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Article

Phylogenetic Analyses of Pristipomoides (Perciformes: Lutjanidae) Based on New Mitochondrial Genomes

by
Chunhui Liu
1,
Dezhao Li
1,
Yue Zhang
1,
Maxime Péré
1,
Zhibo Zhuang
1,
Jingyu Liu
1,
Haolang Zhou
2 and
Xiao Chen
1,*
1
College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China
2
Guangxi Mangrove Research Center, Beihai 536000, China
*
Author to whom correspondence should be addressed.
Fishes 2023, 8(7), 343; https://doi.org/10.3390/fishes8070343
Submission received: 20 May 2023 / Revised: 22 June 2023 / Accepted: 25 June 2023 / Published: 28 June 2023
(This article belongs to the Special Issue Molecular Ecology and Genetic Diversity of Fish)
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Abstract

:
The species in the genus Pristipomoides are commercial fishes with high economic value. There are few studies on the phylogenetic relationship within the genus Pristipomoides at present. mtDNA has the characteristics of maternal inheritance, close gene arrangement, and a high evolutionary rate, which is an excellent tool to study the phylogeny of the species. In this study, the mitochondrial genomes of five species in the genus Pristipomoides were sequenced using the Sanger sequencing method and analyzed for their features. The mitochondrial genome length of the five species ranged from 16,499 to 16,530 bp. The start codon was ATG\GTG, and the stop codon was TAA\TAG\T--\AGG. The ratio of Ka and Ks for protein-coding genes ranged from 0 to 0.117, suggesting a strong purifying selection acting on the 13 protein-coding genes (PCGs). The gene with the highest diversity was nd2 (NADH dehydrogenase subunit 2), suggesting the highest evolutionary rate. Phylogenetic analysis of five Pristipomoides species with other species of Lutjanidae was conducted using maximum likelihood (ML) and Bayesian Inference (BI). The results showed that P. zonatus and P. auricilla were closely related to P. argyrogrammicus and P. sieboldii, respectively, and P. filamentosus and P. multidens clustered together. Furthermore, A. rutilans is deeply nested within the Pristipomoides genus, indicating a close phylogenetic relationship with the species in the Pristipomoides genus. Based on this evidence, we suggest that A. rutilans should be classified under the Pristipomoides genus and recommend a revision in its taxonomy. The molecular data and phylogenetic analysis provided in this study would be helpful for the species identification and phylogenetic studies of the family Lutjanidae.
Keywords:
phylogeny; Pristipomoides; mtDNA; classification; mitochondrial structures; Aphareus
Key Contribution: Based on the mitochondrial genome data; we explored the phylogenetic relationships of the species of the genus Pristipomoides, and constructed a phylogenetic tree of the Lutjanidae using more abundant data sets. The results of phylogenetic analysis suggest that the taxonomy based on morphological characters should be changed.

Graphical Abstract

1. Introduction

The mitochondrial genome (mtDNA) contains a large amount of nucleotide information, which is composed of 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, one control region (CR), and one the origin of L-strand replication (OL) [1]. Due to its maternal inheritance, close gene arrangement, and high evolutionary rate, mitochondrial DNA (mtDNA) serves as a valuable molecular genetic marker widely applied in phylogenetic research, effectively resolving some species classification issues [2]. For instance, Nagl et al. used mitochondrial genomes to investigate the classification and evolutionary relationships of tilapia; Last et al. revised the classification of Dasyatidae by combining morphological and molecular data; Miya et al. reconstructed the phylogenetic relationships of bony fishes based on the complete mitochondrial genome [3,4,5].
Species of the family Lutjanidae belongs to the order Perciformes (Actinopteri), including 113 species, 17 genera, and 4 subfamilies, widely distributed in tropical and subtropical waters such as the Eastern Pacific, Indo-Western Pacific, Eastern Atlantic, and Western Atlantic [6,7]. With the development of molecular sequencing technologies, mitochondrial genes are being increasingly utilized in the phylogenetic analysis of the Lutjanidae family. Chu et al. conducted a phylogenetic analysis of 43 species from the Lutjanidae family using the COI gene and proposed a revision in the classification of Pinjalo pinjalo [8]. Oanh et al. investigated the phylogenetic relationship between Lethrinidae and Lutjanidae using the 16S RNA gene [9]. The majority of these phylogenetic studies primarily focus on the genus Lutjanus within the Lutjanidae family, while species from other genera lack sufficient mitochondrial genome data and are rarely analyzed. The genus Pristipomoides belongs to the Lutjanidae family and comprises 11 species [6]. It is a commercially valuable fish species and is an important food fish in many areas. It primarily inhabits waters ranging from 20 to 500 m in depth [7]. The current classification of the genus Pristipomoides is primarily based on the morphological characteristics of the species. Kami used morphological identification methods to conduct phylogenetic studies of some species of the genus Pristipomoides, indicating that close affinities are found between P. auricilla and P. sieboldii and between P. filamentosus and P. flavipinnis [10]. However, phylogenetic analysis of fish based on traditional morphological features has some limitations, such as the requirement for rich taxonomic knowledge. In recent years, the mitochondrial genome has emerged as a valuable tool for studying the phylogenetic analysis of fish taxa. Moreover, at the molecular level, the phylogenetic analysis of the genus Pristipomoides remains largely unknown. Therefore, we sequenced the mitochondrial genomes of five species within the genus Pristipomoides using the Sanger sequencing method. We conducted a phylogenetic analysis, including other species from the Lutjanidae family, to investigate the phylogenetic relationships within the genus Pristipomoides and the evolutionary relationships between the genus Pristipomoides and other species in the Lutjanidae family. In addition, we conducted some comparative analysis of the mitochondrial genomes of the five species, examining the composition and structure of the mitochondrial genome, secondary structures of 22 tRNA genes, codon preferences, and selection pressures in protein-coding genes, among others. The findings presented in this study can contribute to the understanding of the evolution of the Lutjanidae family. The provided mitochondrial genome data can also aid in species identification of related organisms.

2. Materials and Methods

2.1. Samples, DNA Extraction, Amplification, and Sequencing

Samples of five species in the genus Pristipomoides were collected from Howard Shoal, Penguin Shoal, Dongsha Islands, and Shenhu Shoal, all of which were from China (Table 1). The gill filaments, fins, and other tissues of fish samples were soaked in 95% anhydrous ethanol solution and then stored in a refrigerator at −20 °C. The DNA was extracted from their gills using the Marine animal tissue genome DNA extraction kit (Qiagen, Beijing, China). The extracted DNA was amplified using Premix TaqTM (TaKaRa TaqTM Version 2.0 plus dye) reagent. The mitochondrial genome sequences of the species closely related to the genus Pristipomoides were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 8 July 2022), and the conserved regions were searched to design universal primers (Table S1). Validate the designed primers using Primer Premier v5.0(PREMIER Biosoft International, Palo Alto, CA, USA) [11]. Gaps between sequence fragments were completed by designing specific primers. Mitochondrial genome sequences of the five species were obtained by Long-range PCR, short PCR amplification, and Primer walking. Amplification results were sequenced with the use of Sanger sequencing.

2.2. Sequence Assembly, Annotation, and Analysis

DNAStar v7.1 was used to prune, splice, and assemble the sequenced consequences to obtain a complete mitochondrial genome [12]. Five species of Pristipomoides have been on the morphological identification and were aligned with COI consequences published on NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 12 July 2022). The mitochondrial genome was preliminarily annotated using the MITOS Webserver (http://mitos2.bioinf.uni-leipzig.de/index.pym, accessed on 12 July 2022), and the tRNAs were identified using tRNAscan-SE Search Server v2.0 (http://trna.ucsc.edu/tRNAscan-SE/, accessed on 13 July 2022) and confirmed using ARWEN v1.2 [13,14]. The secondary structure of tRNA was predicted using the MITOS Webserver and tRNAscan-SE Search Server v2.0. The mitochondrial genome sequences of related species were downloaded from NCBI and compared with the assembled mitochondrial genome sequences on DNAman v6 software (Lynnon Biosoft, San Ramon, CA, USA) to determine the gene boundaries of PCGs and rRNAs. Five mitochondrial genome sequences were uploaded to GenBank via the BankIt program (https://submit.ncbi.nlm.nih.gov/about/bankit/, accessed on 9 August 2022 and 4 March 2023), obtaining the accession number of five species of mitochondrial genomes (Table 1).
The mitochondrial genome map was completed by Proksee (https://proksee.ca, accessed on 10 March 2023) [15]. The ratio of Ka (nonsynonymous substitution) to Ks (synonymous substitution) reflects the selective pressure exerted on a protein. Pairwise comparisons of the same protein genes of the two species were performed using KaKs_Calculator 2.0 program and 10 values were obtained for the five species, which were visualized using origin 2021 [16]. DnaSP v6 (Universitat de Barcelona, Barcelona, Spain) software was used to calculate nucleotide polymorphisms of 13 PCGs [17]. Relative synonymous codon usage (RSCU) was calculated using PhyloSuite v1.2.3 software (Bio-Transduction Lab, Wuhan, China) [18]. AT skew as well as GC skew is calculated according to the formulas: AT skew = [(A − T)/(A + T)] and GC skew = [(G − C)/(G + C)] [19].

2.3. Phylogenetic Analysis

We used maximum likelihood (ML) and Bayesian analysis (BI) to construct phylogenetic trees, selecting the species Plectorhinchus lineatus of the family Haemulidae as the outgroup and performed a phylogenetic analysis of the five species in this study together with available mitochondrial whole genome data of species Lutjanidae from NCBI. The start and stop codons of 12 PCGs were manually deleted and compared by MACSE v.2.03 (http://mbb.univ-montp2.fr/macse, accessed on 6 Apirl 2023) [20]. The first and second codons were extracted by MEGA X software(Tokyo Metropolitan University, Tokyo, Japan) after concatenation [21]. MAFFT program aligned the fragments of 12S rRNA and 16S rRNA, the ambiguous sites were deleted using the Gblocks plugin, and the three sequences were concatenated to form the dataset for tree construction, conducted using Phylosuite v1.2.3 software [22,23]. The dataset was divided into three partitions: the first codon position of protein-coding genes (P1), the second codon position (P2), and rRNA genes (P3). Find the optimal evolutionary model for each partition using the ModelFinder lugin (integrated into PhyloSuite) [24]. When maximum likelihood (ML) was used to construct phylogenetic trees, the optimal evolutionary model for P1 and P2 was GTR + F + I + I + R4, and the optimal evolutionary model for P3 was TIM + F + I + I + R2. When the phylogenetic tree was inferred by Bayesian Inference (BI), the optimal evolutionary model of the three partitions was GTR + F + I + G4. The maximum likelihood (ML) was performed using IQ-TREE v1.6.2 software (Medical University of Vienna, Vienna, Austria), and the bootstrap value was 10,000 [25]. The software MrBayes v3.2.6 (Medical University of Vienna, Vienna, Austria) performs Bayesian Inference (BI) and runs twice, each run of 20 million generations and sampling once every 1000 generations [26]. The constructed phylogenetic tree was embellished using iTOL Web Edition (https://itol.embl.de/, accessed on 20 April 2023), and beautified by Adobe Illustrator 2021 (Adobe Systems Incorporated, San Jose, CA, USA) [27].

3. Results and Discussion

3.1. Genome Organization and Base Composition

Their mitochondrial genome structure is the same as the classic vertebrate mitochondrial genome structure (Figure 1), which is a closed circular double-stranded DNA molecule that includes 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, one control region (CR), and one origin of light-strand replication (OL) [28]. Among them, tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser (TGA), tRNA-Glu, tRNA-Pro, and nad6 genes are encoded on the L-strand, while the remaining 28 genes are encoded on the H-strand, consistent with other bony fish [29] (Table 2).
The mitochondrial genome sequence lengths of the five fish range from 16,499 bp (P. filamentosus) to 16,530 bp (P. auricilla). The AT content of the mitochondrial genome sequences for five species was around 54%, exhibiting a clear preference using of A T bases (Figure 2). The AT skew ranges from 0.022 (P. argyrogrammicus) to 0.047 (P. filamentosus), while the GC skew ranges from −0.274 (P. filamentosus) to −0.243 (P. argyrogrammicus). The content of A and C is higher than that of T and G (Figure 3).

3.2. Protein Coding Genes and Codon Usage

The mitochondrial genome contains 13 PCGs (nd1, nd2, cox1, cox2, atp8, atp6, cox3, nd4, nd4L, nd4, nd5, nd6, and cyt b). All genes encode on heavy chains except the nad6 gene. The length of 13 PCGs is similar, ranging from 11,435 bp (P. auricilla, P. filamentosus, and P. sieboldii) to 11,442 bp (P. argyrogrammicus and P. zonatus), accounting for approximately 69% of the mitochondrial genome. The nd6 gene is positive to GC skew, and the other 12 genes are negative to GC skew (−0.514 to −0.181) (Figure 3). The AT content of the protein-coding sequence is higher than the GC content, ranging from 50.2% to 56.5% (Figure 2).
Among the 13 PCGs, except for the COI gene, which uses GTG as the start codon, all PCGs in the five species of Pristipomoides use ATG as the start codon, which is similar to other species in the family Lutjanidae [30]. In the class Actinopterygii, using GTG as the start codon in the COI gene is a normal phenomenon [5] The termination codons mainly include TAA, TAG, T--, and AGG. TAA, TAG, and incomplete stop codon T-- are the stop codon of most protein-coding genes, while AGG is the stop codon of cox1 genes of P. argyrogrammicus and P. zonatus (Table 2). The incomplete termination codon T-- may be transformed into a complete termination codon TAA after connecting poly A tails during RNA processing [31]. In the universal genetic code, AGG is the codon for Arg, but in the mitochondrial genome of metazoans, AGG is coded as Ser or used as a stop codon [32].
Among the five species, the number of codon usage ranges from 3800 (P. auricilla, P. filamentosus, P. sieboldii) to 3803 (P. argyrogrammicus, P. zonatus). Leu (CUU, CUC, CUA, and CUG), Ala (GCU, GCC, GCA, and GCG), and Thr (ACU, ACC, ACA, and ACG) are the most frequently used amino acids, while Cys (UGU and UGC) is the least frequently used, similar to other species of the family Lutjanidae (Figure 4) [33,34]. The analysis of the relative synonymous codon usage (RSCU) indicates a preference for using A and C in the third position of codons (Figure 5).

3.3. Ka/Ks, Nucleotide Diversity

We calculated Ka/Ks (the ratio of nonsynonymous to synonymous substitutions) to analyze selection pressure acting on protein-coding genes [35]. The relative size of Ka/Ks compared to one has different meanings: Ka/Ks > 1 suggests positive selection, Ka/Ks = 1 represents neutral evolution, and Ka/Ks < 1 suggests purifying selection [36]. The Ka/Ks values of the 13 PCGs in the five mitochondrial genomes range from 0 to 0.117, suggesting strong elective constraints on the 13 PCGs. The ten Ka/Ks values of the atp8 gene range from 0 to 0.117, and the atp8 gene sequences of four species except P. argyrogrammicus are very similar, which results in the Ka/Ks value approaching zero. The Ka/Ks ratio of cox1 was relatively small (0–0.017), indicating that cox1 was under stronger selection constraints than other genes, which was consistent with other bony fish. The nd2 gene has a relatively high Ka/Ks ratio and is less subject to purifying selection than other protein-coding genes (Figure 6a).
The average nucleotide diversity of the 13 PCGs encoding five types of fish is generally low, ranging from 0.038 to 0.23. Among them, the nd2 gene has a higher number of variable sites, with a relatively high average nucleotide diversity value of 0.1635. On the other hand, the atp8 gene has the lowest average nucleotide diversity value, which is 0.054 (Figure 6b).
Based on the analysis of both the Ka/Ks ratio and nucleotide diversity, it is inferred that the nd2 gene may have the fastest evolutionary rate among the 13 PCGs. Some related studies suggest that nd2 can be used as a DNA barcoding for identifying certain fish species [37] Cai et al. showed that the nd2 gene is suitable for grouper species identification [38]. The nd2 gene may also be suitable for species identification as DNA barcoding for species of the genus Pristipomoides, but the specific verification needs further exploration and will not be discussed in this paper.

3.4. Transfer RNA and Ribosomal RNA Genes

The secondary structure of tRNA includes an amino acid (AA) stem, a dihydrouridine (D) arm, an anticodon (AC) arm, a thymidine (T) arm, and a variable (V) loop [5]. Except for tRNA-Ser (AGY), which lacks a dihydrouridine loop (D loop) (Figure 7), all other tRNAs have a typical cloverleaf structure. tRNA-Ser (AGY) with incomplete structure is also common in other vertebrates [39]. Comparing the secondary structure of tRNAs among the five Pristipomoides species, tRNA-Asn is the most similar. tRNA-Asp, tRNA-Gly, tRNA-Pro, tRNA-Arg, tRNA-Ser (AGY), tRNA-Ser (UCN), and tRNA-Tyr have different numbers of bases among the five species, and in their secondary structure, they mostly show differences in the length of the D loop. Among the five Pristipomoides species, 22 tRNAs have relatively fixed stem lengths, with the AA stem being 7 bp, the T stem being 4–5 bp, the AC stem being 4–5 bp (except tRNA-Val which is 3–4 bp), and the D stem being 3–4 bp. For some tRNAs (tRNA-Ala, tRNA-Asp, tRNA-Pro, and tRNA-Ser (UCN)), the length of the D stem varies among the five fish species due to differences in base composition. The Discriminator nucleotide of tRNA-Asp, tRNA-Gln, tRNA-Arg, and tRNA-Ser (AGY) differs in base composition among the five species, while there are no significant differences in the secondary structure of other tRNAs (Figure 7).
The mitochondrial genome of the five Pristipomoides species contains 12S rRNA and 16S rRNA, both encoded on the heavy strand and located between the tRNA-Phe, tRNA-Val, and tRNA-Leu (UAA) genes. The total length of these genes ranges from 2622 bp (P. filamentosus) to 2634 bp (P. sieboldii). The length of the 12S rRNA gene ranges from 949 bp (P. filamentosus) to 955 bp (P. sieboldii), while that of the 16S rRNA gene ranges from 1672 bp (P. zonatus) to 1679 bp (P. sieboldii, P. auricilla). The AT content of both rRNA genes is similar among the different species, ranging from 52.6% (P. auricilla) to 53.7% (P. argyrogrammicus), with a consistent preference for AT bases (Figure 2). The AT skew values range from 0.179 (P. sieboldii) to 0.2 (P. filamentosus), with the number of A bases exceeding that of T bases (Figure 3).

3.5. Non-Coding Region and Overlap

In mitochondrial genomes, the non-coding region includes the control region (CR), the origin of L-strand replication (OL), and the gene spacer region. The control region is located between the genes tRNA-Pro and tRNA-Phe, and the lengths of the control regions are similar among the five mitochondrial genomes, with the longest in P. auricilla at 825 bp and the shortest in P. filamentosus at 791 bp. The control region has a high AT content, accounting for 59.3–60.2% of the total bases. The AT skew is positive and the GC skew is negative, indicating a clear preference for using A and C bases in the control region.
The OL is a stem-loop structure located between tRNA-Cys and tRNA-Asn genes. The length of OL in five Pristipomoides species is 35 bp, with a pyrimidine-rich 5′ end and a purine-rich 3′ end. The stem sequences are identical, while the loop sequences of P. auricilla and P. sieboldii differ by only one base, which differs from the loop sequences of the other three Pristipomoides species. The loop sequences of P. zonatus and P. argyrogrammicus are highly similar, with only two bases difference (Figure 8).
The intergenic spacer length among the five fish ranges from 1 to 28 bp. Among the mitochondrial genomes studied, there were 12 overlaps between genes, with overlap region lengths ranging from 1 to 10 bp (Table 2). The length of overlap between atp8 and atp6 genes is 10 bp, which is a normal phenomenon in metazoan mitochondrial genomes [41] A 9 bp overlap region between cox1 and trnS (UCN) is found in the mitochondrial genomes of P. argyrogrammicus and P. zonatus.

3.6. Phylogenetic Analysis

We grouped the five species together with other species of the family Lutjanidae for phylogenetic analysis to investigate the phylogenetic relationships of the genus Pristipomoides. When selecting the phylogenetic dataset, we excluded the nd6 gene because it is encoded on the light chain and has a relatively fast evolutionary rate, which may lead to multiple substitutions at some sites and its poor homology may reduce the resolution of the phylogenetic analysis [42]. Maximum Likelihood (ML) and Bayesian analyses (BI) produced consistent tree topologies, and the nodes of interest showed high bootstrap values and posterior probabilities (Figure 9).
The phylogenetic tree can be divided into two branches. The genus Pristipomoides and Aphareus of the subfamily Etelinae cluster into one branch with high support. The other clade is mainly composed of species of the Lutjanus, Caesio, and Pterocaesio.
In clade 1, P. filamentosus and P. miltidens are sister groups, and they form sister groups with A. utilans. P. zonatus is closer to P. argyrogrammicus, and they form a sister clade to the clade containing P. auricilla and P. sieboldii.
A. rutilans belongs to the Aphareus genus within the Lutjanidae family. Sala et al. previously conducted a phylogenetic analysis of some species within the Lutjanidae family based on the COI gene, which revealed a close relationship between P. multidens and A. rutilans [43]. In this study, we have expanded the analysis to include more species and utilized a more comprehensive dataset. The results indicate that A. rutilans clusters together with species from the Pristipomoides genus and is deeply nested within this genus (clade 1; Figure 9). Based on these findings, we propose that A. rutilans may belong to the Pristipomoides genus and suggest a revision in its taxonomy, previously based on morphological characters.
The NCBI contains different mitochondrial genome data that align with the object of this study. These data were similarly included in the objects of the phylogenetic analysis, and it was observed that the P. zonatus sample from NCBI (GeneBank Accession ID: OP035125, hereafter referred to as P. zonatus (OP035125) did not cluster together with the P. zonatus sample from this study (GeneBank Accession Number: OQ581449). Instead, P. zonatus (OP035125) clustered with P. argyrogrammicus. Sequence alignment revealed a higher sequence similarity between P. zonatus (OP035125) and P. argyrogrammicus (95.97%). Additionally, when comparing the published COI sequences from NCBI with the sequence of P. zonatus (OP035125), the sequence similarity was around 93% (Table S3). Therefore, it is speculated that P. zonatus (OP035125) may not belong to the species P. zonatus.
A major branch formed by the aggregation of species from three genera, Lutjanus, Caesio, and Pterocaesio, is divided into three parts. L. malabaricus and L. sebae are sister taxa that diverged earliest. Fourteen species of the Lutjanus genus cluster together to form a larger branch. The two genera Caesio and Pterocaesio, belonging to the subfamily Caesioninae, are deeply nested within the family Lutjanidae, which is consistent with the phylogenetic analysis based on the 16S rRNA and CYT b genes by Miller et al. [44]. Compared to other species in the Lutjanus genus, the fish species C. cuning, P. tile, and P. digramma are more closely related to L. erythropterus, and these four species form a branch with high support, which is similar with the results of Ha Yeun Song et al. [45]. The family Caesionidae is now recognized as a synonym of the family Lutjanidae [6,46].

4. Conclusions

In this study, the mitochondrial genome sequences of five Pristipomoides species were sequenced. The sequence lengths ranged from 16,499 bp to 16,530 bp, with an average AT content of approximately 54%. The genomic structure was consistent with the classic mitochondrial genome structure. A total of 13 PCGs started with GTG or ATG as the start codon and terminated with TAA, TAG, T--, or AGG as the stop codon. Among the 13 PCGs in the mitochondrial genomes of five species, nd2 showed the fastest evolutionary rate. Except for tRNA-Ser (AGY) lacking D-loop, all other tRNAs exhibited normal cloverleaf structures. The phylogenetic analysis of the Pristipomoides genus revealed that A. rutilans nested within the genus, leading us to conclude that A. rutilans can be assigned to the Pristipomoides genus, suggesting a revision in its taxonomy. Within the Pristipomoides genus, P. zonatus and P. argyrogrammicus showed close phylogenetic affinity. The close relationship between P. auricilla and P. sieboldii was consistent with the results of morphological-based phylogenetic analysis [10]. The presented results will greatly contribute to further phylogenetic analysis of the Pristipomoides genus or the Lutjanidae family, and the provided molecular data will be beneficial for species identification and classification. At the same time, more mitochondrial genome data are also expected to be obtained to go beyond the classification mainly based on morphological features.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fishes8070343/s1, Table S1: Primers for the PCR-amplification of five species in the genus Pristipomoides.; Table S2: Detailed information of the analyzed species in this study; Table S3: P. zonatus (GeneBank Accession ID: OP035125) was aligned to the COI sequence at NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 25 March 2023).

Author Contributions

Conceptualization, methodology, validation, funding acquisition, project administration, supervision, and writing—review and editing, X.C.; formal analysis, C.L. and D.L.; visualization, C.L.; investigation, data curation, Y.Z. and J.L.; writing—original draft preparation, C.L., D.L. and M.P.; supervision, H.Z.; resources, Z.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Guangxi Key Research and Development Programme funding (AB19245045).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Ethics Committee of South China Agriculture University (No: 2022G002).

Data Availability Statement

Mitochondrial genome data for the five species in this study are publicly available from the GenBank of the National Center for Biotechnology Information (NCBI) at https://www.ncbi.nlm.nih.gov (accession number: OP179302, OQ581450, OP179303, OQ581449, and OQ581451).

Acknowledgments

Thanks to Sheng Gao for his assistance in collecting fish samples.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Genetic map of the complete mitochondrial genome of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, and P. sieboldii.
Figure 1. Genetic map of the complete mitochondrial genome of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, and P. sieboldii.
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Figure 2. Base compositions of the whole genomes, 13 PCGs, rRNAs, tRNAs, and Control regions of P. argyrogrammicus, P. auricilla, P. filamentosus, P. sieboldii, and P. zonatus.
Figure 2. Base compositions of the whole genomes, 13 PCGs, rRNAs, tRNAs, and Control regions of P. argyrogrammicus, P. auricilla, P. filamentosus, P. sieboldii, and P. zonatus.
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Figure 3. AT and GC skew of the whole genomes, 13 PCGs, rRNAs and tRNAs of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, and P. sieboldii.
Figure 3. AT and GC skew of the whole genomes, 13 PCGs, rRNAs and tRNAs of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, and P. sieboldii.
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Figure 4. Amino acid usage in the mitochondrial genomes of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, P. sieboldii and other Lutjanidae species.
Figure 4. Amino acid usage in the mitochondrial genomes of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, P. sieboldii and other Lutjanidae species.
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Figure 5. Relative synonymous codon usage (RSCU) of 13 protein-coding genes in the mitogenomes of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, and P. sieboldii. The horizontal axis is the corresponding codon of each amino acid, and the vertical axis is the RSCU value. The different colors correspond to the codon color of the horizontal axis.
Figure 5. Relative synonymous codon usage (RSCU) of 13 protein-coding genes in the mitogenomes of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, and P. sieboldii. The horizontal axis is the corresponding codon of each amino acid, and the vertical axis is the RSCU value. The different colors correspond to the codon color of the horizontal axis.
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Figure 6. The Ka/Ks ratio (a) and Nucleotide diversity (b) of 13 protein-coding genes among P. argyrogrammicus, P. filamentosus, P. auricilla, P. sieboldii, and P. zonatus.
Figure 6. The Ka/Ks ratio (a) and Nucleotide diversity (b) of 13 protein-coding genes among P. argyrogrammicus, P. filamentosus, P. auricilla, P. sieboldii, and P. zonatus.
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Figure 7. Secondary structures of the 22 transfer RNA genes of P. argyrogrammicus, P. filamentosus, P. auricilla, P. sieboldii, and P. zonatus. In five species, if tRNA secondary structure in the same position of different base composition, use IUB code [40]: Y (C, T), W (A, T), R (A, G), K (G, T), M (A, C), S (C, G), D (A, G, T), and H (A, C, T). Loop and stem regions of length variation are annotated with red circles and boxes, and positions annotated with orange boxes represent discordant base pairings in the five species.
Figure 7. Secondary structures of the 22 transfer RNA genes of P. argyrogrammicus, P. filamentosus, P. auricilla, P. sieboldii, and P. zonatus. In five species, if tRNA secondary structure in the same position of different base composition, use IUB code [40]: Y (C, T), W (A, T), R (A, G), K (G, T), M (A, C), S (C, G), D (A, G, T), and H (A, C, T). Loop and stem regions of length variation are annotated with red circles and boxes, and positions annotated with orange boxes represent discordant base pairings in the five species.
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Figure 8. The OL structure of P. argyrogrammicus, P. filamentosus, P. auricilla, P. sieboldii, and P. zonatus. Using P. auricilla as the reference, those with bases different from P. auricilla are marked in orange.
Figure 8. The OL structure of P. argyrogrammicus, P. filamentosus, P. auricilla, P. sieboldii, and P. zonatus. Using P. auricilla as the reference, those with bases different from P. auricilla are marked in orange.
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Figure 9. Phylogenetic tree constructed by the Bayesian inference and maximum likelihood methods based on the concatenated sequences of 12 PCGs and 2rRNAs. Numbers next to nodes are support rates, which are Bayesian posterior probability values (left) and bootstrap support values (right), respectively. The red part is the research object of this paper. Information on species used for tree construction is provided in Table S2.
Figure 9. Phylogenetic tree constructed by the Bayesian inference and maximum likelihood methods based on the concatenated sequences of 12 PCGs and 2rRNAs. Numbers next to nodes are support rates, which are Bayesian posterior probability values (left) and bootstrap support values (right), respectively. The red part is the research object of this paper. Information on species used for tree construction is provided in Table S2.
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Table
Table 1. Information of sampling locations and mtDNA sequences of the five species of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, and P. sieboldii.
Table 1. Information of sampling locations and mtDNA sequences of the five species of P. argyrogrammicus, P. auricilla, P. filamentosus, P. zonatus, and P. sieboldii.
SpeciesLength (bp)GenBank Accession NumberSampling Location
P. argyrogrammicus16,508OP179302Howard Shoal
P. auricilla16,530OQ581450Howard Shoal
P. filamentosus16,499OP179303Penguin Shoal
P. zonatus16,505OQ581449Dongsha Islands
P. sieboldii16,525OQ581451Shenhu Shoal
Table
Table 2. Mitochondrial genome characteristics of P. argyrogrammicus, P. filamentosus, P. auricilla, P. sieboldii, and P. zonatus.
Table 2. Mitochondrial genome characteristics of P. argyrogrammicus, P. filamentosus, P. auricilla, P. sieboldii, and P. zonatus.
GeneStrandLength (bp)Start CodonsStop CodonsAnticodonIntergenic
Nucleotide * (bp)
P. argyrogrammicus/P. filamentosus/P. auricilla/P. sieboldii/P. zonatus
trnFH68/68/69/69/68 GAA0
rrnSH953/949/953/955/953 0/1
trnVH73/73/73/73/73 TAC24/28
rrnLH1673/1673/1679/1679/1672 0
trnL2H74/74/74/74/74 TAA0/2
nad1H975/975/975/975/975ATG/ATG/ATG/ATG/ATGTAA/TAA/TAA/TAA/TAA 2/3
trnIH70/70/70/70/70 GAT−1/0
trnQL71/71/71/71/71 TTG−1
trnMH69/69/69/69/69 CAT0
nad2H1047/1047/1047/1047/1047ATG/ATG/ATG/ATG/ATGTAA/TAA/TAA/TAA/TAA −1
trnWH73/73/73/73/73 TCA0
trnAL69/69/69/69/69 TGC1
trnNL73/73/73/73/73 GTT4
OLH35/35/35/35/35 0
trnCL67/67/67/67/67 GCA0
trnYL70/71/71/71/70 GTA1
cox1H1560/1551/1551/1551/1560GTG/GTG/GTG/GTG/GTGAGG/TAA/TAA/TAA/AGG −9/1
trnS2L71/71/72/72/71 TGA3
trnDH72/72/73/73/72 GTC8
cox2H691/691/691/691/691ATG/ATG/ATG/ATG/ATGT/T/T/T/T 0
trnKH75/75/75/75/75 TTT1
atp8H168/168/168/168/168ATG/ATG/ATG/ATG/ATGTAA/TAA/TAA/TAA/TAA −10
atp6H684/684/684/684/684ATG/ATG/ATG/ATG/ATGTAA/TAA/TAA/TAA/TAA −1
cox3H786/786/786/786/786ATG/ATG/ATG/ATG/ATGTAA/TAA/TAA/TAA/TAA −1
trnGH72/72/72/72/72 TCC0
nad3H351/351/351/351/351ATG/ATG/ATG/ATG/ATGTAG/TAG/TAG/TAG/TAG −2
trnRH70/69/70/71/70 TCG0/1
nad4LH297/297/297/297/297ATG/ATG/ATG/ATG/ATGTAA/TAA/TAA/TAA/TAA −7
nad4H1381/1381/1381/1381/1381ATG/ATG/ATG/ATG/ATGT/T/T/T/T 0
trnHH69/69/69/69/69 GTG0/2
trnS1H67/68/68/68/68 GCT4/5
trnL1H73/73/73/73/73 TAG0
nad5H1839/1839/1839/1839/1839ATG/ATG/ATG/ATG/ATGTAA/TAA/TAA/TAA/TAA −4
nad6L522/522/522/522/522ATG/ATG/ATG/ATG/ATGTAG/TAG/TAG/TAA/TAG 0
trnEL69/69/69/69/69 TTC4/5
cytbH1141/1143/1143/1143/1141ATG/ATG/ATG/ATG/ATGT/TAG/TAG/TAG/T −1/0
trnTH72/72/72/72/72 TGT−1
trnPL71/70/70/70/71 TGG
* As for the adjacent genes, positive number represented spaced, negative number indicated overlap and zero indicated continuous.
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Liu, C.; Li, D.; Zhang, Y.; Péré, M.; Zhuang, Z.; Liu, J.; Zhou, H.; Chen, X. Phylogenetic Analyses of Pristipomoides (Perciformes: Lutjanidae) Based on New Mitochondrial Genomes. Fishes 2023, 8, 343. https://doi.org/10.3390/fishes8070343

AMA Style

Liu C, Li D, Zhang Y, Péré M, Zhuang Z, Liu J, Zhou H, Chen X. Phylogenetic Analyses of Pristipomoides (Perciformes: Lutjanidae) Based on New Mitochondrial Genomes. Fishes. 2023; 8(7):343. https://doi.org/10.3390/fishes8070343

Chicago/Turabian Style

Liu, Chunhui, Dezhao Li, Yue Zhang, Maxime Péré, Zhibo Zhuang, Jingyu Liu, Haolang Zhou, and Xiao Chen. 2023. "Phylogenetic Analyses of Pristipomoides (Perciformes: Lutjanidae) Based on New Mitochondrial Genomes" Fishes 8, no. 7: 343. https://doi.org/10.3390/fishes8070343

APA Style

Liu, C., Li, D., Zhang, Y., Péré, M., Zhuang, Z., Liu, J., Zhou, H., & Chen, X. (2023). Phylogenetic Analyses of Pristipomoides (Perciformes: Lutjanidae) Based on New Mitochondrial Genomes. Fishes, 8(7), 343. https://doi.org/10.3390/fishes8070343

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