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. 2025 Dec 31;15(12):6298–6305. doi: 10.5455/OVJ.2025.v15.i12.13

Utility of large mitochondrial rRNA sequences in phylogenetic inference of some epinephelinae species from the Egyptian Red Sea

Mohammad Allam 1, Ali H Abu Almaaty 2, Layla Omran Elmajdoub 3,*, Maryham M Aziz 2
PMCID: PMC13314014  PMID: 42376501

Abstract

Background:

Grouper fishes have a diverse variety of shapes and colors, making it difficult to accurately perform morphological identification. In addition, certain unique grouper species may be included in the same species description due to morphological similarities.

Aim:

The main purpose of this study was to investigate the evolutionary relationships between various species of the family Epinephelinae using the large mitochondrial rRNA gene.

Methods:

Four Epinephelinae species (Cephalopholis argus, Epinephelus summana, Epinephelus chlorostigma, and Epinephelus latifasciatus) were gathered from the Red Sea and identified (Randall, 1982).

Results:

The accession numbers were obtained by displaying the 16S rRNA sequences in GenBank/NCBI (PQ661169.1—PQ661172.1). The average frequencies of adenine (A), thymine (T), cytosine (C), and guanine (G) were 28,93%, 23.66%, 23.84%, and 23.57%, respectively. Compared with C + G, the average proportion for A + T was more significant.

Conclusion:

Epinephelus summana and E. latifasciatus are closely linked, as evidenced by their low genetic distance.

Keywords: 16S rRNA gene, Cephalopholis, Epinephelus, Grouper

Introduction

The 16S rRNA gene is the ultimate molecular chronometer because of several of its characteristics. It is considered the most prevalent housekeeping genetic marker, making it a valuable target for phylogeny and clinical identification (Woese, 1978; Patel, 2001; Janda and Abbott, 2007). The function of the 16S rRNA has not changed in a long time; therefore, random sequence variations are more likely to represent microbial evolutionary (phylogeny) than selected sequences that change the function of the molecule (Woese, 1978). Moreover, the size of the 16S rRNA gene is approximately 1,500 bp, which is sufficient for informatics applications (Weisburg, 1991; Patel, 2001; Janda and Abbott, 2007).

The presence of several conserved/hypervariable regions that provide a variety of PCR primer design options is a crucial feature that supports the use of the 16S rRNA gene (Van de Peer et al., 1996). The 16S rRNA gene has approximately 50 functional domains, and sequences in other domains are not significantly impacted by the introduction of specific modifications in one domain (Patel, 2001).

To identify genes, regulatory elements, and non-coding RNAs in recently sequenced genomes, classify closely related species sequences, and refine phylogenetic trees, molecular phylogenetics is used to explain both ancient and modern individual genomes (Kellis et al., 2003; Pedersen et al., 2006). Phylogenetic analysis employs molecular techniques based on gene sequence analyses to categorize and comprehend the relationships between closely related species in systematics and taxonomy. This approach has grown in significance across all taxonomy domains due to the abundance of publicly accessible genetic sequence data (Yang and Rannala, 2010).

Consequently, coral reef ecosystems have a high degree of biodiversity, particularly in fish species (Allen and Erdman 2012). Fish habitat is one of the most significant ecological roles of coral reef ecosystems, particularly as a place for spawning, feeding, nidification, and predator protection (Yuliana et al., 2020; Mujiyanto et al., 2021).

Groupers are reef fish that belong to the Epinephelinae subfamily and the Serranidae family. In tropical and subtropical regions, groupers naturally live in shallow-water environments such as coral reefs, estuaries, mangroves, and seagrass (Kamal et al., 2019; Andriyono et al., 2020).

There are 159 species in the Epinephelinae subfamily, including 31 genera of Epinephelus and 15 genera of Cephalopholis (Allen and Adrim, 2003). The Serranidae family has historically been regarded as a convenient pigeonhole for lower percoid fish with ambiguous affinities, despite more recent systematic treatments of the family offering a valid categorization. Our knowledge of the relationships of Serranidae has also been unclear (Craig and Hastings, 2007).

Considering the complexity of the members of the subfamily Epinephelinae (Epinephelus, Serranidae), the limitations of studying lineages among fish in this family remain crucial (Craig and Hastings, 2007).

The morphological characteristics of the Epinephelus and Cephalopholis genera are very similar. Consequently, misidentifications between the two genera may occur during morphological identification. Given the limitations of morphological traits alone, a molecular approach is required to support the use of morphological features in species characterization. Age and other environmental factors affect traits (Becker et al., 2015; Hulley et al., 2018; Dwifajri et al., 2022).

The main purpose of this study was to use the 16S rRNA gene to examine the evolutionary relationships between various species of the Epinephelinae family.

Materials and Methods

Gathering samples and species identification

The Red Sea served as the study sampling location, and four Epinephelinae species (Cephalopholis argus, Epinephelus summana, Epinephelus chlorostigma, and Epinephelus latifasciatus) were gathered and identified (Randall, 1982). To extract DNA, muscle tissue was dissected from the specimens and stored at −20°C.

DNA isolation and polymerase chain reaction amplification

Genomic DNA was extracted from the stored muscles using the Biospin Genomic DNA Extraction Kit following the manufacturer’s instructions. The DNA quality was assessed by Agarose gel electrophoresis. A partial sequence of the mitochondrial 16S rRNA was amplified by PCR using recently reported primers (Simon et al., 1991). The PCR reactions were carried out in a total volume of 50 μl consisting of 25μl of 2X master mix, 1μl of genomic DNA, 1μl of each primer, and 22μl of nuclease-free water. Five minutes of denaturation at 95°C, 30 cycles of denaturation, annealing, and extension at 94°C, 48°C, and 72°C, respectively, for one minute each, followed by a final extension at 72°C for 10 minutes, were the conditions for PCR amplification. Electrophoresis was performed on a 1.5% agarose gel using a 100 bp DNA ladder and ethidium bromide to visualize the outcomes of PCR.

Sequences and phylogenetic analyses

Macrogen (Seoul, South Korea) completed the sequences. The sequences of the 16S rRNA were uploaded to GenBank/NCBI to obtain accession numbers. CLUSTAL W (Thompson et al., 1994) was used to align the sequences with the default settings. Neighbor joining and minimum evolution were the two methods used for phylogenetic structure tree using MEGA software version 7.0 (Kumar et al., 2016). To fully implement the sequence divergences (Felsenstein, 1985), Kimura two-parameter distances with 1,000 bootstrapped iterations (Kimura, 1980) were used.

Ethical approval

The Ethics of Animal Experiments Committee of the Faculty of Science of Port Said University accepted all animal experimentation practices (ERN: PSU. Sci. 79).

Results

This study establishes the evolutionary lineages of Epinephelinae species (C. argus, E. summana, E. chlorostigma, and E. latifasciatus). This was achieved using large subunit ribosomal RNA (16S rRNA) sequences. In all four species, the 16S rRNA-produced bands ranged in length from 566 to 575 bp. The accession numbers were obtained by displaying the 16S rRNA sequences in GenBank/NCBI (PQ661169.1—PQ661172.1). The results indicate that Cephalopholis argus has the longest sequence (575 bp), whereas E. summana has the shortest sequence (566 bp). The average frequencies of adenine (A), thymine (T), cytosine (C), and guanine (G) were 28,93%, 23.66%, 23.84%, and 23.57%, respectively. Compared with C + G, the average proportion for A + T was more significant (Table 1).

Table 1. Four species of the family Epinephelinae, along with their accession numbers, nucleotide frequencies, A+T concentrations, and averages of the 16S rRNA sequence.

Total T C A G A+T
Cephalopholis argus 575 25.57 22.26 28.17 24.00 53.74
Epinephelus summana 566 22.79 24.91 28.80 23.50 51.59
Epinephelus chlorostigmaa 567 23.28 23.81 29.45 23.46 52.73
Epinephelus latifasciatus 570 22.98 24.39 29.30 23.33 52.28
Avg. - 23.66 23.84 28.93 23.57 52.59
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A total of 578 base pairs were included in the final alignments. The conserved and variable locations were 505 and 63, respectively.

Throughout all fish, the P-distances varied from 0.0039% to 1.4114%. The P-distances of the understudied species varied from 0.0039% to 0.0186%. The greatest amount (0.0186) was found between C. argus and E. chlorostigma. The smallest value (0.0039) was found between E. summana and E. latifasciatus (Table 2 and Fig. 1).

Table 2. The pairwise distances between the outgroup and four members of the Epinephelinae family using the 16S rRNA gene.

Table 2.

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Fig. 1. Heatmap representation of the 16S rRNA gene used to establish the P-distances among the four Epinephelinae species.

Fig. 1.

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The sequences gathered from four species in the Epinephelinae family, 17 link sequences, and three species of the out-group from GenBank were employed in this study for a wide phylogenetic research to finalize the phylogenetic tree analysis utilizing the 16S rRNA sequence. Multiple phylogenetic techniques were used for the most compelling phylogenetic analysis employing the 16S rRNA gene: Neighbor joining and minimum evolution. Despite a small variation in the support rate, the approaches produced findings that were fundamentally similar and emphasized two key points: (1) A unique cluster is formed by the outgroup species and (2) With the exception of Epinephelus goreensis, all Epinephelus species were clustered close to one another (Figs. 2 and 3).

Fig. 2. The phylogenetic tree of Neighbor joining between species of the Epinephelinae family and the outgroup using the 16S rRNA gene.

Fig. 2.

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Fig. 3. Minimum evolution phylogenetic tree between species of the Epinephelinae family and the outgroup using the 16S rRNA gene.

Fig. 3.

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Discussion

Heino (2014) stated that the morphological qualities of a living organism can vary according to the ecological conditions in which it lives. Fish exhibit a great deal of variation in their physical attributes, both between and within groups (Brraich and Akhter, 2015). The morphological changes in fish symbolize a kind of environmental adaptation (Hossain et al., 2010).

Because conventional taxonomy frequently uses random morphometric datasets and lacks standards for character selection or coding, species identification can be challenging. In some situations, genetic analysis may be used as an additional method of determining taxonomic identification (Basheer et al., 2015).

Owing to the diversity of shapes and colors in grouper fishes (Tapilatu et al., 2021), this study investigated the evolutionary relationships between various species of the Epinephelinae family using 16S rRNA sequences.

Mitochondrial 16S rRNA has a slower rate of mutation and a lower rate of substitution than other mtDNA genes; thus, it is valuable for studying species, communities, and families (Garland and Zimmer, 2002). Furthermore, the 16S rRNA gene may be used to assess fish phylogenetic connections at both the species and generic levels (Moyer et al., 2004; Chakraborty and Iwatsuki, 2006). Thus, 16S rRNA is advised for the reconstruction of pertinent phylogenetic relationships and as an appropriate identification technique in fish evolutionary studies (Saad et al., 2019).

Using 16S rRNA, nucleotide sequences from the understudied fish had an average length of approximately 550 bp. According to Simon et al. (1991), a 500–650 base fragment is amplified by the 16Sar and 16Sbr primers; therefore, this length was within expectations. Furthermore, several studies found the same outcome (Mar'ie and Allam, 2019; Alyamani et al., 2023; Allam et al., 2024; Eisa et al., 2025).

Contrary to (C + G), this investigation revealed that the average bidder for the understudied fish was (A + T). This finding was consistent with further research. Bo et al. (2013) found that the whole 16S rRNA gene exhibits A+T affluence as opposed to C+G affluence. Basheer et al. (2015) discovered that 16S rRNA had a smaller C+G score than A+T in their study of Rastrelliger species. Furthermore, Mar’ie and Allam (2019) found that two puffer fish had a higher A+T ratio than C+G. Mahrous and Allam (2022) found that the A+T rate in various catfish species was higher than the C+G rate. Eisa et al. (2025) reported that the average content of the A+T sequence of the 16S rRNA gene in two Serranid species (Epinephelus aneus and Mycteroperca rubra) was higher than that of the C+G sequence.

Complete alignments of incomplete 16S rRNA sequences in each of the four species of the Epinephelinae family showed high levels of conserved sites. Basheer et al. (2015) found 575 consistent locations of 590 bp in three Rastrelliger species utilizing 16S rRNA aligned sequences. Sokefun (2017) found 337 conserved regions totaling 463 bp of alignment in a phylogenetic study of Cichlids using the 16S gene. Aligning the incomplete 16S rRNA gene sequences of some Carangid fishes reveals several highly conserved areas (Alyamani et al., 2023). According to Ramadan et al. (2023), the (A + T) median value in the four Lutjanus fish species was greater than the (C + G) median value.

According to Kaleshkumar et al. (2015), highly related species exhibited low genetic distance values, whereas the greatest genetic distance is responsible for situations with considerable genetic divergence. Epinephelus summana and E. latifasciatus were closely linked, as evidenced by their low genetic distance.

Acknowledgments

None.

Conflict of interest

There are no conflicts of interest.

Funding

Not applicable.

Authors' contributions

All authors contributed equally to this study.

Data availability

All data are provided in the manuscript.

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Data Availability Statement

All data are provided in the manuscript.

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