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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Mar. Sci.</journal-id>
<journal-title>Frontiers in Marine Science</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Mar. Sci.</abbrev-journal-title>
<issn pub-type="epub">2296-7745</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fmars.2024.1484198</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Marine Science</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Mitogenomic analysis reveals the phylogenetic placement of monotypic <italic>Parachelon grandisquamis</italic> and distinctive structural features of control regions in mullets</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Yoon</surname>
<given-names>Tae-Ho</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2823070/overview"/>
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<contrib contrib-type="author">
<name>
<surname>Kang</surname>
<given-names>Hye-Eun</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Aini</surname>
<given-names>Sarifah</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Wujdi</surname>
<given-names>Arief</given-names>
</name>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
</xref>
<xref ref-type="aff" rid="aff5">
<sup>5</sup>
</xref>
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</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Kim</surname>
<given-names>Hyun-Woo</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="aff" rid="aff6">
<sup>6</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
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</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Kundu</surname>
<given-names>Shantanu</given-names>
</name>
<xref ref-type="aff" rid="aff7">
<sup>7</sup>
</xref>
<xref ref-type="aff" rid="aff8">
<sup>8</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
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</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>KNU G-LAMP Project Group, KNU Institute of Basic Sciences, Kyungpook National University</institution>, <addr-line>Daegu</addr-line>, <country>Republic of Korea</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Institute of Marine Life Science, Pukyong National University</institution>, <addr-line>Busan</addr-line>, <country>Republic of Korea</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Department of Marine Biology, Pukyong National University</institution>, <addr-line>Busan</addr-line>, <country>Republic of Korea</country>
</aff>
<aff id="aff4">
<sup>4</sup>
<institution>Interdisciplinary Program of Marine and Fisheries Sciences and Convergent Technology, Pukyong National University</institution>, <addr-line>Busan</addr-line>, <country>Republic of Korea</country>
</aff>
<aff id="aff5">
<sup>5</sup>
<institution>Research Center for Fishery, National Research and Innovation Agency (BRIN)</institution>, <addr-line>Bogor</addr-line>, <country>Indonesia</country>
</aff>
<aff id="aff6">
<sup>6</sup>
<institution>Marine Integrated Biomedical Technology Center, National Key Research Institutes in Universities, Pukyong National University</institution>, <addr-line>Busan</addr-line>, <country>Republic of Korea</country>
</aff>
<aff id="aff7">
<sup>7</sup>
<institution>Ocean and Fisheries Development International Cooperation Institute, College of Fisheries Science, Pukyong National University</institution>, <addr-line>Busan</addr-line>, <country>Republic of Korea</country>
</aff>
<aff id="aff8">
<sup>8</sup>
<institution>International Graduate Program of Fisheries Science, Pukyong National University</institution>, <addr-line>Busan</addr-line>, <country>Republic of Korea</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Ana Laura Iba&#xf1;ez, Autonomous Metropolitan University, Mexico</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Jiao Cheng, Chinese Academy of Sciences (CAS), China</p>
<p>Khaled Mohammed Geba, Menoufia University, Egypt</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Hyun-Woo Kim, <email xlink:href="mailto:kimhw@pknu.ac.kr">kimhw@pknu.ac.kr</email>; Shantanu Kundu, <email xlink:href="mailto:shantanu1984@pknu.ac.kr">shantanu1984@pknu.ac.kr</email>; <email xlink:href="mailto:shantanu1984@gmail.com">shantanu1984@gmail.com</email>
</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>19</day>
<month>12</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>11</volume>
<elocation-id>1484198</elocation-id>
<history>
<date date-type="received">
<day>21</day>
<month>08</month>
<year>2024</year>
</date>
<date date-type="accepted">
<day>29</day>
<month>11</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Yoon, Kang, Aini, Wujdi, Kim and Kundu</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Yoon, Kang, Aini, Wujdi, Kim and Kundu</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<sec>
<title>Introduction</title>
<p>The large-scale mullet, <italic>Parachelon grandisquamis</italic> (Teleostei: Mugilidae), is a monotypic species endemic to the eastern Atlantic Ocean, playing a crucial role in tropical ecosystems. Despite its ecological significance, the systematic classification of Mugilidae remains unresolved, largely due to their diverse morphology, which necessitates the integration of molecular data.</p>
</sec>
<sec>
<title>Methods</title>
<p>This study aimed to achieve a comprehensive molecular characterization of the species and establish its matrilineal taxonomic placement using complete mitogenome data. Next-generation sequencing was employed to generate the <italic>de novo</italic> mitogenome of <italic>P. grandisquamis</italic>, which spans 16,859 bp and includes 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNAs, and a non-coding AT-rich control region (CR).</p>
</sec>
<sec>
<title>Results</title>
<p>Most PCGs use ATG as the start codon, with the exception of <italic>COI</italic>, which begins with GTG. Analysis of amino acids abundance revealed high frequencies for leucine, serine, proline, threonine, and alanine with distinctive codon usage. The proportion of nonsynonymous and synonymous substitutions suggests strong purifying selection in most PCGs, except for <italic>ND4L</italic>, <italic>ND5</italic>, and <italic>ND6</italic>. Most transfer RNAs exhibited typical cloverleaf secondary structures, with the exception of <italic>tRNA-Ser1</italic> (GCT), which lacks base pairing in the DHU arm. Mitogenome-based phylogenetic analysis using the Bayesian approach revealed that the monotypic <italic>P. grandisquamis</italic> is closely related to the genera <italic>Chelon</italic> and <italic>Planiliza</italic> within Mugilidae. Furthermore, analysis of the CRs with polymorphic nucleotides in conserved blocks provides additional insight into the development of distinct molecular markers for species identification and population structure analysis of mullets.</p>
</sec>
<sec>
<title>Discussion</title>
<p>Overall, this study provides a comprehensive analysis of the mitogenomic structure and variation of <italic>P. grandisquamis and other mullets</italic>, confirming its maternal evolutionary relationships and offering valuable insights for advancing SNP-based species discrimination within the Mugilidae lineage.</p>
</sec>
</abstract>
<kwd-group>
<kwd>Atlantic Ocean</kwd>
<kwd>mugilids</kwd>
<kwd>endemic species</kwd>
<kwd>mitochondrial genome</kwd>
<kwd>evolution</kwd>
<kwd>conservation</kwd>
</kwd-group>
<contract-num rid="cn001">No. RS-2023-00301914, 2021R1A6A1A03039211</contract-num>
<contract-sponsor id="cn001">National Research Foundation of Korea<named-content content-type="fundref-id">10.13039/501100003725</named-content>
</contract-sponsor>
<counts>
<fig-count count="5"/>
<table-count count="2"/>
<equation-count count="0"/>
<ref-count count="86"/>
<page-count count="15"/>
<word-count count="6197"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Marine Evolutionary Biology, Biogeography and Species Diversity</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<label>1</label>
<title>Introduction</title>
<p>The grey mullets, a group of ray-finned fishes within the order Mugiliformes and family Mugilidae, are widely distributed across tropical, subtropical, and temperate regions globally (<xref ref-type="bibr" rid="B74">Thomson, 1966</xref>). They play a vital ecological role in aquatic ecosystems and serve as an important food resource. Mugilids currently encompasses 76 valid species across 25 genera, with two new species described within the last decade (<xref ref-type="bibr" rid="B16">Fricke et&#xa0;al., 2024</xref>). Despite their ecological significance, the taxonomy and evolutionary relationships of mugilids remain largely unresolved. This is primarily due to the morphological dimorphism observed among species, which complicates their systematic classification (<xref ref-type="bibr" rid="B24">Harrison et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B63">Schultz, 1946</xref>; <xref ref-type="bibr" rid="B75">Thomson, 1997</xref>). As a result, the number of recognized species may be overestimated, as taxonomic evaluations have often been based on local specimens without adequate comparison to those from other regions (<xref ref-type="bibr" rid="B73">Thomson, 1954</xref>).</p>
<p>The first comprehensive taxonomic revision of the Mugilidae family, based on mouth anatomy, validated 13 genera (<xref ref-type="bibr" rid="B63">Schultz, 1946</xref>; <xref ref-type="bibr" rid="B19">Ghasemzadeh et&#xa0;al., 2004</xref>). However, subsequent studies have indicated that the systematics of this group remain incomplete (<xref ref-type="bibr" rid="B48">Nelson, 2006</xref>). Within the extant species, three genera&#x2014;<italic>Chelon</italic>, <italic>Mugil</italic>, and <italic>Planiliza</italic>&#x2014;are particularly species-rich, encompassing 41 valid species and accounting for 53.94% of the family&#x2019;s total diversity. The remaining genera exhibit less diversity, with 16 being monotypic (<xref ref-type="bibr" rid="B16">Fricke et&#xa0;al., 2024</xref>). The high proportion of monotypic genera may reflect the challenges in their classification due to the limited presence of diagnostic or synapomorphic characters, potentially indicating a prolonged period of static ancestral radiation (<xref ref-type="bibr" rid="B13">Durand et&#xa0;al., 2012</xref>). These morpho-anatomical phylogenetic hypotheses have consistently encountered difficulties in resolving the cladistic relationships among mugilid species (<xref ref-type="bibr" rid="B23">Harrison and Howes, 1991</xref>; <xref ref-type="bibr" rid="B63">Schultz, 1946</xref>; <xref ref-type="bibr" rid="B18">Ghasemzadeh, 1998</xref>; <xref ref-type="bibr" rid="B65">Senou, 1988</xref>; <xref ref-type="bibr" rid="B75">Thomson, 1997</xref>).</p>
<p>Over the past two decades, DNA-based research has significantly advanced the resolution of systematic challenges in fish taxonomy across various levels (<xref ref-type="bibr" rid="B9">Chen and Mayden, 2010</xref>). Initially, due to shared plesiomorphic characteristics, mugilids were positioned in an intermediate spot within the Acanthomorph phylogeny (<xref ref-type="bibr" rid="B71">Stiassny, 1993</xref>). However, more refined teleost cladistic analyses have since clarified that mugilids are evolutionarily closer to other advanced teleosts within the Percomorpha (<xref ref-type="bibr" rid="B8">Chen et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B45">Miya et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B10">Chen et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B41">Mabuchi et&#xa0;al., 2007</xref>). The phylogenetic placement of mugilids within the Acanthomorph lineage has been further investigated using molecular data (<xref ref-type="bibr" rid="B66">Setiamarga et&#xa0;al., 2008</xref>; <xref ref-type="bibr" rid="B37">Li et&#xa0;al., 2009</xref>). Despite this, phylogenetic analyses specifically targeting mugilids have often relied on partial molecular markers, both nuclear and mitochondrial, sourced from various geographical regions (<xref ref-type="bibr" rid="B5">Caldara et&#xa0;al., 1996</xref>; <xref ref-type="bibr" rid="B57">Rossi et&#xa0;al., 1998</xref>; <xref ref-type="bibr" rid="B77">Turan et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B15">Fraga et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B50">Papasotiropoulos et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B64">Semina et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B4">Blel et&#xa0;al., 2008</xref>; <xref ref-type="bibr" rid="B14">Erguden et&#xa0;al., 2010</xref>; <xref ref-type="bibr" rid="B34">Lee et&#xa0;al., 1995</xref>). These efforts have predominantly focused on species-rich genera such as <italic>Mugil</italic>, <italic>Chelon</italic>, and <italic>Planiliza</italic> to elucidate their evolutionary relationships and lineage diversification (<xref ref-type="bibr" rid="B1">Aurelle et&#xa0;al., 2008</xref>; <xref ref-type="bibr" rid="B25">Heras et&#xa0;al., 2009</xref>). Additionally, molecular data have been effectively employed in phylogeographic studies of mugilids, with particular emphasis on the species <italic>Mugil cephalus</italic> and <italic>Mugil curema</italic> (<xref ref-type="bibr" rid="B11">Crosetti et&#xa0;al., 1994</xref>; <xref ref-type="bibr" rid="B27">Jamandre et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B28">Ke et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B40">Livi et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B68">Shen et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B55">Rocha-Olivares et&#xa0;al., 2000</xref>).</p>
<p>Recently, the integration of next-generation sequencing (NGS) into biodiversity research has greatly enhanced the resolution of teleost phylogeny, including that of mugilids, through complete mitogenome-based assessments (<xref ref-type="bibr" rid="B43">Miya et&#xa0;al., 2001</xref>, <xref ref-type="bibr" rid="B45">2003</xref>; <xref ref-type="bibr" rid="B83">Yamanoue et&#xa0;al., 2007</xref>). Ichthyologists worldwide have analyzed the complete mitogenomes of numerous fish species, leveraging their genetic traits to reconstruct evolutionary hypotheses and trace lineage diversification across time and space (<xref ref-type="bibr" rid="B26">Iwasaki et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B62">Satoh et&#xa0;al., 2016</xref>). Currently, the global GenBank database includes mitogenomes from 24 Mugilidae species across 16 genera, including eight monotypic taxa. However, this taxonomic coverage is skewed by the inclusion of just three species (<italic>Chelon labrosus</italic>, <italic>Oedalechilus labeo</italic>, and <italic>Mugil curema</italic>), which are all distributed in the eastern Atlantic Ocean. Notably, the monotypic and sympatric mugilids <italic>Neochelon falcipinnis</italic> and <italic>Parachelon grandisquamis</italic>, which are endemic to the eastern Atlantic, have yet to be assessed from this unique marine environment. As a result, mitogenome-based phylogenetic hypotheses may offer a biased perspective of mugilid evolution, highlighting the critical need to include the remaining 50% of monotypic taxa on a global scale.</p>
<p>The large-scaled mullet, <italic>Parachelon grandisquamis</italic>, is distributed along the eastern Atlantic coast, from Senegal south to Congo, including the islands of Bioko (Equatorial Guinea), S&#xe3;o Tom&#xe9;, and Pr&#xed;ncipe (<xref ref-type="bibr" rid="B16">Fricke et&#xa0;al., 2024</xref>; <xref ref-type="bibr" rid="B17">Froese and Pauly, 2024</xref>). This species inhabits shallow coastal waters, estuaries, and brackish lagoons, including mangroves, creeks, inundated mudflats, and freshwater rivers (<xref ref-type="bibr" rid="B21">Harrison, 2008</xref>). The taxonomic classification of <italic>P. grandisquamis</italic> has undergone several revisions; originally described under the genus <italic>Mugil</italic>, it was later placed into the genus <italic>Liza</italic> due to key morphological features, including its large sized scale count, the presence of two pharyngobranchial valves, and the yellowish colorization of its anal and lower caudal fin lobe (<xref ref-type="bibr" rid="B22">Harrison, 2016</xref>; <xref ref-type="bibr" rid="B76">Trape et&#xa0;al., 2012</xref>). More recently, phylogenetic analyses based on mitochondrial DNA placed the species in a monotypic genus, <italic>Parachelon</italic>, distinguishing it from other mugilids (<xref ref-type="bibr" rid="B13">Durand et&#xa0;al., 2012</xref>). Beyond taxonomy, molecular data from several nuclear genes (<italic>RAG1</italic>, <italic>ENC1</italic>, <italic>Myh6</italic>, <italic>Ptr</italic>, <italic>Glyt</italic>, <italic>SH3PX3</italic>, <italic>Sidkey</italic>, <italic>Rhodopsin</italic>, <italic>TMO</italic>-<italic>4C4</italic>, <italic>plagl2</italic>, <italic>serb2</italic>, <italic>RNF213</italic>) and mitochondrial genes (<italic>12S rRNA</italic>, <italic>16S rRNA</italic>, <italic>COI</italic>, and <italic>Cytb</italic>) have been generated for <italic>P. grandisquamis</italic> to elucidate the multilocus-based phylogeny of mugilids and address persistent taxonomic challenges (<xref ref-type="bibr" rid="B13">Durand et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B82">Xia et&#xa0;al., 2016</xref>). However, to achieve a comprehensive matrilineal phylogeny of mugilids, the generation and characterization of the complete mitogenome are crucial for clarifying the taxonomic placement of this monotypic taxon. This study aims to generate and characterize the complete mitogenome of <italic>P. grandisquamis</italic> from the eastern Atlantic Ocean to illuminate its maternal evolutionary relationships with other mugilids. In addition to provide evolutionary insights, this genetic data will be critical for understanding the population structure and evolutionary patterns of this species, contributing to conservation genetics. Such studies are essential for all extant Mugiliformes species globally, offering new perspectives on the roles of various mitochondrial genes in the evolution of these enigmatic fish across their marine habitats.</p>
</sec>
<sec id="s2" sec-type="materials|methods">
<label>2</label>
<title>Materials and methods</title>
<sec id="s2_1">
<label>2.1</label>
<title>Sampling and species identification</title>
<p>A single specimen of the large-scaled mullet, P<italic>. grandisquamis</italic>, was collected from the muddy seabed along the coast of Cameroon, Africa (3.6322&#xb0;N, 9.8005&#xb0;E) (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>). The identification was confirmed based on taxonomic characteristics described in previous studies, as these often overlap with its closest congener, <italic>Chelon bandialensis</italic>. However, <italic>P. grandisquamis</italic> can be distinguished by having large size of scales, 25-30 scales in the longitudinal series and 8-10.5 scales in the transverse series (<xref ref-type="bibr" rid="B76">Trape et&#xa0;al., 2012</xref>). Additionally, the buccal teeth of <italic>P. grandisquamis</italic> are slightly smaller, and its dorsal, anal, and caudal fins are less distinctly yellowish compared to those of <italic>C. bandialensis</italic>. Muscle tissue was collected from the ventral thoracic region for molecular analysis and novel mitogenome generation. To validate the morphology-based species identification, we amplified a partial fragment of the mitochondrial COI gene using previously published primer pairs and protocols (<xref ref-type="bibr" rid="B81">Ward et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B30">Kundu et&#xa0;al., 2019</xref>). The amplified sequence showed a 99.79% nucleotide similarity in a BLAST search with the GenBank sequence (Accession number JQ060476) generated in the previous study where the genus and species were reassigned (<xref ref-type="bibr" rid="B13">Durand et&#xa0;al., 2012</xref>). The specimen was stored in 10% formaldehyde at the Fisheries and Animal Industries (MINEPIA) facility in Yaound&#xe9;, Cameroon. The experimental protocol was approved by the host institute Animal Care and Use Committee (PKNUIACUC-2022-72). The global distribution map of <italic>P. grandisquamis</italic> was obtained from FishBase and generated using the CMAR c-squares mapper, hosted at <ext-link ext-link-type="uri" xlink:href="https://www.obis.org.au">https://www.obis.org.au</ext-link> (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>).</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Global distribution pattern and complete mitogenome of <italic>P. grandisquamis</italic>. The sampling locality is denoted by a blue pin in Cameroon, eastern Atlantic Ocean. The spherical representation of gene structure and arrangement is provided and annotated by the MitoAnnotator. Various color arcs delineate the occurrence of PCGs, tRNAs, rRNAs, and CR.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1484198-g001.tif"/>
</fig>
</sec>
<sec id="s2_2">
<label>2.2</label>
<title>DNA extraction and sequencing</title>
<p>The total genomic DNA was extracted by using an AccuPrep<sup>&#xae;</sup> DNA isolation kit with standardized protocol (Bioneer in Daejeon, Republic of Korea). Both the quantity and quality of the DNA were checked through a NanoDrop Microvolume spectrophotometer (Thermo Fisher Scientific D1000, Waltham, MA, USA). The complete mitogenome of <italic>P. grandisquamis</italic> was sequenced through a next-generation sequencing approach on the NovaSeq platform (Macrogen, Daejeon, Republic of Korea). The TruSeq Nano DNA high-throughput library preparation kit was used to formulate the sequencing library (Illumina, Inc., San Diego, CA, USA). Primarily, 100 ng of genomic DNA was fragmented through an adaptive-focused acoustic tool (Covaris, Woburn, MA, USA). Further, abiding by the end-repair process, DNA fragments were chosen via a bead-based process, adapted by inserting an &#x2018;A&#x2019; base, and later ligated through TruSeq DNA UD indexing adapters. To yield the final library, the resulting products endured purification and subsequent PCR enrichment. The quantification of the library was measured using qPCR, ensuing the typical procedure (KAPA Quantification Kits for Illumina Sequencing), and quality judgment was accomplished through the 4200 TapeStation D1000 screentape (Agilent Technologies, Santa Clara, CA, USA). Lastly, the paired-end (2 &#xd7; 150 bp) sequencing was directed through the NovaSeq platform (Illumina, Inc., San Diego, CA, USA).</p>
</sec>
<sec id="s2_3">
<label>2.3</label>
<title>Mitogenome assembly and annotation</title>
<p>More than 20 million raw reads were screened using the Cutadapt tool (<ext-link ext-link-type="uri" xlink:href="http://code.google.com/p/cutadapt/">http://code.google.com/p/cutadapt/</ext-link>), applying a Phred quality score threshold (Q score &gt; 20). The target mitogenome was assembled using high-quality paired-end reads with the Geneious Prime version 2023.0.1 software. The complete mitogenome of <italic>M. cephalus</italic> (Accession No. AP002930) served as the reference sequence during assembly, utilizing default mapping algorithms (<xref ref-type="bibr" rid="B43">Miya et&#xa0;al., 2001</xref>). Gene boundaries and strand orientations were validated with MitoAnnotator (<ext-link ext-link-type="uri" xlink:href="http://mitofish.aori.u-tokyo.ac.jp/annotation/input/">http://mitofish.aori.u-tokyo.ac.jp/annotation/input/</ext-link>) (<xref ref-type="bibr" rid="B26">Iwasaki et&#xa0;al., 2013</xref>) and MITOS Version 1.1.6, integrated with the Galaxy Version 1.1.6 online servers (<ext-link ext-link-type="uri" xlink:href="https://mitos.bioinf.uni-leipzig.de/">https://mitos.bioinf.uni-leipzig.de/</ext-link>) (<xref ref-type="bibr" rid="B2">Bernt et&#xa0;al., 2013</xref>). Overlapping regions were further scrutinized using MEGA X to confirm the accuracy of the assembled mitogenome (<xref ref-type="bibr" rid="B29">Kumar et&#xa0;al., 2018</xref>). The putative amino acid sequences for each protein-coding gene (PCG) were identified using the Open Reading Frame Finder web tool (<ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/orffinder/">https://www.ncbi.nlm.nih.gov/orffinder/</ext-link>), employing the vertebrate mitochondrial genetic code.</p>
</sec>
<sec id="s2_4">
<label>2.4</label>
<title>Validation of control region</title>
<p>To obtain the full length of the control region (CR), a new primer pair (5&#x2032;-CGTCGTAATTCTTACCTGAATTGG-3&#x2032; and 5&#x2032;-GCCTGATACCAGCTCCTTGTC-3&#x2032;) was designed from the segment between <italic>Cytb</italic> and <italic>12S rRNA</italic> genes. The targeted CR was amplified using a TaKaRa Verity Thermal Cycler with a reaction mixture containing 1 &#xb5;L of <italic>P. grandisquamis</italic> DNA template, 1X PCR buffer, 1 U Taq polymerase, 10 pmol of each primer, and 2.5 mM dNTPs. The amplicon was purified using the AccuPrep<sup>&#xae;</sup> PCR/Gel Purification Kit (Bioneer, Daejeon, Republic of Korea). Subsequently, the purified amplicon was amplified using the BigDye<sup>&#xae;</sup> Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and sequenced bidirectionally on an ABI PRISM 3730XL DNA analyzer via the Sanger sequencing method (Macrogen, Daejeon, Republic of Korea). The consensus sequence was screened using SeqScanner version 1.0 (Applied Biosystems Inc., Foster City, CA, USA) to eliminate noisy base pairs, and CR validation was confirmed by aligning overlapping regions with MEGA X. The final mitochondrial genome sequence of <italic>P. grandisquamis</italic> was deposited in the global GenBank database, where it received an accession number.</p>
</sec>
<sec id="s2_5">
<label>2.5</label>
<title>Genomic characterization and comparative analyses</title>
<p>In this study, a circular representation of the assembled mitochondrial genome was generated using the MitoAnnotator online tool (<ext-link ext-link-type="uri" xlink:href="http://mitofish.aori.u-tokyo.ac.jp/annotation/input/">http://mitofish.aori.u-tokyo.ac.jp/annotation/input/</ext-link>). Intergenic spacers between adjacent genes and overlapping sections were manually identified. The nucleotide composition of PCGs, ribosomal RNA (rRNA), transfer RNA (tRNA), and CR was determined using MEGA X. Base composition skew was calculated using the formulas AT-skew = [A &#x2212; T]/[A + T] and GC-skew = [G &#x2212; C]/[G + C] (<xref ref-type="bibr" rid="B51">Perna and Kocher, 1995</xref>). The mitochondrial genetic code of vertebrates was utilized in MEGA X to verify the initiation and termination codons of each PCG. Additionally, DnaSP 6.0 was employed for comparative analysis, including the calculation of relative synonymous codon usage (RSCU) and the assessment of amino acid abundance (<xref ref-type="bibr" rid="B59">Rozas et&#xa0;al., 2017</xref>). The boundaries of tRNA and rRNA genes were confirmed using ARWEN 1.2 and tRNAscan-SE Search Server 2.0 (<xref ref-type="bibr" rid="B7">Chan et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B33">Laslett and Canb&#xe4;ck, 2008</xref>). To investigate critical components such as PCG substitution patterns and the structure of conserved blocks in CRs, comparative analyses were performed with P. grandisquamis and other mugilids (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table S1</bold>
</xref>). The DnaSP 6.0 program was also used to estimate pairwise substitution patterns of PCGs, including non-synonymous (Ka) and synonymous (Ks) substitutions. As detailed in previous studies, CLUSTAL X alignment was used to identify structural features within the conserved blocks in CRs (<xref ref-type="bibr" rid="B72">Thompson et&#xa0;al., 1997</xref>; <xref ref-type="bibr" rid="B31">Kundu et&#xa0;al., 2024</xref>).</p>
</sec>
<sec id="s2_6">
<label>2.6</label>
<title>Dataset preparation and phylogenetic analyses</title>
<p>To elucidate the evolutionary relationships among species in the Mugilidae family, including <italic>P. grandisquamis</italic>, we conducted a phylogenetic analysis using the mitochondrial genomes of 21 species representing 15 genera. The Black Mouth Cameroon Tilapia<italic>, Coptodon camerunensis</italic> (Cichliformes: Cichlidae) was included as an outgroup taxon (Access number OQ696044) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table S1</bold>
</xref>). The phylogenetic tree was constructed using a dataset comprising 13 PCGs, assembled with iTaxoTools 0.1 (<xref ref-type="bibr" rid="B78">Vences et&#xa0;al., 2021</xref>). The optimal substitution model, &#x2018;GTR + G + I,&#x2019; was identified based on the Bayesian information criterion (BIC) using PartitionFinder 2 and JModelTest v2 (<xref ref-type="bibr" rid="B12">Darriba et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B32">Lanfear et&#xa0;al., 2016</xref>). Bayesian analysis (BA) was performed with MrBayes 3.1.2, employing a model with nst = 6, one cold and three hot chains of the Metropolis-coupled Markov chain Monte Carlo (MCMC). The analysis ran for 1,000,000 generations, with tree samples collected every 100 generations and 25% of the initial samples discarded as burn-in (<xref ref-type="bibr" rid="B56">Ronquist and Huelsenbeck, 2003</xref>). The resulting Bayesian tree was visualized using the iTOL v4 web server (<ext-link ext-link-type="uri" xlink:href="https://itol.embl.de/login.cgi">https://itol.embl.de/login.cgi</ext-link>) (<xref ref-type="bibr" rid="B36">Letunic and Bork, 2007</xref>).</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<label>3</label>
<title>Results</title>
<sec id="s3_1">
<label>3.1</label>
<title>Mitogenome structure and organization</title>
<p>In this study, we illustrated the mitogenome of <italic>P. grandisquamis</italic>, revealing a length of 16,859 base pairs (bp) with access numbers in GenBank OR487150 (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>). The mitogenome for <italic>P. grandisquamis</italic> contains 37 genes, consisting of 13 PCGs, 22 tRNAs, two rRNAs, and AT-rich CR. The positive strand contains 12 PCGs, 14 tRNAs, and two rRNAs, while the negative strand contains a single PCG (<italic>ND6</italic>) as well as eight tRNAs (<italic>tRNA-Gln</italic>, <italic>tRNA-Ala</italic>, <italic>tRNA-Asn</italic>, <italic>tRNA-Cys</italic>, <italic>tRNA-Tyr</italic>, <italic>tRNA-Ser, tRNA-Glu</italic>, and <italic>tRNA-Pro</italic>) (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>). The overall mitogenome base composition of <italic>P. grandisquamis</italic> showed an AT bias of 55.08% (A = 28.30%, T = 26.70%, G = 15.42%, and C = 29.43%), resulting an AT-skew of 0.030 and a GC-skew of -0.312 (<xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>). Further investigation on the gene boundaries and arrangement revealed 15 intergenic spacers with a total of 122 bp and five overlapping regions, totaling of 23 bp in the mitogenome of <italic>P. grandisquamis</italic>. The longest intergenic spacer with 32 bp was identified between <italic>tRNA-Thr</italic> (T) and <italic>tRNA-Pro</italic> (P), while the most extensive overlap region of 10 bp was shared between the genes <italic>ATP8</italic> and <italic>ATP6</italic> (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>).</p>
<table-wrap id="T1" position="float">
<label>Table&#xa0;1</label>
<caption>
<p>List of annotated mitochondrial genes, including their boundaries, sizes, and intergenic nucleotides for <italic>P. grandisquamis</italic>.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="middle" align="left">Genes</th>
<th valign="middle" align="left">Start</th>
<th valign="middle" align="left">End</th>
<th valign="middle" align="left">Strand</th>
<th valign="middle" align="left">Size (bp)</th>
<th valign="middle" align="left">Intergenic Nucleotide</th>
<th valign="middle" align="left">Anti-codon</th>
<th valign="middle" align="left">Start Codon</th>
<th valign="middle" align="left">Stop Codon</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Phe (F)</italic>
</td>
<td valign="middle" align="center">1</td>
<td valign="middle" align="center">69</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">69</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">GAA</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>12S rRNA</italic>
</td>
<td valign="middle" align="center">70</td>
<td valign="middle" align="center">1025</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">956</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Val (V)</italic>
</td>
<td valign="middle" align="center">1026</td>
<td valign="middle" align="center">1097</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">72</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">TAC</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>16S rRNA</italic>
</td>
<td valign="middle" align="center">1098</td>
<td valign="middle" align="center">2786</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">1689</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Leu (L2)</italic>
</td>
<td valign="middle" align="center">2787</td>
<td valign="middle" align="center">2860</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">74</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">TAA</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ND1</italic>
</td>
<td valign="middle" align="center">2861</td>
<td valign="middle" align="center">3835</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">975</td>
<td valign="middle" align="center">4</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">TAA</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Ile (I)</italic>
</td>
<td valign="middle" align="center">3840</td>
<td valign="middle" align="center">3909</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">70</td>
<td valign="middle" align="center">-1</td>
<td valign="middle" align="center">GAT</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Gln (Q)</italic>
</td>
<td valign="middle" align="center">3909</td>
<td valign="middle" align="center">3979</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">71</td>
<td valign="middle" align="center">-1</td>
<td valign="middle" align="center">TTG</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Met (M)</italic>
</td>
<td valign="middle" align="center">3979</td>
<td valign="middle" align="center">4048</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">70</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">CAT</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ND2</italic>
</td>
<td valign="middle" align="center">4049</td>
<td valign="middle" align="center">5093</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">1045</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">T--</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Trp (W)</italic>
</td>
<td valign="middle" align="center">5094</td>
<td valign="middle" align="center">5165</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">72</td>
<td valign="middle" align="center">1</td>
<td valign="middle" align="center">TCA</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Ala (A)</italic>
</td>
<td valign="middle" align="center">5167</td>
<td valign="middle" align="center">5235</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">69</td>
<td valign="middle" align="center">1</td>
<td valign="middle" align="center">TGC</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Asn (N)</italic>
</td>
<td valign="middle" align="center">5237</td>
<td valign="middle" align="center">5309</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">73</td>
<td valign="middle" align="center">30</td>
<td valign="middle" align="center">GTT</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Cys (C)</italic>
</td>
<td valign="middle" align="center">5340</td>
<td valign="middle" align="center">5405</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">66</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">GCA</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Tyr (Y)</italic>
</td>
<td valign="middle" align="center">5406</td>
<td valign="middle" align="center">5472</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">67</td>
<td valign="middle" align="center">7</td>
<td valign="middle" align="center">GTA</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>COI</italic>
</td>
<td valign="middle" align="center">5480</td>
<td valign="middle" align="center">7051</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">1572</td>
<td valign="middle" align="center">26</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">GTG</td>
<td valign="middle" align="center">TAA</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Ser (S2)</italic>
</td>
<td valign="middle" align="center">7078</td>
<td valign="middle" align="center">7148</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">71</td>
<td valign="middle" align="center">3</td>
<td valign="middle" align="center">TGA</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Asp (D)</italic>
</td>
<td valign="middle" align="center">7152</td>
<td valign="middle" align="center">7224</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">73</td>
<td valign="middle" align="center">4</td>
<td valign="middle" align="center">GTC</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>COII</italic>
</td>
<td valign="middle" align="center">7229</td>
<td valign="middle" align="center">7919</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">691</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">T--</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Lys (K)</italic>
</td>
<td valign="middle" align="center">7920</td>
<td valign="middle" align="center">7993</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">74</td>
<td valign="middle" align="center">1</td>
<td valign="middle" align="center">TTT</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ATP8</italic>
</td>
<td valign="middle" align="center">7995</td>
<td valign="middle" align="center">8162</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">168</td>
<td valign="middle" align="center">-10</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">TAA</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ATP6</italic>
</td>
<td valign="middle" align="center">8153</td>
<td valign="middle" align="center">8835</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">683</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">TA-</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>COIII</italic>
</td>
<td valign="middle" align="center">8836</td>
<td valign="middle" align="center">9619</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">784</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">T--</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Gly (G)</italic>
</td>
<td valign="middle" align="center">9620</td>
<td valign="middle" align="center">9692</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">73</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">TCC</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ND3</italic>
</td>
<td valign="middle" align="center">9693</td>
<td valign="middle" align="center">10041</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">349</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">T--</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Arg (R)</italic>
</td>
<td valign="middle" align="center">10042</td>
<td valign="middle" align="center">10110</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">69</td>
<td valign="middle" align="center">1</td>
<td valign="middle" align="center">TCG</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ND4L</italic>
</td>
<td valign="middle" align="center">10112</td>
<td valign="middle" align="center">10408</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">297</td>
<td valign="middle" align="center">-7</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">TAA</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ND4</italic>
</td>
<td valign="middle" align="center">10402</td>
<td valign="middle" align="center">11782</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">1381</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">T--</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-His (H)</italic>
</td>
<td valign="middle" align="center">11783</td>
<td valign="middle" align="center">11853</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">71</td>
<td valign="middle" align="center">1</td>
<td valign="middle" align="center">GTG</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Ser (S1)</italic>
</td>
<td valign="middle" align="center">11855</td>
<td valign="middle" align="center">11922</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">68</td>
<td valign="middle" align="center">4</td>
<td valign="middle" align="center">GCT</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Leu (L1)</italic>
</td>
<td valign="middle" align="center">11927</td>
<td valign="middle" align="center">11999</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">73</td>
<td valign="middle" align="center">3</td>
<td valign="middle" align="center">TAG</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ND5</italic>
</td>
<td valign="middle" align="center">12003</td>
<td valign="middle" align="center">13850</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">1848</td>
<td valign="middle" align="center">-4</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">TAA</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>ND6</italic>
</td>
<td valign="middle" align="center">13847</td>
<td valign="middle" align="center">14368</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">522</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">T--</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Glu (E)</italic>
</td>
<td valign="middle" align="center">14369</td>
<td valign="middle" align="center">14437</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">69</td>
<td valign="middle" align="center">4</td>
<td valign="middle" align="center">TCC</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>Cyt b</italic>
</td>
<td valign="middle" align="center">14442</td>
<td valign="middle" align="center">15582</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">1141</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">ATG</td>
<td valign="middle" align="center">T--</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Thr (T)</italic>
</td>
<td valign="middle" align="center">15583</td>
<td valign="middle" align="center">15653</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">71</td>
<td valign="middle" align="center">32</td>
<td valign="middle" align="center">TGT</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">
<italic>tRNA-Pro (P)</italic>
</td>
<td valign="middle" align="center">15686</td>
<td valign="middle" align="center">15756</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">71</td>
<td valign="middle" align="center">0</td>
<td valign="middle" align="center">TGG</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
<tr>
<td valign="middle" align="left">Control region</td>
<td valign="middle" align="center">15757</td>
<td valign="middle" align="center">16859</td>
<td valign="middle" align="center">+</td>
<td valign="middle" align="center">1103</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
<td valign="middle" align="center">.</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="T2" position="float">
<label>Table&#xa0;2</label>
<caption>
<p>Nucleotide composition of mitochondrial genomes in <italic>P. grandisquamis</italic>.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="bottom" align="center">Locus</th>
<th valign="bottom" align="center">Size (bp) (bp)</th>
<th valign="bottom" align="center">A%</th>
<th valign="bottom" align="center">T%</th>
<th valign="bottom" align="center">G%</th>
<th valign="bottom" align="center">C%</th>
<th valign="bottom" align="center">A+T%</th>
<th valign="bottom" align="center">AT-Skew</th>
<th valign="bottom" align="center">GC- Skew</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="middle" align="left">Complete Mitogenome</td>
<td valign="middle" align="right">16,859</td>
<td valign="bottom" align="right">28.38</td>
<td valign="bottom" align="right">26.70</td>
<td valign="bottom" align="right">15.42</td>
<td valign="bottom" align="right">29.43</td>
<td valign="middle" align="right">55.08</td>
<td valign="bottom" align="right">0.030</td>
<td valign="bottom" align="right">-0.312</td>
</tr>
<tr>
<td valign="middle" align="left">Protein-Coding Genes (PCGs)</td>
<td valign="middle" align="right">11,456</td>
<td valign="bottom" align="right">26.65</td>
<td valign="bottom" align="right">27.66</td>
<td valign="bottom" align="right">13.98</td>
<td valign="bottom" align="right">31.70</td>
<td valign="middle" align="right">54.31</td>
<td valign="bottom" align="right">-0.019</td>
<td valign="bottom" align="right">-0.388</td>
</tr>
<tr>
<td valign="middle" align="left">Ribosomal RNAs (rRNAs)</td>
<td valign="bottom" align="right">2,645</td>
<td valign="bottom" align="right">31.95</td>
<td valign="bottom" align="right">21.97</td>
<td valign="bottom" align="right">20.23</td>
<td valign="bottom" align="right">25.86</td>
<td valign="middle" align="right">53.91</td>
<td valign="bottom" align="right">0.185</td>
<td valign="bottom" align="right">-0.122</td>
</tr>
<tr>
<td valign="middle" align="left">Transfer RNAs (tRNAs)</td>
<td valign="middle" align="right">1,556</td>
<td valign="bottom" align="right">31.62</td>
<td valign="bottom" align="right">25.58</td>
<td valign="bottom" align="right">18.96</td>
<td valign="bottom" align="right">23.84</td>
<td valign="middle" align="right">57.20</td>
<td valign="bottom" align="right">0.106</td>
<td valign="bottom" align="right">-0.114</td>
</tr>
<tr>
<td valign="middle" align="left">Control Region (CR)</td>
<td valign="middle" align="right">1,103</td>
<td valign="bottom" align="right">33.27</td>
<td valign="bottom" align="right">29.83</td>
<td valign="bottom" align="right">13.87</td>
<td valign="bottom" align="right">21.85</td>
<td valign="middle" align="right">63.10</td>
<td valign="bottom" align="right">0.055</td>
<td valign="bottom" align="right">-0.223</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec id="s3_2">
<label>3.2</label>
<title>Features of protein-coding genes</title>
<p>The mitogenomes of <italic>P. grandisquamis</italic> contain 13 PCGs with a total length of 11,456 bp (68% of the complete mitogenome). Among that, the <italic>ND5</italic> gene has the longest length with 1,824 bp, while the shortest was <italic>ATP8</italic> with 168 bp (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>). Additionally, the PCG of <italic>P. grandisquamis</italic> has an AT bias of 54.31% with an AT-skew of -0.019 and a GC-skew of -0.388. In the mitogenome of this species, most PCGs start codons beginning with ATG, except in <italic>COI</italic> which starts with GTG. Notably, five PCGs (<italic>ND1</italic>, <italic>COI</italic>, <italic>ATP8</italic>, <italic>ND4L</italic>, <italic>ND5</italic>) ended with TAA, while the remaining PCGs terminated by incomplete stop codon either TA- or T&#x2013; (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table S1</bold>
</xref>).</p>
<p>The analysis of Relative Synonymous Codon Usage (RSCU) revealed that the codons in the 13 PCGs were conserved and specifically translated to amino acids. The total number of codon transcriptions for the 20 amino acids was 3,808 for <italic>P. grandisquamis</italic>, excluding the termination codons. The amino acid composition of <italic>P. grandisquamis</italic> PCGs was predominantly characterized by two hydrophobic amino acids, leucine (14.29%) and alanine (5.86%), along with three neutral amino acids, serine (10.87%), proline (10.06%), and threonine (8.46%). In contrast, three hydrophobic amino acids&#x2014;tryptophan (0.63%), cysteine (1.02%), and methionine (1.08%)&#x2014;one hydrophilic amino acid, aspartic acid (1.73%), and one neutral amino acid, glycine (2.42%), were found to be less abundant (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2A</bold>
</xref>, <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table S2</bold>
</xref>). Notably, the RSCU analysis revealed a noteworthy six variation of codon in Arginine, Leucine and Serine, enabling an increasing its frequency. Moreover, several codons showed its augmentation (&gt; 1.5) to translate a specific amino acid: GGC in Glycine, TCC in Serine, CCC in Proline, and GCC in Alanine, indicating as the most frequent compared to the other codons (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2B</bold>
</xref>, <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table S3</bold>
</xref>).</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>Structural characteristics of <italic>P. grandisquamis</italic> Protein-coding genes. <bold>(A)</bold> Amino acids abundance, <bold>(B)</bold> Relative Synonymous codon usage pattern of PCGs in mitogenomes of <italic>P. grandisquamis</italic>, and <bold>(C)</bold> Box plot displays the pairwise divergence in the Ka/Ks ratio for each PCG across all available Mugilids species mitogenomes.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1484198-g002.tif"/>
</fig>
<p>The calculation of the nonsynonymous (Ka) and synonymous (Ks) substitution proportions demonstrated that PCGs of <italic>P. grandisquamis</italic> and its relative species within the family Mugilidae are subject to the different experience of selection pressure. The average pairwise Ka/Ks ratios ranged from the lowest of 0.0124 &#xb1; 0.0034 for <italic>COI</italic> to a high of 1.2976 &#xb1; 0.1017 for <italic>ND5</italic>, following the order: <italic>COI</italic> &lt; <italic>COII</italic> &lt; <italic>COIII</italic> &lt; <italic>Cytb</italic> &lt; <italic>ATP6</italic> &lt; <italic>ND3</italic> &lt; <italic>ND1</italic> &lt; <italic>ND4</italic> &lt; <italic>ND2</italic> &lt; <italic>ATP8</italic> &lt; <italic>ND4L</italic> &lt; <italic>ND6</italic> &lt; <italic>ND5</italic>. Most of PCGs showed Ka/Ks ratio value less than one, except <italic>ND4L</italic>, <italic>ND5</italic>, and <italic>ND6</italic> that have value greater than one (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2C</bold>
</xref>, <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table S4</bold>
</xref>).</p>
</sec>
<sec id="s3_3">
<label>3.3</label>
<title>Ribosomal RNA and transfer RNA genes</title>
<p>The mitogenome in this study has two ribosomal RNA molecules, explicitly 12S rRNA of 956 bp and 16S rRNA of 1,689 bp which collectively contribute 16% of the complete mitogenome. The rRNA gene displayed an AT bias of 53.91% with AT-skew and GC-skew contributing 0.185 and -0.122, respectively (<xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>). The mitogenome of <italic>P. grandisquamis</italic> also contained 22 tRNA dispersedly in between rRNA and PCGs. It has a cumulative length of 1,556 bp, contributing 9% of total mitogenome. The tRNA genes of this species exhibited an AT bias of 57.20% with AT-skew and GC-skew of 0.106 and -0.114, respectively (<xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>). Furthermore, most of tRNA showed conventional form of cloverleaf secondary structure, except for <italic>tRNA-Ser1</italic> due to lack of nucleotide bond in DHU arm. Fifteen tRNA genes (<italic>tRNA-Phe</italic>, <italic>tRNA-Gln</italic>, <italic>tRNA-Met</italic>, <italic>tRNA-Trp</italic>, <italic>tRNA-Ala</italic>, <italic>tRNA-Asn</italic>, <italic>tRNA-Cys</italic>, <italic>tRNA-Tyr</italic>, <italic>tRNA-Ser2</italic>, <italic>tRNA-Asp</italic>, <italic>tRNA-Gly</italic>, <italic>tRNA-Arg</italic>, <italic>tRNA-Ser1</italic>, <italic>tRNA-Glu</italic>, and <italic>tRNA-Pro</italic>) were constructed through a combination of conventional Watson-Crick base pairs (A=T and G&#x2261;C) and wobble base pairs (G-T), while the other seven tRNA genes exclusively use Watson-Crick base pairs. Among those 15 tRNA genes, wobble base pairing was observed prominently in <italic>tRNA-Ala</italic> and <italic>tRNA-Glu</italic>. Moreover, Wobble base pairings were identified in the DHU stem of six tRNAs, the T&#x3c8;C stem of eight tRNAs, the anticodon stem of five tRNAs, and the acceptor stem of nine tRNAs (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>).</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>Secondary structures of 22 transfer RNAs (tRNAs) displaying the structural variation in <italic>P. grandisquamis</italic>. The tRNAs are represented by full names and IUPAC-IUB single letter amino acid codes. Watson-Crick and wobble base pairing are shown by black and red color bars respectively. The last structure shows the nucleotide positions and details of stem-loop of tRNAs.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1484198-g003.tif"/>
</fig>
</sec>
<sec id="s3_4">
<label>3.4</label>
<title>Structural features of control region</title>
<p>The total length of control region in <italic>P. grandisquamis</italic> was 1,103 bp in length, contributing 6.5% of the total mitogenome. The control region of this species showed an AT bias of 57.20%, with an AT-skew of 0.106 and a GC-skew of -0.114 (<xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>). The CR of <italic>P. grandisquamis</italic> consists of four conserved blocks (CSB-D, CSB-1, CSB-2, and CSB-3), as observed in other fish mitogenomes, including other Mugilids (<italic>M. cephalus</italic>, Accession No. AP002930 and <italic>Moolgarda crenilabis</italic>, Accession No. JF911707). Among these conserved blocks, CSB-III is the longest at 31 bp, compared to other regions ranging from 18 bp (CSB-D and CSB-II) to 21 bp (CSB-I) (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>). Comparative analysis of the conserved blocks with other mugilids species demonstrated two groups in CSB-D; the first group with most of species including <italic>P. grandisquamis</italic>, showed a similarity on conserved nucleotides, however two species (<italic>Rhinomugil nasutus</italic> and <italic>Chaenomugil proboscideus</italic>) showed nucleotide similarity as second group. In CSB-I, three groups were observed indicating conserve nucleotides: the first group comprising with four species (<italic>Planiliza haematocheilus, Aldrichetta forsteri, R. nasutus</italic>, and <italic>Dajaus monticola</italic>), the second group with two species (<italic>M. crenilabis</italic> and <italic>Osteomugil engeli</italic>), and the third group with two species (<italic>Planiliza carinata</italic> and <italic>Planiliza lauvergnii</italic>). Similarly, the conserved nucleotides were also found in CSB-II, showing two different groups: the first group comprising six species and the other group with four species. While in CSB-III, three species (<italic>P. carinata, P. haematocheilus</italic>, and <italic>P. lauvergnii</italic>) were detected have similar conserved nucleotide, while the other 17 species have different nucleotide patterns, which may be diverged due to the deletion of base pairs in their CRs (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>). In addition, several mugilidis species allowed tandem repeats in the extended termination-associated sequences (ETAS) region, including 2.3 copy of 44 bp consensus sequence in <italic>Plicomugil labiosus</italic>, 6.8 copies of 79 bp consensus sequence in <italic>O. labeo</italic>, 2.6 copies of 44 bp consensus sequence in <italic>C. proboscideus</italic>, and 15.6 copies of 22 bp and 17.4 copies of 22 bp consensus sequences in <italic>Minimugil cascasia</italic> (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>).</p>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>Schematic representation of the different conserved blocks in the control region of <italic>P. grandisquamis</italic> and other Mugilids species. General linearized representation is showed in the top. The conserved nucleotides pattern in multiple species are marked by different color boxes. Highly conserved bases are denoted by black star across all mugilids.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1484198-g004.tif"/>
</fig>
</sec>
<sec id="s3_5">
<label>3.5</label>
<title>Phylogenetic relationship of mullets</title>
<p>A mitogenome-based phylogenetic analysis was performed using 13 PCGs from <italic>P. grandisquamis</italic> and other related species within the Mugilidae family. The BA phylogenetic tree clearly resolved the species into distinct classifications with high posterior probability support. <italic>P. grandisquamis</italic> was found to be closely related to species in the genera <italic>Chelon</italic> and <italic>Planiliza</italic> (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Additionally, the phylogenetic tree confirmed the matrilineal relationships of 10 monotypic species, including <italic>P. grandisquamis</italic>. The analysis also addressed taxonomic placement of ambiguous species within the Mugilidae family, such as <italic>M. cascasia</italic> (formerly <italic>Sicamugil cascasia</italic>), <italic>D. monticola</italic> (formerly <italic>Agonostomus monticola</italic>), <italic>M. crenilabis</italic> (formerly <italic>Crenimugil crenilabis</italic>), <italic>P. labiosus</italic> (formerly <italic>Oedalechilus labiosus</italic>), <italic>R. nasutus</italic> (formerly <italic>Squalomugil nasutus</italic>), <italic>Pseudomyxus capensis</italic> (formerly <italic>Myxus capensis</italic>), two species of <italic>Osteomugil</italic> (formerly under the genus <italic>Moolgarda</italic>), and four species of <italic>Planiliza</italic> (formerly under the genus <italic>Liza</italic>) (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Notably, the cladistic analysis revealed a non-monophyletic pattern for the genus <italic>Moolgarda</italic>, with <italic>M. crenilabis</italic> and <italic>M. cunnesius</italic> showing distinct lineages, suggesting the need for further investigation.</p>
<fig id="f5" position="float">
<label>Figure&#xa0;5</label>
<caption>
<p>The Bayesian (BA) phylogeny constructed by the concatenated 13 PCGs clearly describe the placement of <italic>P. grandisquamis</italic> and other mugilids species with high posterior probability supports in each node. The cladogram infers the insights into the revised taxonomical classification and cladistic pattern of mugilids. The environment, climate zone, and distribution range of each species is remarked by different color boxes representing to their distinctive clade.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1484198-g005.tif"/>
</fig>
</sec>
</sec>
<sec id="s4" sec-type="discussion">
<label>4</label>
<title>Discussion</title>
<sec id="s4_1">
<label>4.1</label>
<title>Mitogenomic signature of large-scaled mullet</title>
<p>The mitochondrial genome of <italic>P. grandisquamis</italic> comprises 37 genes, with 28 genes located on the heavy strand and nine on the light strand. The nucleotide composition of the genome exhibits an A-T bias, which is consistent with the hydrophobic nature of mitochondrial proteins (<xref ref-type="bibr" rid="B47">Naylor et&#xa0;al., 1995</xref>). The gene arrangement and strand organization of <italic>P. grandisquamis</italic> align with those observed in other teleost mitogenomes (<xref ref-type="bibr" rid="B45">Miya et&#xa0;al., 2003</xref>, <xref ref-type="bibr" rid="B44">2005</xref>). Our findings suggest that the mitochondrial genome of <italic>P. grandisquamis</italic> is conserved relative to other species within the Mugilidae family (<xref ref-type="bibr" rid="B43">Miya et&#xa0;al., 2001</xref>). Given the general conservation of mitochondrial genomes, gene rearrangements are an important focus in organismal systematics (<xref ref-type="bibr" rid="B20">Gong et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B85">Zhang et&#xa0;al., 2020</xref>). Therefore, understanding gene arrangement and nucleotide composition across species is essential for refining species classification. The arrangement of mitochondrial genes can influence various aspects of fish physiology, molecular mechanisms, life histories, and genomic evolutionary processes (<xref ref-type="bibr" rid="B46">Monta&#xf1;a-Lozano et&#xa0;al., 2022</xref>).</p>
<p>In PCGs, most genes use ATG as the initiation codon, with the exception of <italic>COI</italic>, which terminates with either complete or incomplete stop codons. This pattern of initiation and termination codon usage in <italic>COI</italic> is consistent with that observed in other mugilid species (<xref ref-type="bibr" rid="B43">Miya et&#xa0;al., 2001</xref>; <xref ref-type="bibr" rid="B67">Shen et&#xa0;al., 2016a</xref>, <xref ref-type="bibr" rid="B70">2016</xref>). In this study, amino acids with neutral hydropathy characteristics were found to be more abundant than those with hydrophobic or hydrophilic properties. This observation aligns with findings from previous studies on other teleost species (<xref ref-type="bibr" rid="B80">Wang et&#xa0;al., 2022a</xref>). The hydropathy characteristics of amino acids play a crucial role in the evolution of mitochondrial proteins, which are essential for cellular respiration and energy production that are vital for the survival and adaptation of organisms in dynamic environments (<xref ref-type="bibr" rid="B3">Berthelot et&#xa0;al., 2019</xref>). Given the frequent fluctuations in salinity, temperature, and oxygen levels experienced by marine fishes, particularly in the Atlantic Ocean (<xref ref-type="bibr" rid="B60">Rutterford et&#xa0;al., 2023</xref>), detailed studies on hydropathy-related modifications in mitochondrial proteins could offer valuable insights into their role in optimizing protein conformation and function under such environmental stressors. These adaptations may enhance thermal tolerance and hypoxia resilience, enabling marine species to survive and thrive in highly variable and challenging habitats.</p>
<p>Codon usage was further assessed using RSCU values, revealing that most RSCU values deviated from &#x2018;1&#x2019;, indicating varying levels of codon bias for different amino acids. Codons for leucine (Leu) and serine (Ser), which have six codon combinations each, were the most frequently used, whereas methionine (Met) and tryptophan (Trp), each represented by a single codon (ATG and TGG, respectively), were less prevalent. Further, the Ka/Ks ratio, a well-established metric for assessing selective pressure and evolutionary associations at the molecular level (<xref ref-type="bibr" rid="B84">Yang and Nielsen, 2000</xref>; <xref ref-type="bibr" rid="B86">Zhao et&#xa0;al., 2022</xref>). Most PCGs exhibited Ka/Ks ratios less than &#x2018;1&#x2019;, suggesting strong negative selection in <italic>P. grandisquamis</italic> and its relatives within the Mugilidae family, with mutations predominantly replaced by synonymous substitutions. This pattern indicates the role of natural selection in reducing deleterious mutations with adverse selective coefficients, positioning with universal patterns detected in other teleosts (<xref ref-type="bibr" rid="B31">Kundu et&#xa0;al., 2024</xref>). However, three PCGs (<italic>ND4L</italic>, <italic>ND5</italic>, and <italic>ND6</italic>) showed Ka/Ks ratios greater than &#x2018;1&#x2019;, indicating potential positive selection. These genes may be more sensitive to genetic mutations, potentially influenced by environmental adaptations or new functional developments (<xref ref-type="bibr" rid="B38">Liao et&#xa0;al., 2024</xref>). The Ka/Ks ratio analysis provides valuable insights into how natural selection shapes speciation and evolutionary trajectories in mugilids across diverse aquatic environments. Previous studies have reported that NADH dehydrogenase genes have evolved under positive selection in anadromous salmonids and other deep-sea fishes, suggesting their adaptation to diverse ecosystems influenced by environmental factors such as salinity, temperature, low oxygen levels, darkness, and high-water pressure (<xref ref-type="bibr" rid="B79">Wang et&#xa0;al., 2022b</xref>; <xref ref-type="bibr" rid="B69">Shen et&#xa0;al., 2019</xref>). However, the specific roles of these genes in mullets remain unclear. The incorporation of this novel mitogenome data of <italic>P. grandisquamis</italic> into global databases, along with a comparative analysis of substitution patterns with other mugilids, provides baseline information to better understand the environmental adaptations of mullets. This foundational data is crucial for monitoring their protein-level evolutionary dynamics and adaptive strategies.</p>
<p>Additionally, the rRNAs in <italic>P. grandisquamis</italic> are located on the heavy strand and are separated by <italic>tRNA-Val</italic>, a pattern consistent with other fish species (<xref ref-type="bibr" rid="B61">Satoh et&#xa0;al., 2006</xref>; <xref ref-type="bibr" rid="B38">Liao et&#xa0;al., 2024</xref>). Ribosomes, as conserved ribonucleoproteins, play a crucial role in translating genetic information from mRNA into proteins. The structural association of rRNA genes, including their conserved loops, delivers valuable insights into the catalytic developments that are essential for protein synthesis (<xref ref-type="bibr" rid="B35">Lehman, 2004</xref>; <xref ref-type="bibr" rid="B61">Satoh et&#xa0;al., 2006</xref>). Our analysis also reveals that most tRNAs exhibit a cloverleaf secondary structure, with the exception of <italic>tRNA-Ser1</italic>. This cloverleaf pattern is common in the mitogenomes of many bony fish, although the absence of the DHU arm in some tRNAs can serve as a distinguishing feature (<xref ref-type="bibr" rid="B38">Liao et&#xa0;al., 2024</xref>). The tRNA molecules function as adaptors that translate genetic information into proteins by delivering amino acids during translation. The proper arrangement of tRNAs, particularly in the WANCY region, is crucial for effective mitochondrial gene expression (<xref ref-type="bibr" rid="B6">Cantatore et&#xa0;al., 1987</xref>; <xref ref-type="bibr" rid="B53">Ponce et&#xa0;al., 2008</xref>). Thus, the analysis of rRNAs and tRNAs in <italic>P. grandisquamis</italic>, especially through the evaluation of secondary structures, provides important insights into genetic mechanisms and mitochondrial function.</p>
<p>In the mitogenome of <italic>P. grandisquamis</italic>, the pronounced AT bias in the CR is consistent with observations in other fish species, which typically exhibit a preference for adenine (A) and thymine (T) bases (<xref ref-type="bibr" rid="B43">Miya et&#xa0;al., 2001</xref>, <xref ref-type="bibr" rid="B45">2003</xref>, <xref ref-type="bibr" rid="B44">2005</xref>). The CR is particularly significant due to its dynamic nature, as it is the most variable region in the mitochondrial genome. Notably, the repeat-rich ETAS region within the CR is characterized by specific motifs likely forming stable hairpin loops. These structures act as sequence-specific signals to regulate mitochondrial DNA replication termination (<xref ref-type="bibr" rid="B62">Satoh et&#xa0;al., 2016</xref>). Understanding the complex mechanisms controlling the CR&#x2014;such as random loss, dimer formation of mitogenomes, non-random loss, and genomic rearrangement through double replications&#x2014;is essential for grasping the structural diversity of mitogenomes and the evolutionary dynamics of mitochondrial genomes (<xref ref-type="bibr" rid="B31">Kundu et&#xa0;al., 2024</xref>). Thus, our analysis of the CR, including the identification of polymorphic nucleotides in conserved blocks, provides valuable insights for developing distinct molecular markers for species identification and understanding population structure.</p>
<p>The present mitogenome-based phylogenetic assessment provides valuable insights into the evolutionary relationships of <italic>P. grandisquamis</italic>, particularly in relation to other monotypic species within the Mugilidae family. This analysis supports the revision of classifications and highlights the non-monophyletic clustering of the <italic>Moolgarda</italic> genus within the Mugilidae family. Molecular phylogenetics offers robust, independent insights into species evolutionary relationships based on maternal lineage (<xref ref-type="bibr" rid="B5">Caldara et&#xa0;al., 1996</xref>; <xref ref-type="bibr" rid="B25">Heras et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B39">Liu et&#xa0;al., 2010</xref>). Integrating multi-dimensional research, including mitogenomic data alongside morphological information, is essential for elucidating ancestral and descendant lineages and resolving evolutionary patterns that have long been debated among ichthyologists due to morphological contradictions (<xref ref-type="bibr" rid="B24">Harrison et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B13">Durand et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B54">Rajan et&#xa0;al., 2023</xref>). Given the species richness and broad distribution of mugilids across diverse aquatic environments, it is crucial to expand mitogenomic research to enhance our understanding of their speciation and adaptation processes.</p>
</sec>
<sec id="s4_2">
<label>4.2</label>
<title>Future perspectives for conservation framework of mullets</title>
<p>The large-scale mullet, <italic>P. grandisquamis</italic> demonstrates considerable versatility and adaptability across various habitats within the coastal ecosystems of the eastern Atlantic region. However, this adaptability may render it susceptible to genetic mutations and increased heterozygosity in response to environmental changes (<xref ref-type="bibr" rid="B42">Martinez et&#xa0;al., 2018</xref>). Coastal and riverine habitat alterations often negatively impact fish populations, potentially creating physical barriers to migration, which can lead to population fragmentation and disruptions in gene flow, thereby increasing the risk of inbreeding in restricted ecosystems (<xref ref-type="bibr" rid="B52">Pimentel et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B49">Ovidio et&#xa0;al., 2020</xref>). This fragmentation can reduce genetic diversity, threaten population viability, and heighten the risk of local extinction (<xref ref-type="bibr" rid="B58">Rourke et&#xa0;al., 2019</xref>). Therefore, integrating traditional species knowledge with molecular data is crucial for assessing genetic variation, population structure, and connectivity within specific ecosystems. Mitogenome-based studies are essential for observing adaptive evolution in <italic>Parachelon</italic> and other mugilids, which are currently underrepresented. The mitogenome characterization provided here serves as a foundational baseline for monitoring the genetic diversity of this migratory fish in the eastern Atlantic. Our findings highlight the vulnerability of <italic>P. grandisquamis</italic> to positive selection pressures and unique structural variations in its mitochondrial genome. Notably, mitochondrial PCGs such as <italic>ND4L</italic>, <italic>ND5</italic>, and <italic>ND6</italic> exhibit heightened sensitivity to gene mutations, offering a basis for further research into the adaptive evolution of this endemic and monotypic species. Understanding how specific genes influence physiological adaptation is crucial for evaluating their adaptability to environmental changes and climate variability (<xref ref-type="bibr" rid="B79">Wang et&#xa0;al., 2022b</xref>; <xref ref-type="bibr" rid="B69">Shen et&#xa0;al., 2019</xref>). Given that <italic>P. grandisquamis</italic> is typically catadromous, with a tendency to migrate into freshwater systems, this migratory behavior may have a significant impact on mitochondrial gene evolution, as reflected in their Ka/Ks ratios. These insights will be instrumental in developing effective management strategies and conducting population genetic analyses of this species in near future. Additionally, given the restricted distribution of <italic>P. grandisquamis</italic> in the eastern Atlantic, this mitogenomic data will aid in elucidating lineage diversification patterns through time tree analyses incorporating multiple mitogenomic datasets from the Mugiliformes lineage.</p>
</sec>
</sec>
<sec id="s5" sec-type="conclusions">
<label>5</label>
<title>Conclusion</title>
<p>In this study, mitochondrial genome sequence analysis was performed to investigate the monotypic <italic>P. grandisquamis</italic>, which is endemic to the eastern Atlantic. The complete mitochondrial genome provided detailed insights into the genetic configuration and matrilineal evolutionary patterns of <italic>P. grandisquamis</italic>, confirming its evolutionary relationships with other mugilids. Given the potential conservation implications for mugilids in the eastern Atlantic, this research underscores the necessity of a thorough understanding of both mitogenomic and demographic information within marine ecosystems. Overall, the analysis of the mitochondrial genome and genetic characteristics of the large-scaled mullet provides valuable insights for future advancements in single nucleotide polymorphism (SNP)-based species discrimination, particularly within the Mugilidae family. Additionally, it offers a better understanding of the regulation of mitochondrial genes and potential environmental adaptations of this catadromous species in the eastern Atlantic Ocean.</p>
</sec>
</body>
<back>
<sec id="s6" sec-type="data-availability">
<title>Data availability statement</title>
<p>The mitogenome of the study species <italic>Parachelon grandisquamis</italic> (GenBank Accession Number: OR487150) is now publicly accessible at <uri xlink:href="https://www.ncbi.nlm.nih.gov/nuccore/OR487150">https://www.ncbi.nlm.nih.gov/nuccore/OR487150</uri>.</p>
</sec>
<sec id="s7" sec-type="ethics-statement">
<title>Ethics statement</title>
<p>The animal study was approved by Pukyong National University Animal Care and Use Committee (PKNUIACUC-2022-72). The study was conducted in accordance with the local legislation and institutional requirements.</p>
</sec>
<sec id="s8" sec-type="author-contributions">
<title>Author contributions</title>
<p>T-HY: Conceptualization, Formal Analysis, Funding acquisition, Validation, Writing &#x2013; original draft. H-EK: Formal Analysis, Investigation, Software, Validation, Writing &#x2013; original draft. SA: Data curation, Methodology, Software, Visualization, Writing &#x2013; original draft. AW: Data curation, Methodology, Software, Visualization, Writing &#x2013; original draft. H-WK: Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing &#x2013; review &amp; editing. SK: Conceptualization, Project administration, Resources, Supervision, Writing &#x2013; review &amp; editing.</p>
</sec>
<sec id="s9" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This research was supported by Global-Learning &amp; Academic research institution for Master&#x2019;s, Ph.D. students, and Postdocs (LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (No. RS-2023-00301914). This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1A6A1A03039211).</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>The authors would like to thank to Fantong Zealous Gietbong from the Ministry of Livestock, Fisheries and Animal Industries (MINEPIA), Yaounde, Cameroon, for invaluable assistance in conducting sampling.</p>
</ack>
<sec id="s10" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s11" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s12" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fmars.2024.1484198/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fmars.2024.1484198/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="SupplementaryFile1.docx" id="SM1" mimetype="application/vnd.openxmlformats-officedocument.wordprocessingml.document"/>
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