Research Article |
Corresponding author: Jinqing Ye ( jqye@nmemc.org.com ) Corresponding author: Junsheng Zhong ( jszhong@shou.edu.cn ) Corresponding author: Chaopeng Jiang ( cpjiang@shou.edu.cn ) Academic editor: Nina Bogutskaya
© 2024 Jiajie Chen, Xiaodong Wang, Sheng Zeng, Wei Tian, Deyuan Yang, Jinqing Ye, Junsheng Zhong, Chaopeng Jiang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Chen J, Wang X, Zeng S, Tian W, Yang D, Ye J, Zhong J, Jiang C (2024) Morphometric and phylogenetic analysis of a commercial fish Leiognathus equula (Teleostei, Leiognathidae). ZooKeys 1219: 249-270. https://doi.org/10.3897/zookeys.1219.130546
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The complete mitochondrial genome and phylogenetic analysis for Leiognathus equula from the South China Sea was performed using shallow genome skimming. For accurate species identification and redescription, morphometric and meristic characters were examined and compared with previous descriptions. To facilitate the identification of species and to enable comparison with the mitochondrial genome phylogeny, molecular comparisons were conducted using three mitochondrial genes: large ribosomal RNA (16S rRNA), cytochrome c oxidase subunit 1 (COX1), and NADH dehydrogenase (ND5). The mitogenome (16 398 bp) comprised 38 mitochondrial genes, similar to most bony fishes: 13 protein-coding genes (PCGs), 2 rRNA and 22 transfer RNA genes, and 1 non-coding region. The complete mitogenome comprised 30.7% A, 24.2% T, 15.0% G, and 30.1% C. The A+T content (54.9%) was higher than the G+C content (45.1%). All PCGs started with the typical ATG codon, except COX1, which started with GTG. Seven PCGs ended with incomplete stop codons (TA or T). The Ka/Ks ratios of all PCGs were < 1, indicating purifying selection. The phylogenies of Leiognathidae, both based on the amino acid sequences of the 13 PCGs and the single genes 16S RNA and COX1, were monophyletic with high nodal support (> 75%). L. brevirostris (NC 026232) is believed to be a Nuchequula species, whereas L. ruconius (NC 057225) is not classified under Leiognathus in the NCBI database. The phylogenetic trees divided the Leiognathidae family into three clades. The mitogenome phylogeny suggested that the Leiognathidae and Chaetodontidae clades are sister groups. These findings provide important genetic data for population genetics research and a phylogenetic analysis of Leiognathidae.
Leiognathidae, Leiognathus equula, mitochondrial genome, phylogenetic tree, South China Sea
Leiognathids (family Leiognathidae), commonly known as ponyfishes or slipmouths, are characterized by their highly protractile mouths that extend dorsorostrally, rostrally, or ventrorostrally. The relationship of Leiognathidae—an ex-perciform—with the order Perciformes is currently under debate. In the new taxonomical classification, Leiognathidae were reclassified from Perciformes to Chaetodontiformes (
All members of the family Leiognathidae possess a distinctive circumesophageal light organ that houses bioluminescent bacteria belonging to the genus Photobacterium. Males possess a larger light organ and associated features that intensify light during sexual displays for photic communication (
Leiognathus equula is a tropical inshore bottom-dwelling panfish with a large, robust, and rhomboid-shaped body (
The species is fished commercially in South China; it is one of the most economically important species in Hainan and is often sold together with silver pomfret (Pampus argenteus) but at approximately half the price of the latter (Suppl. material
In recent studies, some species previously classified as Leiognathus have been reclassified as belonging to other genera (
The objective of this paper is to provide a description of the mitochondrial hologeny of L. equula, a basal species of Leiognathidae (
To achieve this objective, eight specimens were collected from the South China Sea and identified as L. equula following a process of careful documentation and morphometric comparison. To identify the species accurately, we compared the mitochondrial DNA sequence data of 16S rRNA, COX1, and ND5 fragments and compared the results of the phylogenetic relationships among Leiognathidae species. The genetic relationships within Leiognathidae were analyzed by assembling the mitochondrial genome of L. equula and selecting one of the three mitochondrial genome datasets for description. The present findings enhance the understanding of the mitochondrial genome features of the Leiognathidae and its taxonomic classification. Furthermore, they provide crucial genetic data for phylogenetic and population genetic studies of the family Leiognathidae.
Eight specimens morphologically identified as L. equula were collected from various sources in China, including landing points, fish markets, and onboard commercial and research vessels (Suppl. material
Left lateral view of L. equula specimen DHS19056 (SL 105.8 mm) A showing color in life (photo by Weiyue Zhang) and B radiograph. The whitish area below the dorsal fin indicates where the muscle tissue was sampled. Body depth A was measured at the anal-fin origin, and body depth B was measured at the dorsal-fin origin. Scale bars: 10 mm.
Morphological identification was performed following the original description (
The assembled sequence was subjected to BLAST using the NCBI BLAST similarity search tool. Subsequently, the 16S, COX1, and ND5 sequences from NCBI (Suppl. material
The DNA library was prepared by the Illumina TruseqTM DNA Sample Preparation Kit (Illumina, San Diego, USA) using the manufacturer’s instructions. The prepared library was sequenced by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) using the DNBSEQ-T7 platform to generate 150 bp paired-end reads. In total, ~ 5 Gb of raw sequence data were generated for each sample. Data cleaning was performed using Fastp v. 0.23.2 with default parameters (
The mitochondrial genome was annotated using MITOS2 (
In order to guarantee the greatest possible accuracy in species identification, a random selection of three regions was tested from a sample of eight. The results were consistent across the three sample assemblies, and we selected OR344340 for description. In order to ascertain the phylogenetic position of species within the Leiognathidae family, we reconstructed a phylogeny of the family Leiognathidae using the mitogenome sequences from the GenBank database (https://ncbi.nlm.nih.gov/) for 36 species (accessed 6 February 2024), including Lagocephalus gloveri (
The ML analysis was performed in IQ-TREE v. 2.2.2 (
Description based on eight specimens ranging 81.33–144.02 mm in standard length (SL; Suppl. material
Comparison of morphometric and meristic characters of L. equula in the present study and previous studies.
Counts and measurements | Present study (n = 8) |
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Standard length (mm) | 105.99 (81.33–144.02) | 110.6 (69.0–177.8) | 92–128.8 | 60–232 |
Counts | ||||
Dorsal fin rays | VIII 16 | VII–VIII 12–17 | — | VIII 16 |
Anal fin rays | III 14 | III 14 | — | III 14 |
Pectoral fin rays | 20 | 19 | — | 20 |
Pored scales in lateral line | 63–70 | 56–65 | 50–60 | 58–67 |
Vertebrae (precaudal+caudal) | 9+14 = 23 | 9+14 = 23 | — | — |
Measurements | ||||
As % of SL | ||||
Head length | 33.21 (31.47–35.91) | 30.3 (28.6–33.1) | 31.8 (29.8–34.5) | 28.74–31.65 |
Body depth A (origin anal fin) | 55.61 (52.95–59.56) | 56.2 (51.2–60.5) | — | 51.81–57.80 |
Body depth B (origin dorsal fin) | 57.08 (54.76–60.38) | 55.1 (49.5–58.3) | 57.3 (53.9–61.8) | — |
Head width (max.) | 12.29 (11.52–13.22) | — | 16.1 (15.2–17.6) | — |
Caudal peduncle length | 5.52 (4.66–6.73) | 11.1 (9.4–13.7) | 8.3 (6.8–10.6) | — |
Caudal peduncle depth | 7.57 (7.08–8.25) | 6.7 (5.9–7.4) | 6.9 (6.5–7.1) | — |
Caudal peduncle width | 4.26 (3.42–5.10) | 4.0 (3.3–4.7) | 4.2 (3.6–4.7) | — |
Pectoral fin length | 25.73 (23.20–27.18) | 23.9 (21.2–26.2) | 22.0 (20.1–24.1) | — |
Pelvic fin length | 15.64 (12.58–17.24) | 15.5 (11.4–16.8) | 16.6 (15.0–18.1) | — |
Dorsal fin base length | 57.79 (56.34–59.70) | 56.2 (53.2–59.1) | — | — |
Anal fin base length | 47.08 (45.05–50.79) | 45.6 (42.3–48.8) | — | — |
Predorsal length | 52.49 (50.22–54.32) | 50.8 (48.4–53.3) | 52.5 (50.5–54.0) | — |
Prepelvic length | 41.04 (37.62–44.85) | 37.3 (35.1–40.2) | 43.1 (37.2–50.5) | — |
Preanal length | 57.16 (54.71–59.36) | 54.1 (50.2–58.5) | 58.2 (56.6–60.2) | — |
As % of HL | ||||
Snout length | 36.37 (33.26–40.37) | 37.0 (33.7–39.6) | 34.9 (33.3–37.3) | 26.11–32.79 |
Head width (max.) | 37.05 (35.02–42.00) | 50.9 (47.9–54.4) | — | — |
Upper jaw length | 40.48 (38.26–43.04) | 25.4 (23.1–27.6) | 38.9 (35.0–41.3) | — |
Lower jaw length | 36.85 (34.90–38.84) | 52.0 (48.0–57.4) | 40.0 (25.7–60.0) | — |
Interorbital width | 32.02 (25.31–38.48) | 34.3 (31.7–36.6) | 36.8 (25.7–43.5) | — |
Orbital diameter | 31.12 (28.58–33.54) | 37.3 (33.3–41.7) | 34.6 (30.0–37.6) | 29.67–35.34 |
Preorbital depth | 54.00 (48.60–58.99) | 22.7 (19.6–25.9) | — |
Body robust and large, laterally compressed, rhomboid and deep. Dorsal profile more convex than ventral profile. Greatest body depth at vertical from dorsal fin origin to abdomen. Dorsal fin origin posterior to pelvic fin origins. Anal fin origin vertical through first dorsal fin ray (Fig.
Mouth small and terminal, directed slightly downward, forming tube when protruded. Cleft slightly sloping downward. Lower jaw strongly concave, forming 45° angle when mouth closed. Gape horizontal with inferior eye margin. Lips fleshy but thin. Maxilla exposed, through orbital anterior margin.
Eye moderately large, placed high, lower margin above body axis. Preorbital spine with ridge serrated. Adipose eye lid underdeveloped. Interorbital slightly convex. One short spine on anterior superior margin of eye and posterior of nostril. Nostrils above eyes, two on each side. Anterior nasal pore small and round, posterior large and oblong.
Fins: Dorsal fin 1, with eight spines and 16 rays. First dorsal fin spine very short; second dorsal fin spine longest; 80.35 (77.50–86.39) %HL and 25.26 (15.09–27.83) %SL. Third and fourth dorsal fin spine margin anteriorly serrate. Anal fin with three spines and 14 rays. Second anal fin spine longest; 59.06 (51.79–65.10) %HL and 19.61 (16.98–21.43) %SL. The anal fin spine margin anteriorly serrate. Both dorsal and anal fin base anteriorly covered by membranous sheaths. Pectoral fins rounded and wide. Subthoracic ventral fin shorter than pectoral fin; large axillary scale on pelvic fins; spines retract when laid flat. Terminal ventral fin reached anal fin origin in juveniles (DHS14500, DHS22489, DHS22490). Caudal fin forked, tips of both lobes round and blunt.
Squamation: Lateral line slightly arched posteriorly from the pectoral fin base to the caudal peduncle, continuing horizontally along the caudal peduncle. Head and chest asquamate, body cycloid scales. Lateral line slightly curved and complete, includes 63–70 pored scales.
Dentition: Teeth pointed and bristled, with three or four tooth rows on upper and lower jaw, with incurve, banded arrangement. Vomer, palatine, and tongue toothless.
Fresh coloration: Body greyish to silvery, with compact grey-black narrow band on back and sides; a dark brown saddle on caudal peduncle. Axil of pectoral fins grey to black. Pelvic fins white. Margin of soft dorsal fin black; no dark spots. Margin of soft anal fin yellow. Caudal fin yellowish with black margin. Concentration of melanophores on snout side (Suppl. material
Coloration in preservative: Body yellowish. Color pattern of fins similar to fresh coloration but with a yellowish tinge.
Owing to the limited availability of mitochondrial genome data in Leiognathidae, single mitochondrial genes were chosen as the basis for molecular identification. Phylogenetic reconstruction of the family Leiognathidae on the basis of mitochondrial genes 16S rRNA, COX1, and ND5 was performed using BI and ML methods. The sequences of L. equula from different waters clustered together in all three single-gene phylogenetic trees (Suppl. material
The Hongsha (PP551518) sequences clustered with those from the Philippines (AY541653) and Malaysia (EU366341) in the 16S rRNA BI tree and with those from the Philippines in the 16S rRNA ML tree (Suppl. material
The mitochondrial genome of L. equula (GenBank accession number: OR344340) was 16,398 bp (Fig.
The L. equula mitogenome showed a slight bias towards A and T nucleotides, which comprised 54.9% of the total base composition (A = 30.7%, T = 24.2%, G = 15.0%, and C = 30.1%). This was accompanied by a positive A+T skew (0.117) and a negative G+C skew (−0.334) (Suppl. material
The total length of PCGs was 11,421 bp, with gene length ranging from 177 bp (ATP8) to 1830 bp (ND5). In total, 12 PCGs had canonical ATG start codons, whereas the COX1 gene had a GTG start codon. A complete stop codon was observed in seven PCGs, whereas the remaining six PCGs exhibited an incomplete stop codon (TA or T) at their respective termini—ATP6 and COX3 were terminated by TA and COX2, CYTB, ND3, and ND4 were terminated by T (Suppl. material
Suppl. material
The selection pressure on 13 PGCs of six Leiognathidae species was quantified by computing the ratio of non-synonymous substitutions to synonymous substitutions (Ka/Ks). The Ka/Ks ratios of all PCGs were significantly lower than one (Fig.
The mitogenome of L. equula contained 22 tRNA genes ranging from 67 to 75 bp in size, representing 9.5% (1552 bp) of the entire mitogenome (Suppl. material
The two rRNA genes, 12S and 16S rRNA genes, were 949 bp and 1694 bp in length, respectively (Suppl. material
The control region of L. equula is located between trnP (ugg) and trnF (gaa), with a total length of 727 bp (Fig.
The position of Leiognathidae in the molecular phylogenetic trees was reconstructed on the basis of 13 concatenated PCGs using the ML and BI methods. Phylogenetic analyses conducted using BI and ML yielded a consistent topology. The phylogenetic trees divided the Leiognathidae family into three distinct clades (Fig.
The position of Leiognathidae in the Bayesian inference (BI) and maximum likelihood (ML) phylogenetic tree based on the amino acid sequences of 13 protein-coding genes of the mitochondrial genome and their groupings, clades, ecotypes, and lengths. The numbers above or below branches indicate ML bootstrap values and Bayesian posterior probabilities, respectively; “*” indicates absence from the maximum clade credibility tree; “-” indicates no support value. L. brevirostris (NC 026232) should be a species of Nuchequula, and L. ruconius (NC 057225) is not classified under Leiognathus in the NCBI database.
The morphometric and meristic values recorded in the present study were similar to those reported in previous studies on this species. But the number of pored scales in the lateral line (Table
The genus Leiognathus currently includes L. equula and L. robustus (
Leiognathus equula, the type species of Leiognathus, differs from L. robustus, which has been described in various reports (
The COX1 gene had a GTG start codon. Other Leiognathidae species have also been reported to use this non-standard start codon (
Previous phylogenetic studies on Leiognathidae did not use the mitochondrial genome. Studies on the mitochondrial genome of Leiognathidae species have been limited. The mitochondrial genome data of only five Leiognathidae species are available in the NCBI database (acquisition number: AB355911, MG677547, NC_026232, NC_057225, NC_062376; accessed February 6, 2024). There are two entries for Leiognathus: L. ruconius (acquisition number: NC_057225) and L. brevirostris (acquisition number: NC_062376). The former was identified as Deveximentum ruconius (
Previous single-gene phylogenetic studies suggest that most genera within the family Leiognathidae are monophyletic (
In the present study, the phylogenies based on mitochondrial genome sequences showed that Leiognathidae are most closely related to Chaetodontidae, forming a sister group. This finding is consistent with the osteological evidence (
In the present study, samples of L. equula were collected from different regions of China. The species were identified using both morphological and molecular characteristics. Phylogenies based on the amino acid sequences of 13 protein-coding genes and two single gene sequences (16S RNA and COX1) but not that based on the single gene ND5, indicated that Leiognathidae is a monophyletic family. The phylogenetic trees show that the family Leiognathidae is divided into three clades. Notably, the family Leiognathidae formed was placed as a sister group to the family Chaetodontidae. In the present study, the mitochondrial genome sequence of L. equula in the family Leiognathidae was obtained using shallow genome skimming. L. equula occupied a basal branch of the Leiognathidae phylogenetic tree; thus, the study provides essential data for the study of the complete mitochondrial genome phylogeny of Leiognathidae.
During the process of specimen collection, we thank Qi Chu and Weiyue Zhang from the National Marine Environment Monitoring Center for their assistance in collecting marine specimens. We are also grateful to Yuange Chen from the East China Sea Fisheries Research Institute for his contribution to sample collection. We are grateful to the reviewers and editors for their many helpful comments and questions, which significantly improved the final version of the manuscript.
The authors have declared that no competing interests exist.
No ethical statement was reported.
No funding was reported.
Conceptualization: JC, DY. Data curation: JC, SZ, DY. Formal analysis: JC, SZ, DY. Funding acqui-sition: JZ, JY, CJ. Investigation: JC, JY, WT. Project administration: JY, XW, JC. Resources: JY, CP, DY. Supervision: JZ, CP. Validation: DY, JC. Visualization: SZ, JC. Writing - original draft: JC, XW, SZ. Writing - review and editing: JC, XW, DY.
Jiajie Chen https://orcid.org/0000-0001-7487-231X
Xiaodong Wang https://orcid.org/0000-0001-9424-7896
Sheng Zeng https://orcid.org/0009-0001-0943-3772
Wei Tian https://orcid.org/0009-0008-5272-0700
Deyuan Yang https://orcid.org/0000-0003-3735-9909
Jinqing Ye https://orcid.org/0009-0009-6503-1477
Junsheng Zhong https://orcid.org/0000-0002-9145-7473
Chaopeng Jiang https://orcid.org/0009-0004-0957-231X
All of the data that support the findings of this study are available in the main text or Supplementary Information.
Supplementary file
Data type: zip
Explanation note: figure S1. Leiognathus equula was on sale at the landing ports (above) and fish markets (below) in Qinglan, Hainan, on November 9, 2023. figure S2. Dorsal head slightly triangular, L. equula DHS14327. Arrow indicates the nuchal spine. figure S3. Phylogenetic trees of Leiognathidae based on the 16S rRNA dataset constructed using the Bayesian inference and maximum likelihood methods. figure S4. Phylogenetic trees of Leiognathidae based on the COX1 dataset constructed using the Bayesian inference and maximum likelihood methods. figure S5. Phylogenetic trees of Leiognathidae based on the ND5 dataset constructed using the Bayesian inference and maximum likelihood methods. table S1. Collection data for eight specimens of L. equula. table S2. Taxa used in the 16S rRNA and COX1 molecular phylogenetic analysis. table S3. Taxa sampled for the ND5 phylogenetic analysis. table S4. Information on raw data and cleaned data from three specimens. table S5. List of mitogenomes included in the present study and their base composition and GenBank accession numbers. table S6. Morphometric and meristic data of L. equula in this study. table S7. Information on each gene fragment of L. equula. table S8. Base composition of the L. equula mitochondrial genome. table S9. Codon number and relative synonymous codon usage of L. equula mitochondrial protein-coding genes.