with TreeAnnotator v. 2.5.1 and visualized in FigTree v 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/, access on 1 November 2021). The Newick ultrametric tree was uploaded to the Exelixis Lab web interface for GMYC modeling (https://species.h-its.org/gmyc/, access on 1 September 2021).
bPTP modeling was also performed on the Exelixis Lab web interface (https://species.h-its.org/, access on 1 September 2021). The bPTP model is an updated version of the original maximum likelihood PTP model, with both the implementation of maximum likelihood searches and Bayesian analyses. Similar to GMYC modeling, the bPTP model delimitates speciation events based on a shift in the number of substitutions between internal nodes instead of time [38,39]. It requires a distance-based phylogram instead of a time-based ultrametric tree [40], and thus might eliminate an error-prone step of divergence time inference that potentially affects the previous method. The Newick tree file for bPTP modeling was generated in MrBayes v 3.2 using the concatenated dataset of COI and Cytb. The settings for MrBayes v 3.2 were the same as those described in Section 2.3.

3. Results

3.1. Sequence Variation Indices and Barcoding Gaps of COI and Cytb

For COI and Cytb, 582 and 1077 bp sequences were retrieved from each specimen collected in this study, respectively; no indel was found in either dataset. The two datasets were concatenated and formed a 1659 bp dataset. Average base compositions (A:G:C:T) of the COI and Cytb datasets were 0.248:0.175:0.246:0.330 and 0.266:0.131:0.292:0.311. Among the seven assessed Pampus species, the 582 bp COI dataset contained 167 polymorphic sites, including 132 parsimony informative sites. The 1077 bp Cytb dataset contained 361 polymorphic sites, including 284 parsimony informative sites. Pairwise COI K2P distances among the seven Pampus species (i.e., interspecific distances) ranged from 0.0034 to 0.1823, and pairwise COI K2P distances within each species (i.e., intraspecific distances) ranged from 0.0000 to 0.0052 (Table 2). COI barcoding gaps have been well identified in five species, i.e., P. argenteus, P. candidus, P. chinensis, P. minor, and P. punctatissimus (Figure 2), with their values ranging from 0.0104 to 0.1221 (Table 2). In contrast, the COI barcoding gaps of P. cinereus and P. liuorum were found to be very small (0.0017 and 0.0000, respectively; Figure 2 and Table 2), which resulted from smaller pairwise K2P distances comparing sequences of P. cinereus and P. liuorum (0.0034–0.0069). For the Cytb dataset, interspecific K2P distances among the seven species ranged from 0.0237 to 0.1850, whereas intraspecific K2P distances ranged from 0.0000 to 0.0065. The Cytb barcoding gaps have been well identified in all seven species, with the values being 0.0200–0.1452. The smallest Cytb barcoding gap (0.0200) has been observed in P. cinereus and P. liuorum.

3.2. Phylogenetic Inference

Maximum likelihood and BI trees retrieved from COI and Cytb datasets singly recovered well-supported clades, corresponding to the morphologically identified species. In the COI tree (Figure 3A), five well-supported clades (BS = 81–100; posterior probabilities, PP = 1) can be identified, which are, based on their morphological identification, P. argenteus, P. minor, P. chinensis, P. punctatissimus, and a mix clade of P. liuorum, P. cinereus, and P. candidus. The COI sequences of P. liuorum, P. cinereus, and P. candidus do not form monophyla reciprocally. Instead, sequences of the three species form a single well-supported (BS = 81; PP = 1) clade, with the COI sequences of P. candidus and P. cinereus being two monophyla nested within it (Figure 3A). For Cytb trees (Figure 3B), the sequences of the seven morphological species, i.e., P. argenteus, P. minor, P. chinensis, P. punctatissimus, P. liuorum, P. cinereus, and P. candidus, form monophyla reciprocally, which are well supported by BS values of 93–100, and a PP value of 1 (Figure 3B). Pampus liuorum has been resolved as a sister species of P. cinereus (BS = 74; PP = 0.84), whereas P. candidus is closely linked to the two species (BS = 100; PP = 1, Figure 3B). A sister relationship between P. argenteus and P. minor is indicated in the Cytb tree, although it is supported by a relatively low PP value (PP = 0.87, Figure 3B).
Similar to the Cytb trees, phylogenetic trees retrieved from concatenated datasets of COI and Cytb well support the monophyly of all seven morphological species (BS = 98–100; PP = 1, Figure 4). The topology of the ML and BI trees is almost identical, except for the different relationships of P. candidus, P. liuorum, and P. cinereus. In the ML tree, Pampus liuorum is a sister to P. cinereus (BS = 60), whereas P. candidus is closely linked to the two species (Figure 4). In the BI tree, Pampus candidus is resolved as a sister species of P. cinereus (PP = 0.51). In both the ML and BI trees, Pampus argenteus is resolved as a sister of P. minor (BS = 78, PP = 0.64, Figure 4).

3.3. Species Delimitation

Species delimitation with the ABGD, GMYC, and bPTP methods using the concatenated dataset consensually concluded seven operational taxonomic units (OTUs) among the analyzed Pampus specimens, which are congruent with the seven morphological species (Figure 4). The ABGD method indicated that the first detected significant barcoding gap was 0.0166. The number of OTUs was reduced from seven to five when applying a larger prior maximum intraspecific K2P distance, e.g., the next maximum intraspecific K2P distance value scanned by the ABGD, 0.0184, which suggested that seven putative species were delimitated with the first barcoding gap detected. The GMYC model delimited seven OTUs as the maximum likelihood solution, which was also the only solution in the confidence interval. The likelihood ratio test of the GMYC model showed highly significant differences (p < 0.001) between the maximum likelihood (-Log LGMYC-max = 671.454) of the GMYC model and likelihood (-Log LNull = 649.49) of the null model (i.e., assuming only one species among all analyzed specimens). The likelihood ratio test therefore refuted the null model and supported the alternative hypothesis, i.e., the seven species delimitation. The bPTP modeling detected the seven most supported partitions among all analyzed specimens. The delimitation support values for each morphological species are as follows: P. argenteus, 0.95; P. minor, 0.91; P. chinensis, 0.97; P. punctatissimus, 0.88; P. candidus, 0.82; P. cinereus, 0.96; and P. liuorum, 0.92.

4. Discussion

4.1. Pampus cinereus, Pampus liuorum, and Pampus candidus as Distinct Valid Species

Both phylogenetic inferences of COI and Cytb implied a relatively closer evolutionary relationship of P. candidus, P. liuorum, and P. cinereus. The K2P distances between each of these three species (COI, 0.0034–0.0210; Cytb, 0.0237–0.0277) were relatively small compared with those of other species pairs (COI, 0.0580–0.1572; Cytb, 0.0555–0.1850), which might imply a close phylogenetic relationship and more recent origin of these three species. Phylogenetic trees retrieved from COI, Cytb, and the concatenated dataset of the two genes congruently resolved the three species as a monophyletic group, well supported by BS values of 81–100 and PP values of 1 (Figure 3 and Figure 4). A close relationship of P. cinereus and P. candidus was also supported by the phylogenetic tree in Radhakrishnan et al. [9]. However, our phylogenetic inference is based on only two mitochondrial gene fragments, which might account for the low support values in the trees and the inconsistency between the BI and ML trees (Figure 4). The phylogeny of the genus Pampus needs to be clarified with larger genetic datasets in the future.
Despite their close genetic relationships, the three species are clearly delineated as different species in the ABGD, GMYC, and bPTP models (Figure 4). Liu and Li [2] illustrated that P. liuorum could be distinguished from P. cinereus by the following characteristics: shorter pectoral fins [31.5–41.7% standard length (SL) vs. 42.0–47.2% SL]; more vertebrae (38 vs. 36); when alive, with golden bronze or yellowish blue color on its back (vs. P. cinereus, whole body silvery grey, anal fin and ventral side sometimes yellow). Although the total vertebral counts of P. cinereus and P. liuorum were claimed to be identical (37 vertebrae) in Jawad and Liu [8], the actual numbers of total vertebrae counted from their radiographs were 36 (P. cinereus, Figure 3C in Jawad and Liu [8]) and 38 (P. liuorum, Figure 1A in Jawad and Liu [8]), which agrees with the descriptions in Liu and Li [2]. The recently resurrected P. candidus possesses an intermediate number of total vertebrae (37 vertebrae) between P. cinereus (36 vertebrae) and P. liuorum (38 vertebrae) [9]. It could also be discriminated from P. liuorum by having fewer vertebrae (14 vs. 15) between the first pterygiophore of dorsal and anal fins [9]. Therefore, the total vertebral count is an exclusive and conservative characteristic in identifying the three species. Yin et al. [7] proposed a synonymy of P. cinereus and P. liuorum, because their phylogenetic analysis using numerous nuclear genes indicated a mixing clade of P. cinereus with P. liuorum. In fact, the mixing clade of P. liuorum and P. cinereus contains three well-supported clades (BS = 100), i.e., a clade of P. cinereus, a clade of P. liuorum, and a mixed clade formed of two “P. cinereus” and “P. liuorum” specimens. The genetic distances among the three clades (approximately 0.0056–0.0100) were similar to those between P. chinensis and P. punctatissimus (approximately 0.0073–0.0144, Figure 1 in Yin et al. [7]), implying that the three clades might contain three species. The total vertebral counts of P. cinereus (36–37) and P. liuorum (36–38) varied between the estimated specimens in Yin et al. [7], which was incongruent with those recorded in Liu and Li [2]. Yin et al.’s [7] conclusion on the synonymy of P. cinereus and P. liuorum might be based on misidentified specimens, and might therefore be incorrect. Our analyses indicate that the well-identified specimens of P. liuorum, including the paratypes of the species (i.e., IOCAS120541, 0542), are delineated as a single species, which is clearly distinct from P. cinereus and P. candidus (Figure 4). It supports that P. liuorum described in Liu and Li [2] is a valid species. On this basis, the genus Pampus now contains eight recognized valid species: Pampus argenteus (Euphrasen, 1788), P. candidus (Cuvier, 1829), Pampus chinensis (Euphrasen, 1788), Pampus cinereus (Bloch, 1795), Pampus liuorum Liu & Li, 2013, Pampus minor Liu & Li, 1998, Pampus nozawae (Ishikawa, 1904), and Pampus punctatissimus (Temminck & Schlegel, 1845); however, P. nozawae needs further taxonomic revision in order to clarify its validity.

4.2. Species Delimitation and Validity of Pampus argenteus

Our phylogenetic trees and species delimitation analyses (ABGD, GMYC, and bPTP model) also support the validity of P. argenteus (Figure 4). The identity of P. argenteus used to be disputed because of its lack of holotype and the vague original morphological description upon its first publication, which could be applied to multiple known pomfret species [6,10,14]. To solve this taxonomic problem, Liu et al. [6] redescribed the species and designated its neotype—the neotype is assigned as the new name-bearing type for P. argenteus. Concurrently, Liu et al. [6] listed a series of non-type specimens identified as P. argenteus, which are alternative morphological references of P. argenteus. In our genetic analyses (Figure 3 and Figure 4), three of these non-type specimens (i.e., IOCAS120413, IOCAS120423, and IOCAS120435) formed a well-supported clade with the other P. argenteus specimens, which were delineated as a single species in ABGD, GMYC and bPTP modeling (Figure 4). Pampus argenteus could be distinguished from its congeners by having a combination of the following characters: mouth subterminal (vs. mouth terminal, P. chinensis and P. punctatissimus); eyes small, with an eye diameter 24.6–27.1% of head length (vs. 27.3–36.4% of head length, P. minor); more vertebrae, a total vertebral count of 40 (vs. 32–38, other Pampus species); and dorsal and anal fins with short falcate lobes (vs. fins with long falcate lobes, P. cinereus, P. liuorum, P. candidus, and P. punctatissimus) [2,6,9,11]. Pampus argenteus redescribed in Liu et al. [6] is thus a valid species with exclusive morphological and genetic characteristics.

4.3. Verification of COI and Cytb as Potential DNA Barcodes for Pomfret Identification

In this study, both COI and Cytb exhibited certain abilities to identify species of the genus Pampus, although the shorter fragment of COI failed to distinguish the closely related species P. candidus, P. liuorum, and P. cinereus. The anterior region of COI (~600 bp, amplified from universal primer pairs for fish, e.g., FishF1 and Fish R1; Fish F2 and Fish R2 [20]; VF1 and VR1 [41]) is a common DNA barcode for fish identification [42,43]. It has widely been applied in various areas, including fishery management [42,44,45] and the forensic investigation of smuggled fish products [46]. Barcoding gaps between intraspecific and interspecific genetic distance have frequently been reported in mitochondrial barcodes among a vast number of fish taxa, with the intraspecific genetic difference rarely exceeding 2% [47,48,49]. The 2% genetic difference in mitochondrial genes could thus be empirically accepted as a general boundary and standard for distinguishing interspecific and intraspecific divergence [42,50,51]. In our study, 582 bp of the common COI barcodes were obtained for Pampus species using the two primer pairs from Ward et al. [20]. Explicit barcoding gaps (Figure 2) were found in five of the analyzed species, i.e., P. argenteus, P. candidus, P. chinensis, P. minor, and P. punctatissimus. Nevertheless, our result showed no obvious COI barcoding gap for P. cinereus and P. liuorum, although there were only 2–4 bp differences among their COI sequences. The shorter traditional COI barcode (586 bp) could contain insufficient variant information to distinguish the two species. The fragments of Cytb have recently been applied as alternative barcodes for fish identification [52,53]. Comparative analyses of COI, Cytb, 16S rRNA, and 18S rRNA suggested that Cytb possesses a higher level of sequence variation among fish species [53]. In this study, analyses on 1077 bp partial Cytb sequences clearly verified barcoding gaps for all seven pomfret species, with the maximum intraspecific K2P distance and minimum interspecific K2P distance being 0.0065 and 0.0237, respectively (Figure 2 and Table 2). Phylogenetic inference using Cytb sequences supported the monophyly of each analyzed species (Figure 3B). This suggests that the longer fragments of Cytb could provide more variant information than the traditional barcoding region of COI in identifying Pampus species. Therefore, adopting longer fragments of Cytb as the DNA barcode could be a recommended strategy to ascertain the accurate identification of pomfret species.

5. Conclusions

In this study, we have evaluated partial sequences of the COI (582 bp) and Cytb (1077 bp) of seven Pampus species as their potential DNA barcodes. Cytb barcoding gaps have been identified in all assessed species, whereas COI barcoding gaps were not identified in P. cinereus and P. liuorum, which suggests that the longer fragment of Cytb would be a more suitable barcode for the genus Pampus. Species delimitations have been performed with GMYC and bPTP models to assess the validities of the seven collected species. Both delimitation methods identified seven OTUs, which were congruent with the seven morphological species. Therefore, we proposed the seven analyzed species, including the controversial species Pampus liuorum Liu & Li, 2013, as valid species.

Author Contributions

Conceptualization, J.L. and R.W.; software, J.W. and R.W.; formal analysis, J.W., R.W. and H.Z.; data curation, J.W., R.W., J.L. and M.A.A.-M.; investigation, R.W., Y.X., L.A.J., J.L. and M.A.A.-M.; writing—original draft preparation, J.W. and R.W.; writing—review and editing, J.W., R.W., Y.X., L.A.J., Y.W., J.L. and M.A.A.-M.; visualization, J.W.; supervision, J.L. and Y.W.; funding acquisition, R.W., Y.W. and J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study is funded by National Natural Science Foundation of China (Nos. 31872195 and 31772869), Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB42000000), and the Science and Technology Planning Project of Guangdong Province, China (No. 2017A030303077).

Institutional Review Board Statement

The experimental animal protocols in the present study have been reviewed and approved by the Animal Experimental Ethics Committee of Institute of Oceanology, Chinese Academy of China (approval number: 0928-2021). Experiment procedures were performed in accordance with the Provisions and Regulations for the National Experimental Animal Management Regulations (China, July 2013).

Informed Consent Statement

Not applicable.

Data Availability Statement

All of the COI and Cytb data used in this study have been deposited in NCBI GenBank (https://www.ncbi.nlm.nih.gov, access on 1 November 2021) with the accession numbers MK300954–MK301093, MZ604279–MZ604282, and MZ6042560–MZ6042563. The COI and Cytb sequences have also been deposited in BOLD under project code IOCAS and the title of this study (https://www.boldsystems.org/index.php/Public_SearchTerms?query=IOCAS, access on 1 November 2021).

Acknowledgments

The authors are thankful to Chun-sheng Li of the Institute of Oceanology, Chinese Academy of Sciences, China, for sharing part of his Pampus collections and useful information on species identification with us.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Photographs of the seven studied Pampus species of this study. (A) P. argenteus (PA-IOCAS120435); (B) P. candidus (PCA-2015004); (C) P. chinensis (PCH-201006003); (D) P. cinereus (PCI-20120520); (E) P. liuorum (PL-IOCAS120542); (F) P. minor (PM-2012504); (G) P. punctatissimus (PP-2013129).
Figure 1. Photographs of the seven studied Pampus species of this study. (A) P. argenteus (PA-IOCAS120435); (B) P. candidus (PCA-2015004); (C) P. chinensis (PCH-201006003); (D) P. cinereus (PCI-20120520); (E) P. liuorum (PL-IOCAS120542); (F) P. minor (PM-2012504); (G) P. punctatissimus (PP-2013129).
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Figure 2. Boxplot showing the COI (A) and Cytb (B) barcoding gaps of the studied species in genus Pampus. Pairwise interspecific and intraspecific K2P distances of each species are annotated with blue and yellow. Mean and median values of the interspecific and intraspecific distances are indicated with circles and lines, respectively.
Figure 2. Boxplot showing the COI (A) and Cytb (B) barcoding gaps of the studied species in genus Pampus. Pairwise interspecific and intraspecific K2P distances of each species are annotated with blue and yellow. Mean and median values of the interspecific and intraspecific distances are indicated with circles and lines, respectively.
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Figure 3. Maximum likelihood tree of the Pampus species inferred from COI (A) and Cytb (B) datasets. Bootstrap values (before slash) and posterior probabilities (after slash) are shown on each node; “-” on the node indicates that the node was not included in the maximum likelihood or Bayesian analyses. Specimens in purple are specimens derived from Liu and Li [2] and Liu et al. [3].
Figure 3. Maximum likelihood tree of the Pampus species inferred from COI (A) and Cytb (B) datasets. Bootstrap values (before slash) and posterior probabilities (after slash) are shown on each node; “-” on the node indicates that the node was not included in the maximum likelihood or Bayesian analyses. Specimens in purple are specimens derived from Liu and Li [2] and Liu et al. [3].
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Figure 4. Maximum likelihood tree of the Pampus species inferred from concatenated dataset of COI and Cytb sequences. Bootstrap values (before slash) and posterior probabilities (after slash) are shown on each node; “-” on the node indicates that the node was not included in the maximum likelihood or Bayesian analyses. Results of the two species delimitation methods, i.e., the GYMC and bPTP model, are shown on the left. Specimens in purple are specimens derived from Liu and Li [2] and Liu et al. [3].
Figure 4. Maximum likelihood tree of the Pampus species inferred from concatenated dataset of COI and Cytb sequences. Bootstrap values (before slash) and posterior probabilities (after slash) are shown on each node; “-” on the node indicates that the node was not included in the maximum likelihood or Bayesian analyses. Results of the two species delimitation methods, i.e., the GYMC and bPTP model, are shown on the left. Specimens in purple are specimens derived from Liu and Li [2] and Liu et al. [3].
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Table
Table 1. Sampling data, BOLD sample IDs, and GenBank accession numbers of the Pampus species used in this study. The“✓” sign indicates that the specimen photo is available on BOLD.
Table 1. Sampling data, BOLD sample IDs, and GenBank accession numbers of the Pampus species used in this study. The“✓” sign indicates that the specimen photo is available on BOLD.
SpeciesSampling DateSampling Location
(Number of Specimens) 		BOLD Specimen VoucherGenBank Accession Number
Museum IDPhoto ReferenceCOICytb
Pampus argenteusApril 2012Zhuhai, Guangdong, China (3)PA-IOCAS120413✓MK300954MK301024
PA-IOCAS120423✓MK300957MK301027
PA-IOCAS120435✓MK300958MK301028
April–May 2012Shenzhen, Guangdong, China (6)PA-20120418✓MK300955MK301025
PA-20120419✓MK300956MK301026
PA-20120443✓MK300959MK301029
PA-20120444✓MK300960MK301030
PA-20120445✓MK300961MK301031
PA-20120447✓MK300962MK301032
May 2012Zhanjiang, Guangdong, China (3)PA-20120531✓MK300963MK301033
PA-20120532✓MK300964MK301034
PA-20120533✓MK300965MK301035
January 2014Weihai, Shandong, China (4)PA-201401001 MK300988MK301058
PA-201401002 MK300989MK301059
PA-201401003 MK300990MK301060
PA-201401004 MK300991MK301061
April 2012Qingdao, Shandong, China (7)PA-20120401✓MK300981MK301051
PA-20120402✓MK300982MK301052
PA-20120403✓MK300983MK301053
PA-20120404✓MK300984MK301054
PA-20120405 MK300985MK301055
PA-20120406 MK300986MK301056
PA-20120409 MK300987MK301057
May 2012Zhoushan, Zhejiang, China (3)PA-EZ2012003✓MK300992MK301062
PA-EZ2012004✓MK300993MK301063
PA-EZ2012005✓MK300994MK301064
Pampus candidusJanuary 2015Iraq (4)PCA-2015004✓MZ604279MZ604560
PCA-2015005 MZ604280MZ604561
PCA-2015006 MZ604281MZ604562
PCA-2015007 MZ604282MZ604563
Pampus chinensisAugust 2009Xiamen, Fujian, China (1)PCH-200908009✓MK300966MK301036
May 2010Zhuhai, Guangdong, China (5)PCH-2010050025✓MK301037MK300967
PCH-2010050027✓MK301038MK300968
PCH-201006001✓MK301039MK300969
PCH-201006002✓MK301040MK300970
PCH-201006003✓MK301041MK300971
Pampus cinereusApril–May 2012Shenzhen, Guangdong, China (3)PCI-20120457✓MK300972MK301042
PCI-20120459✓MK300973MK301043
PCI-20120460✓MK300974MK301044
April–May 2012Zhuhai, Guangdong, China (3)PCI-20120464✓MK300975MK301045
PCI-20120465✓MK300976MK301046
PCI-20120481✓MK300977MK301047
May 2012Zhanjiang, Guangdong, China (3)PCI-20120520✓MK300978MK301048
PCI-20120521✓MK300979MK301049
PCI-20120522✓MK300980MK301050
Pampus liuorumMay 2012Zhuhai, Guangdong, China (2)PL-IOCAS120541✓MK300995MK301065
PL-IOCAS120542✓MK300996MK301066
July–August 2013Dongshan, Fujian, China (9)PL-20130726061 MK300997MK301067
PL-20130726062 MK300998MK301068
PL-20130726063 MK300999MK301069
PL-20130726064 MK301000MK301070
PL-20130726065 MK301001MK301071
PL-20130810031 MK301002MK301072
PL-20130726066 MK301003MK301073
PL-20130810029 MK301004MK301074
PL-20130810030 MK301005MK301075
Pampus minorOctober 2013Zhoushan, Zhejiang, China (1)PM-2013159 MK301013MK301083
April 2012Shenzhen, Guangdong, China (1)PM-20120430✓MK301006MK301076
May 2010Zhuhai, Guangdong, China (2)PM-S20-098 MK301014MK301084
PM-S20-102✓MK301015MK301085
May 2012Zhanjiang, Guangdong, China (3)PM-20120503✓MK301007MK301077
PM-20120504✓MK301008MK301078
PM-20120513✓MK301009MK301079
April 2013Beihai, Guangxi, China (3)PM-2013065 MK301010MK301080
PM-2013066 MK301011MK301081
PM-2013067 MK301012MK301082
Pampus punctatissimusJune 2013Zhoushan, Zhejiang, China (2)PP-20130618✓MK301017MK301087
PP-20130619✓MK301018MK301088
October 2013Xiamen, Fujian, China (5)PP-2013129✓MK301019MK301089
PP-2013138 MK301020MK301090
PP-2013139 MK301021MK301091
PP-2013146 MK301022MK301092
PP-2013154 MK301023MK301093
April 2012Zhuhai, Guangdong, China (1)PP-20120427✓MK301016MK301086
Table
Table 2. Interspecific and intraspecific K2P distances of the seven analyzed Pampus species.
Table 2. Interspecific and intraspecific K2P distances of the seven analyzed Pampus species.
SpeciesCOICytb
InterspecificIntraspecificBarcoding GapInterspecificIntraspecificBarcoding Gap
Pampus argenteus0.1273–0.15720.0000–0.00520.12210.1508–0.18090.0000–0.00560.1452
Pampus candidus0.0139–0.15560.0000–0.00340.01050.0355–0.18490.0009–0.00650.0290
Pampus chinensis0.0580–0.18230.0000–0.00340.05450.0555–0.17900.0000–0.00280.0527
Pampus cinereus0.0034–0.17990.0000–0.00170.00170.0237–0.18500.0000–0.00370.0200
Pampus liuorum0.0034–0.15720.0000–0.00340.00000.0237–0.18110.0000–0.00370.0200
Pampus minor0.1318–0.15720.0000–0.00340.12830.1698–0.18500.0000–0.00470.1651
Pampus punctatissimus0.0580–0.14270.0000–0.00340.05450.0555–0.17770.0000–0.00560.0499
Overall0.0034–0.18230.0000–0.0052−0.00180.0237–0.18500.0000–0.00650.0172
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Wei, J.; Wu, R.; Xiao, Y.; Zhang, H.; Jawad, L.A.; Wang, Y.; Liu, J.; Al-Mukhtar, M.A. Validity of Pampus liuorum Liu & Li, 2013, Revealed by the DNA Barcoding of Pampus Fishes (Perciformes, Stromateidae). Diversity 2021, 13, 618. https://doi.org/10.3390/d13120618

AMA Style

Wei J, Wu R, Xiao Y, Zhang H, Jawad LA, Wang Y, Liu J, Al-Mukhtar MA. Validity of Pampus liuorum Liu & Li, 2013, Revealed by the DNA Barcoding of Pampus Fishes (Perciformes, Stromateidae). Diversity. 2021; 13(12):618. https://doi.org/10.3390/d13120618

Chicago/Turabian Style

Wei, Jiehong, Renxie Wu, Yongshuang Xiao, Haoran Zhang, Laith A. Jawad, Yajun Wang, Jing Liu, and Mustafa A. Al-Mukhtar. 2021. "Validity of Pampus liuorum Liu & Li, 2013, Revealed by the DNA Barcoding of Pampus Fishes (Perciformes, Stromateidae)" Diversity 13, no. 12: 618. https://doi.org/10.3390/d13120618

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