Nothing Special   »   [go: up one dir, main page]
Next Article in Journal
Expanding the Clinical Spectrum Associated with the Recurrent Arg203Trp Variant in PACS1: An Italian Cohort Study
Previous Article in Journal
The Impact of the Competition on miRNA, Proteins, and Metabolites in the Blood Exosomes of the Yili Horse
Previous Article in Special Issue
Complete Mitochondrial Genomes of Pluvialis fulva and Charadrius dubius with Phylogenetic Analysis of Charadriiformes
You seem to have javascript disabled. Please note that many of the page functionalities won't work as expected without javascript enabled.
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 16
Issue 2
10.3390/genes16020226
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Characterization of the Complete Mitochondrial Genome of Dwarf Form of Purpleback Flying Squid (Sthenoteuthis oualaniensis) and Phylogenetic Analysis of the Family Ommastrephidae

by
Wenjuan Duo
1,†,
Lei Xu
2,3,†,
Mohd Johari Mohd Yusof
1,
Yingmin Wang
4,
Seng Beng Ng
5 and
Feiyan Du
2,*
1
Faculty of Design and Architecture, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia
2
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China
3
Sanya Tropical Fisheries Research Institute, Sanya 572018, China
4
School of Environmental Sciences, James Cook University, Singapore 387380, Singapore
5
Department of Multimedia, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2025, 16(2), 226; https://doi.org/10.3390/genes16020226
Submission received: 16 January 2025 / Revised: 13 February 2025 / Accepted: 14 February 2025 / Published: 15 February 2025
(This article belongs to the Special Issue Molecular Evolution, Mitochondrial Genomics and Mitochondrial Genome Expression in Animals: 2024–2025)
Browse Figures
Figure 1
<p>Annotated mitochondrial genome of dwarf form of <span class="html-italic">S. oualaniensis</span>. Blue bars denote protein-coding genes, virescent bars represent rRNA genes, and lavender bars indicate tRNA genes. The direction of transcription is shown by the orientation of gene arrows: arrows pointing to the right indicate the heavy strand, while those pointing to the left denote the light strand. The black circle represents GC content, with outward projections indicating GC content above the average level and inward projections indicating below-average content. The GC skew is depicted using purple and green circles, where green represents negative GC skew and deep purple indicates positive GC skew.</p> "> Figure 2
<p>Relative synonymous codon usage (RSCU) patterns in the medium-sized and dwarf forms of <span class="html-italic">S. oualaniensis</span>.</p> "> Figure 3
<p>The ratio of nonsynonymous to synonymous substitutions (Ka/Ks) across 13 protein coding genes in two forms of <span class="html-italic">S. oualaniensis</span>.</p> "> Figure 4
<p>Bayesian inference (BI) (<b>A</b>) and maximum likelihood (ML) (<b>B</b>) phylogenetic trees illustrating the evolutionary relationships among cephalopod species based on mitochondrial genome sequences. The tree is rooted with <span class="html-italic">A. dux</span> as the outgroup. Posterior probabilities and support value are displayed at the nodes. Notable clades include multiple mitochondrial haplotypes of <span class="html-italic">S. oualaniensis</span> forming a well-supported cluster, and a close relationship between <span class="html-italic">D. gigas</span> and <span class="html-italic">Eucleoteuthis luminosa</span>. The longer branch lengths of <span class="html-italic">T. pacificus</span> and <span class="html-italic">I. argentinus</span> indicate greater genetic divergence compared to other taxa. The scale bar represents genetic distance.</p> ">
Versions Notes

Abstract

:
Background: The Ommastrephidae family of cephalopods is important in marine ecosystems as both predators and prey. Species such as Todarodes pacificus, Illex argentinus, and Dosidicus gigas are economically valuable but are threatened by overfishing and environmental changes. The genus Sthenoteuthis, especially S. oualaniensis, shows significant morphological and genetic variation, including medium-sized and dwarf forms found in the South China Sea. Methods: Specimens of S. oualaniensis were collected from the South China Sea, their genomic DNA sequenced, and phylogenetic relationships analyzed using mitochondrial genomes from various Ommastrephidae species. Results: The study presents the complete mitochondrial genome of the dwarf form of S. oualaniensis (20,320 bp) and compares it with the medium-sized form, revealing a typical vertebrate structure with 13 protein-coding genes, 21 tRNA genes, and 2 rRNA genes, along with a strong AT bias. Nucleotide composition analysis shows a 12% genetic divergence between the two forms, suggesting a recent common ancestor and potential cryptic speciation, with all protein-coding genes exhibiting purifying selection based on Ka/Ks ratios below 1. Conclusions: The mitochondrial genome of the dwarf form of S. oualaniensis shows a close evolutionary relationship with the medium-sized form and a 12% genetic divergence, suggesting potential cryptic speciation. These findings underscore the importance of mitochondrial analysis in understanding speciation and guiding future conservation efforts.

1. Introduction

The family Ommastrephidae, a prominent group of cephalopods, plays a crucial ecological role and possesses significant biological value in marine ecosystems. Widely distributed across tropical, subtropical, and certain temperate waters, ommastrephid squids occupy surface to midwater zones, acting as vital conduits for energy transfer within marine food webs. Their rapid growth rates, high reproductive potential, and remarkable adaptability to environmental changes underpin their ecological importance and contribute to the dynamic balance of marine ecosystems [1,2,3]. As mid-to-high trophic level predators, they feed on small fish, crustaceans, and other cephalopods, while simultaneously serving as key prey for numerous fish species, marine mammals, and seabirds. This dual role positions them as integral components of marine food webs, playing a critical role in sustaining ecosystem dynamics and ensuring ecological stability [4,5,6]. Species of the family Ommastrephidae are considered ideal models for studying ecological adaptation, population dynamics, and evolutionary mechanisms, owing to their complex population structures, remarkable morphological diversity, and rapid life cycles [7,8]. Their heightened sensitivity to environmental fluctuations further underscores their value as indicator species for assessing the health of marine ecosystems and monitoring the effects of climate change [9]. These research advancements not only enhance our understanding of biodiversity within the family but also offer critical theoretical support for the development of evidence-based fisheries management strategies [10,11]. Additionally, the family Ommastrephidae, as a highly valuable marine biological resource, occupies a critical position in global fisheries. Representative species such as the jumbo squid (D. gigas), the Argentinean short-finned squid (I. argentinus), and the Japanese common squid (T. pacificus) are among the primary targets in global cephalopod fisheries. These species are distinguished by their broad distribution, high yields, and the use of advanced fishing techniques, making them essential resources for both coastal and distant-water fisheries. However, the exploitation of Ommastrephidae resources is subject to considerable spatiotemporal variability, with factors such as population size, distribution range, and resource abundance being influenced by both environmental fluctuations and human fishing activities [12,13,14]. In recent years, the development of Ommastrephidae fisheries has become increasingly globalized, with a notable expansion in fishing grounds such as the Northwest Pacific, South Atlantic, and Eastern Pacific Oceans. Species within this family exhibit rapid growth, high reproductive rates, and short life cycles, enabling them to withstand certain fishing pressures [15,16,17]. Nevertheless, their sensitivity to environmental fluctuations, including marine surface temperature (MST), oceanic currents, and basal productivity, often leads to substantial variability in resource abundance. For instance, climatic phenomena such as El Niño and La Niña significantly influence the distribution and yield of D. gigas [18,19,20]. Additionally, overfishing presents a considerable threat to certain populations, particularly in unregulated high-seas regions. These challenges underscore the urgent need for comprehensive scientific assessments and the adoption of robust management approaches to safeguard the long-term viability of these crucial fishery resources [12,21,22].
The family Ommastrephidae comprises five subfamilies (Ommastrephinae, Ornithoteuthinae, Todarodinae, Todaropsinae, and Illicinae) and includes several genera such as Todarodes, Illex, Dosidicus, Nototodarus, and Sthenoteuthis. Each genus contains species uniquely adapted to specific ecological niches [23,24,25,26]. For instance, the genus Todarodes includes economically significant species like T. pacificus, while Illex encompasses highly migratory species such as I. argentinus and I. illecebrosus. Among the members of Dosidicus, D. gigas, commonly known as the jumbo squid, is particularly noteworthy as one of the largest and most ecologically influential species, exerting a dominant role in the Eastern Pacific ecosystem [2,27].
S. oualaniensis Lesson 1830 from the genus Sthenoteuthis, commonly known as the purpleback flying squid, belonging to the family Ommastrephidae, is extensively found in the equatorial and subtropical areas of both the Pacific and Indian Oceans [28]. Its range extends from 30° N to 25° S, with occasional occurrences as far north as 40° N and south to 47° S, and recent reports have documented its presence as far north as 60° N. While it is absent from temperate Pacific waters, the species is most prevalent in the South China Sea and the northwestern part of the Indian Ocean [2,28,29]. Similar to other species in the Ommastrephidae family, S. oualaniensis displays several forms that differ in mature size and the presence of a prominent large dorsal photophore. This species includes two major morphs in the South China Sea: a dwarf form and a medium-sized form. The dwarf form, which does not possess a dorsal photophore, exhibits a mantle length between 9 and 12 cm. In contrast, the medium-sized form, distinguished by the presence of a photophore, features females maturing at a mantle length of 15 to 40 cm, while males mature at a length of 12 to 24 cm. The current taxonomic classification of S. oualaniensis is complicated by the limited understanding of its life cycles, foraging behaviors, and reproductive patterns, leading to ongoing debates regarding its classification [1,30]. Previous molecular analyses of S. oualaniensis revealed four genetically distinct lineages in the Indian Ocean, highlighting significant phylogeographic structuring and uncovering five evolutionary units, with evidence suggesting sympatric speciation of the dwarf form [31]. Xu et al. [32] investigated the genetic and morphological variation of S. oualaniensis in the South China Sea, identifying two distinct lineages: a dwarf form confined to equatorial waters and a medium-sized form with a broader distribution. These results suggest a pseudo-cryptic species complex, supported by both genetic markers and morphology, with the medium-sized form further subdivided into two clades. However, these studies are limited by the small number of species analyzed and the use of short sequence fragments, underscoring the necessity for further research incorporating a larger species sample and more comprehensive sequence data to achieve more robust and reliable phylogenetic insights. Mitochondrial DNA (mtDNA) is widely used in phylogenetic studies due to its relatively high mutation rate, absence of recombination, and maternal inheritance, which make it a useful tool for tracing evolutionary relationships and population structure [33,34]. However, it is essential to acknowledge the limitations of mtDNA. Since it is inherited maternally, it does not capture paternal genetic contributions, which could limit our understanding of the full genetic diversity and evolutionary history of species. Furthermore, the lack of recombination in mtDNA results in a more static genetic marker, potentially overlooking the finer-scale genetic variation that could be present in more dynamic genomic regions [35,36]. In this study, we conducted the sequencing, assembly, and annotation of the complete mitochondrial genome for the dwarf form of S. oualaniensis and compared it with that of the medium-sized form for the first time. Additionally, we analyzed the phylogenetic relationships among 7 out of the 23 recognized species within the family Ommastrephidae using complete mitochondrial genomes. Specifically, our objective is to elucidate the phylogenetic position of S. oualaniensis and to investigate the phylogenetic relationships between species across different subfamilies within the Ommastrephidae family.

2. Materials and Methods

2.1. Sample Collection and Identification of Morphology

Specimens of S. oualaniensis were obtained from the South China Sea (11°26′ N, 114°01′ E) on 11 April 2017, using a light falling net deployed by the commercial fishing vessel “Guibeiyu 96886”. The collected samples were immediately stored in a freezer at −20 °C after morphological identification of the genus Sthenoteuthis. This study adhered to the guidelines established by the International Union for Conservation of Nature (IUCN) for studies involving endangered species, along with the rules set forth by the Convention on Biological Diversity (CBD) and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). S. oualaniensis was classified as “Least Concern” on the IUCN Red List of Threatened Species [37]. The different forms were identified at the Guangdong Provincial Key Laboratory of Fishery Ecology and Environment based on the following morphological characteristics. Mantle Length (ML): The medium-sized form exhibits significantly greater body size compared to the dwarf form, with mantle lengths ranging from 120 mm to 400 mm, whereas the dwarf form typically measures between 90 mm and 120 mm. Arm Length (AL): Distinct differences in arm length are evident, particularly in the first pair of arms (AL1), which serve as a critical morphological feature for distinguishing the two forms. Tentacle Length (TL): Tentacle length also varies, with the dwarf form having noticeably shorter tentacles compared to the medium-sized form. Fin Length (FL) and Fin Width (FW): The size and shape of fins differ between the forms, offering another distinguishing morphological characteristic. Gonad Maturity: The medium-sized form typically exhibits delayed gonadal maturation, while the dwarf form tends to mature earlier, highlighting a significant reproductive difference. Photophore Presence: Photophore development is a key differentiator. The medium-sized form usually possesses photophores on its dorsal side, while individuals from the dwarf form lack photophores or exhibit degenerated ones [2,30,38]. The specimens in this study had a small mantle length (ML) of only 10 cm, tentacle lengths of 4 cm, lacked photophores on the dorsal side, and exhibited mature gonads.

2.2. DNA Extraction, Sequencing and Annotation

Genomic DNA was isolated from the body muscle tissue of a dwarf form of S. oualaniensis specimen using the TIANamp Marine Animals DNA Kit (TIANGEN, Beijing, China), in accordance with the manufacturer’s protocol specifically designed for extracting DNA from marine animal muscle tissue. The specimen and the extracted DNA have been stored at the Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, registered under the voucher number SCS2017-S10-450. Genomic DNA samples, characterized by an A260/A280 ratio between 1.8 and 2.1, were utilized as templates for PCR amplification. DNA sequencing was conducted using an ABI 3730xl DNA automatic sequencer with genomic DNA as the template. The PCR primers were developed based on highly conserved regions of tRNA sequences from related species. Additionally, species-specific primers were designed as required, drawing from previously acquired sequence data [39] (Table S1). PCR amplification was carried out using TopTaq DNA polymerase (Qiagen, Hilden, Germany), following the manufacturer’s protocol with annealing temperatures varying between 45 °C and 55 °C. Sanger sequencing was conducted on the PCR products in both directions using the primer walking method to ensure comprehensive and accurate sequence coverage. The COI sequence from the dwarf form of S. oualaniensis served as the reference seed for iterative assembly using MITObim version 1.8 [40]. The mitogenome assembly and annotation were performed using SeqMan version 7.1.0, followed by manual verification [41]. Transfer RNA genes were predicted using tRNAScan-SE 1.21 via its online platform, utilizing the default search parameters and selecting “Mito/chloroplast” as the genetic source [42]. The mitochondrial genome of the species was mapped using assembly and annotation data. A diagram was generated through the GCView Server to effectively visualize the genomic information [43]. Nucleotide composition skewness was determined using the following equations: A / T − s k e w = A − T A + T and G / C − s k e w = G − C G + C [44]. The relative synonymous codon usage (RSCU) for the 13 protein-coding genes (PCGs) was calculated using MEGA software, version 6.0 [45]. This analysis provides insights into codon preference, which is essential for understanding genetic coding patterns and evolutionary adaptations within the mitochondrial genome. The mitochondrial genome of the dwarf form of S. oualaniensis was compared to that of the medium-sized form of S. oualaniensis, as documented in our previous study (MT661575) [46]. This comparative analysis aimed to identify differences and similarities in genomic features between the two forms, providing insights into their evolutionary relationship and potential species differentiation. Additionally, non-synonymous (Ka) and synonymous (Ks) substitution rates were calculated using DnaSP v6, offering valuable insights into the evolutionary dynamics of the mitochondrial genome [47].

2.3. Phylogenetic Analysis

A total of eight mitochondrial genomes from the Ommastrephidae family, retrieved from the NCBI database, were analyzed to investigate phylogenetic relationships, with Architeuthis dux (KC701744) used as the outgroup (Table S2). A. dux was selected as the outgroup due to its well-characterized mitochondrial genome and its phylogenetic position within Oegopsida, making it an appropriate reference for rooting the tree. Other deep-sea cephalopods were not included primarily due to the limited availability of high-quality, complete mitochondrial genomes in public databases. The sequences underwent preliminary processing for visualization and assembly within BioEdit, after which they were automatically aligned utilizing the default settings. To ensure the robustness of our alignment, gaps and missing data were carefully treated [48].
Phylogenetic analyses were conducted using PhyloSuite v1.2.1 [49] through both maximum likelihood (ML) and Bayesian inference (BI) methods. ModelFinder was used to select the best-fit partition model (edge-unlinked) using BIC criterion [50]. Maximum likelihood (ML) phylogenies were constructed using IQ-TREE [51] with the edge-linked partition model, employing 5000 ultrafast bootstrap replicates and the Shimodaira–Hasegawa-like approximate likelihood ratio test [52] to assess branch support. Bayesian inference (BI) phylogenies were generated using MrBayes 3.2.6 [53] under the partition model (2 parallel runs, 7,711,800 generations). The BI analysis consisted of two parallel runs over 10,000 generations, with the initial 25% of sampled data discarded as burn-in to ensure convergence and reliability.

3. Results

3.1. Mitogenome Structure

The mitochondrial genome of the dwarf form of S. oualaniensis is a closed circular molecule measuring 20,320 bp (GenBank accession number MW542205). The mitochondrial genome exhibits an overall base composition of A (37.23%), T (32.78%), G (10.53%), and C (19.46%), resulting in a pronounced AT bias of 70.01%. Its structure is highly conserved, adhering to the typical vertebrate mitochondrial genome organization. The genome comprises 2 rRNA genes (12S rRNA and 16S rRNA), 21 tRNA genes, and 13 protein-coding genes (PCGs). These PCGs encompass the cytochrome b gene (Cytb), two ATP synthase subunits (ATP6, ATP8), three cytochrome c oxidase subunits (COX1, COX2, COX3), and seven NADH dehydrogenase subunits (ND1, ND2, ND3, ND4, ND4L, ND5, ND6). The light strand (L-strand) of the mitochondrial genome encodes thirteen transfer RNA genes (tRNA-Met, tRNA-Tyr, tRNA-Trp, tRNA-Gly, tRNA-Glu, tRNA-Phe, tRNA-Val, tRNA-Cys, tRNA-Gln, tRNA-His, tRNA-Ser, tRNA-Pro, and tRNA-Leu), six protein-coding genes (ND1, ND4, ND4L, ND5, ND6, and Cytb), and two rRNA genes (12S rRNA and 16S rRNA). Conversely, the remaining genes are encoded on the heavy strand (H-strand) of the mitochondrial genome (Figure 1). The ribosomal RNA genes have lengths of 1425 bp for the 16S rRNA and 990 bp for the 12S rRNA, respectively. All 21 tRNA genes display a full cloverleaf secondary structure, with their lengths varying between 65 and 74 bp. The mitochondrial genome additionally contains seven duplicated genes (ATP6, ATP8, COX1, COX2, COX3, tRNA-Leu, and tRNA-Asp), a feature that is characteristic of Oegopsid cephalopod mitogenomes [39]. The mitochondrial genome of the dwarf form of S. oualaniensis demonstrated a positive A/T skew of 0.0636 and a negative G/C skew of −0.2978, indicating a higher proportion of cytosine compared to guanine.
The protein-coding genes in the mitochondrial genome of the dwarf form of S. oualaniensis collectively cover a total length of 15,041 bp, representing 74.02% of the entire mitochondrial genome. This region displayed a significant AT bias, with A + T content comprising 68.32%, and collectively encoded 4788 amino acid residues. Within the set of protein-coding genes, ND1, ND4, ND4L, ND5, ND6, and Cytb were positioned on the light strand (L-strand), whereas the other genes were located on the heavy strand (H-strand). Of these, the ND5 gene was the longest, spanning 1698 bp and encoding 565 amino acids, while the ATP8 gene was the shortest, at only 156 bp, encoding 51 amino acids. In the mitochondrial genome of the dwarf form of S. oualaniensis, the majority of protein-coding genes (PCGs) employ the initiation codon ATG, with the exception of ND4 and Cytb, which utilize ATA. All thirteen PCGs have complete termination codons: seven genes (COX3, ND3, COX1, ATP8, ND2, ND1, and ND5) terminate with TAA, five genes (ATP6, ND4L, ND6, Cytb, and ND4) with TAG, and one gene (COX2) with AAT. In comparison, the mitochondrial genome of the medium-sized form of S. oualaniensis shows some differences in codon usage. The initiation codon ATG is used by most PCGs, except for ND1 (which uses ATA) and ND5 and ND2 (both of which use ATT). Like the dwarf form, all 13 PCGs in the medium-sized form possess complete termination codons. Six genes (ND2, ND3, ND5, COX2, COX3, and ATP8) terminate with TAA, while seven genes (Cytb, COX1, ND1, ND4, ND4L, ND6, and ATP6) use TAG as their termination codon (Table 1).
To investigate codon usage patterns and amino acid distribution, the amino acid composition and relative synonymous codon usage (RSCU) of the 13 protein-coding genes were analyzed in the mitochondrial genomes of both the dwarf and medium-sized forms of S. oualaniensis. This comparative analysis provides insights into codon preference and the functional implications of amino acid composition in these two forms. The RSCU analysis revealed that the mitochondrial genomes of both forms of S. oualaniensis exhibited the highest frequency for codons corresponding to the amino acids Leu, Ile, Phe, Met, Val, and Gly, while codons for Cys were less frequent (Figure 2). Two identical long non-coding regions (LNCRs), measuring 561 bp and 562 bp, respectively, were identified on the heavy (H) strand of the mitochondrial genomes in both the medium-sized and dwarf forms of S. oualaniensis. The first long non-coding RNA (LNCR) is situated in the region between the tRNA-Gln gene and the COX3 gene, whereas the second LNCR is found in the interval between the tRNA-Glu gene and the duplicate copy of the COX3 gene. These non-coding regions likely play critical roles in mitochondrial gene regulation, including DNA replication and transcription. The ratio of non-synonymous to synonymous substitution rates (Ka/Ks) was employed to evaluate the selective pressure acting on 13 protein-coding genes (PCGs) across the two forms of S. oualaniensis. The Ka/Ks ratios for all PCGs remained consistently below 1 (Figure 3), indicating strong purifying selection acting on these genes in both forms. Among the PCGs, ND4 exhibited the highest Ka/Ks value, suggesting a relatively faster evolutionary rate compared to other mitochondrial genes. In contrast, COX2 showed the lowest Ka/Ks values, highlighting significant selective constraints and slower evolutionary rates for these genes.

3.2. Phylogenetic Tree

To ascertain the phylogenetic position of the dwarf form of S. oualaniensis, all available mitochondrial genomes from the family Ommastrephidae were retrieved from the NCBI database. This dataset included the mitochondrial genome of the medium-sized form of S. oualaniensis from our previous study [46]. Additionally, the mitochondrial genome of Architeuthis dux was used as an outgroup. Phylogenetic trees were generated using Maximum Likelihood (ML) and Bayesian Inference (BI) approaches, based on the nucleotide sequences from 13 protein-coding genes (PCGs). The phylogenetic analyses revealed congruent topological structures between the Maximum Likelihood (ML) and Bayesian Inference (BI) methods, strongly supporting the delineation of several clades based on the available data (Figure 4). The main clade consists of species from the genera Dosidicus, Eucleoteuthis, Sthenoteuthis, and Ommastrephes, all members of the subfamily Ommastrephinae. In contrast, the remaining clades include species from the genera Illex and Todarodes, which belong to the subfamilies Illicinae and Todaropsinae, respectively. The phylogenetic analyses revealed that the target species, the dwarf form of S. oualaniensis, and the medium-sized form of S. oualaniensis are clustered together as a monophyletic group. This clustering indicates that the two forms share a recent common ancestor and exhibit a closer relationship to each other compared to any other species within the Ommastrephidae family. All these results underscore the reliability of the available data in elucidating the evolutionary relationships among species within this family.

4. Discussion

The mitochondrial DNA of Metazoa serves as an excellent model for investigating evolutionary genomics. With the rapid advancements in DNA sequencing technologies and bioinformatics, mitochondrial genomes of marine organisms have increasingly been utilized in fields such as species identification and phylogenetic research [54,55,56,57,58,59,60,61]. This research provided the full mitochondrial genome sequence of the dwarf form of S. oualaniensis, comprising a total length of 20,320 bp. The structural features of this genome closely resemble those of other Oegopsida species, demonstrating a significant level of conservation within this order. Additionally, the gene lengths observed in S. oualaniensis are largely consistent with those of previously published Oegopsida mitochondrial genomes, further supporting the notion of extensive conservation across species within this group. These findings highlight the structural stability of mitochondrial genomes in Oegopsida and suggest that the essential functions of these genomes are maintained across species [25,62,63,64].
AT-skew and GC-skew values provide crucial insights into the nucleotide composition bias in DNA sequences, particularly within mitochondrial genomes. These values reflect the relative abundance of adenine (A) versus thymine (T) (AT-skew) and guanine (G) versus cytosine (C) (GC-skew) within a genome. They are frequently utilized to assess the asymmetry between the light (L-strand) and heavy (H-strand) strands of mitochondrial DNA. The AT-skew value primarily reflects the asymmetric distribution of A and T, which is often associated with the directionality of transcription and the replication mechanisms within the genome. Conversely, the GC-skew value highlights the asymmetry between C and G, typically correlating with replication origins and the characteristics of the lagging strand [44]. The mitochondrial DNA sequence of the dwarf form of S. oualaniensis exhibits a high AT content, demonstrating a clear AT-bias, a feature also observed in other cephalopod species. In cephalopods, AT/GC-skew values show considerable variation across different species, reflecting distinct mitochondrial genome characteristics. Generally, AT-skew values in cephalopods are positive, indicating an overrepresentation of adenine (A) relative to thymine (T) on the heavy strand. This pattern is consistent with typical replication and transcription mechanisms observed in mitochondrial genomes. In contrast, GC-skew values in cephalopods tend to be negative, indicating a greater proportion of cytosine (C) relative to guanine (G) on the heavy strand. These base preferences may arise from factors such as natural mutations and selective pressures acting during the processes of replication and transcription [25,65,66,67].
The non-coding regions (NCRs) within the mitochondrial genome play a primary role in regulating gene expression and are essential for the replication and transcription of mitochondrial DNA. In the mitochondrial genome of the dwarf form of S. oualaniensis, the non-coding regions are similar to those found in other Oegopsida species, consisting mainly of two copies of long non-coding regions (LNCRs) located on the heavy (H) strand. NCRs harbor critical elements for initiating the replication and transcription of mitochondrial DNA. These regions contain promoters for RNA polymerase and sequences that bind replication proteins, ensuring proper mitochondrial genome maintenance and gene expression. NCRs also are hotspots for genetic recombination and rearrangement, which may drive mitochondrial genome evolution in cephalopods. Tomita, Yokobori, Oshima, Ueda, and Watanabe [65] reported that the mitochondrial genome of Loligo bleekeri contains 19 noncoding regions, three of which (515, 507, and 509 bp) are nearly identical, suggesting they originated from duplication events in an ancestral genome. The study further proposed that the dispersion of tRNA genes in Loligo is associated with the multiplication of noncoding regions, highlighting a potential mechanism underlying mitochondrial genome evolution in cephalopods. In addition, these non-coding regions represent some of the fastest-evolving sequences in mitochondrial DNA, playing a significant role in the molecular evolution of cephalopods. Due to their high evolutionary rate, these regions are widely utilized in studies of cephalopod population genetics and molecular systematics [68,69]. The mitochondrial genome of the dwarf form of S. oualaniensis exhibits seven gene duplications, including ATP6, ATP8, COX1, COX2, COX3, tRNA-Leu, and tRNA-Asp. These duplications are characteristic features of Oegopsid cephalopod mitogenomes [39]. Duplicated genes in cephalopods may arise through mechanisms such as gene duplication, genome rearrangement, or errors in mitochondrial DNA replication, processes potentially linked to their complex life histories and adaptation to environmental pressures. The emergence of duplicated genes holds significant evolutionary importance for cephalopods. On one hand, it provides raw material for gene functional diversification, potentially facilitating the development of new functions to adapt to diverse ecological niches. On the other hand, duplicated genes may enhance the flexibility of the mitochondrial genome, contributing to optimized energy metabolism and adaptation to varying environmental conditions, thereby improving the competitive survival capabilities of cephalopods [70,71]. Later studies have suggested that the mitochondrial genomes of Sepiida and Oegopsida likely evolved through a process of whole-genome duplication followed by random gene loss, originating from the mitochondrial genome of Octopoda [24,72]. In the mitochondrial genome of the dwarf form of S. oualaniensis analyzed in this study, the duplicated gene copies were found to be highly conserved. Among them, only the COX2 gene exhibited five synonymous mutation sites, yet the encoded amino acid sequences remained identical. This observation suggests that the duplicated gene copies retain identical functions. Kawashima et al. [72] reported that in the mitochondrial genome of Bathyteuthis, a single amino acid change from A to V was observed in the duplicated COX1 genes. In contrast, two amino acid substitutions—W to Y and N to D—were identified in the duplicated COX2 genes. Notably, the second copy of the COX3 gene displayed multiple deletions and insertions relative to the first copy. Consequently, they proposed that the second copy of the COX3 gene may have lost its functionality. Understanding how duplicated non-coding regions and functional genes in cephalopod mitochondrial genomes maintain their functional roles, such as supporting replication and transcription, requires further investigation. This knowledge is expected to emerge with the accumulation of mitochondrial genome data from a broader range of cephalopod species. The Ka/Ks ratio, which compares non-synonymous to synonymous substitution rates, is commonly used to assess positive Darwinian selection and evolutionary relationships between species, both homogeneous and heterogeneous, at the molecular level [73]. A Ka/Ks ratio below 1 suggests negative or purifying selection, indicating evolutionary constraints that eliminate harmful mutations. A ratio of 1 represents neutral evolution, where mutations neither offer advantages nor disadvantages. In contrast, a ratio above 1 indicates positive or diversifying selection, pointing to adaptive changes driven by evolutionary pressures. In mollusks, species with low mobility exhibit a higher Ka/Ks ratio than those with high mobility, suggesting a greater accumulation of non-synonymous mutations in the mtDNA of less mobile species [73,74]. In this study, the Ka/Ks ratios of all 13 protein-coding genes (PCGs) in the two forms of S. oualaniensis were consistently below 1, indicating that these genes were under strong purifying selection to preserve their functional integrity. Among the 13 PCGs, ND4 exhibited the highest Ka/Ks ratio, a pattern similarly observed in other Oegopsida species, suggesting a relatively fast evolutionary rate and potential adaptive changes [25,66]. In contrast, COX2 had the lowest Ka/Ks ratio, reflecting strong purifying selection and slower evolutionary rates. These findings highlight the differential selective pressures acting on mitochondrial genes, underscoring their functional importance and evolutionary dynamics.
The phylogenetic analyses yielded well-supported and congruent topologies across both Bayesian Inference (BI) and Maximum Likelihood (ML) approaches, clearly delineating three primary clades within the family Ommastrephidae, and the tree includes branching patterns supported by posterior probabilities and bootstrap value (indicated at the nodes). These clades reflect evolutionary divergence consistent with ecological, morphological, and genetic distinctions among the included genera. This primary clade includes species from the genera Dosidicus, Eucleoteuthis, Sthenoteuthis, and Ommastrephes, all belonging to the subfamily Ommastrephinae. The strong support for this grouping, as evidenced by high bootstrap values and posterior probabilities, validates its monophyletic status. These species are broadly distributed across tropical and subtropical waters and exhibit significant adaptations to pelagic lifestyles. Key traits such as rapid growth rates, high fecundity, and extensive migratory behaviors have likely driven their evolutionary success and diversification. Notably, within the Ommastrephinae clade, the placement of S. oualaniensis highlights its close evolutionary relationships with other genera, corroborating findings from previous molecular and morphological studies [62,75]. The second clade consists of species from the genus Todarodes, belonging to the subfamily Todaropsinae. The relationship between T. pacificus (Japanese flying squid) and the D. gigas + E. luminosa clade exhibits moderate to weak support (posterior probability = 0.939, bootstrap = 56). While these taxa are related, the genetic divergence indicated by branch length suggests that T. pacificus may have experienced unique selective pressures, potentially driven by its temperate and subarctic habitat in contrast to the tropical ranges of Dosidicus and Eucleoteuthis. This highlights the need for additional genomic markers or increased taxon sampling to better resolve this relationship, as it may involve processes such as incomplete lineage sorting or introgression [25,69,76]. Both phylogenetic trees position I. argentinus (Argentine shortfin squid) as an early-diverging lineage within the primary Ommastrephidae clade, reflecting significant genetic differentiation. Its placement underscores the impact of geographic isolation in the southern Atlantic Ocean and adaptation to colder waters on its evolutionary trajectory. This divergence aligns with the species’ distinct ecological and behavioral strategies, including cold-water spawning and dietary specialization, which have likely contributed to its unique evolutionary path [77,78]. The clear separation of these clades underscores the evolutionary bifurcation within Ommastrephidae, likely driven by historical geographic, ecological, and behavioral factors. The strong support for subfamily-level delineations reaffirms the utility of mitochondrial genome analysis for resolving phylogenetic relationships within this family. Furthermore, the inclusion of Architeuthis dux as an outgroup effectively anchored the tree, providing a broader context for interpreting lineage-specific divergence.
The phylogenetic analyses revealed that the multiple forms of S. oualaniensis clustered together, forming a distinct and robustly supported monophyletic clade. This grouping suggested a close evolutionary relationship and indicated that these two forms share a recent common ancestor. Notably, the genetic divergence observed between the dwarf and medium-sized forms aligns with levels typically associated with species differentiation, providing strong evidence for their separation as distinct evolutionary units. Additionally, in this study, the genetic divergence between the dwarf and medium-sized forms of S. oualaniensis across the complete mitochondrial genome reached 12%, which is nearing the highest congeneric distance of 13.9% previously reported for the Ommastrephinae subfamily, suggesting a level of genetic differentiation that may be indicative of cryptic speciation [75]. Additionally, the observed differences in the initiation and termination codon usage of protein-coding genes (PCGs) may reflect subtle variations in mitochondrial genome evolution between the two morphotypes of S. oualaniensis. In our earlier research, we examined genetic and size variations in Sthenoteuthis squids from the South China Sea, providing evidence for the differentiation between the dwarf and medium-sized forms as separate species. The medium-sized form exhibits a broader distribution, whereas the dwarf form is predominantly found in equatorial waters. Both genetic and morphological analyses reveal significant divergence, indicating the existence of pseudo-cryptic species. The observed mitonuclear discordance may be attributed to either hybridization or lineage sorting [32]. This level of divergence highlights potential cryptic speciation within S. oualaniensis, where traditional morphological or ecological distinctions are subtle or absent but genetic data reveal significant evolutionary separation. The clustering pattern further emphasizes the intricate structure of the S. oualaniensis species complex and suggests that these forms may represent separate adaptive responses to differing ecological or environmental conditions, such as variations in water temperature, depth, or prey availability. Further research integrating genetic, ecological, and morphological data is necessary to clarify the mechanisms driving this differentiation. For instance, studies on reproductive isolation, habitat preferences, and life history traits could provide additional evidence to support their classification as distinct species. These insights could also inform conservation strategies and fisheries management plans, ensuring sustainable utilization and conservation of the genetic diversity within the S. oualaniensis species complex. The findings have broader implications for taxonomy and evolutionary biology. The differentiation of these forms may reflect unique evolutionary pressures or adaptive strategies that have facilitated their survival and success in distinct niches. This distinction is also critical for effective resource management and conservation efforts, as treating the two forms as a single species could obscure their individual ecological roles and population dynamics. In addition, incorporating nuclear genome data in future research is crucial, as it would provide a more comprehensive understanding of the genetic divergence and help validate our mitochondrial findings, offering a more robust framework for interpreting the evolutionary relationships within S. oualaniensis.

5. Conclusions

The mitochondrial genome of the dwarf form of S. oualaniensis sheds significant light on the evolutionary dynamics within the Ommastrephidae family. The genome exhibits a highly conserved structure, displaying typical features of Oegopsid cephalopod mitogenomes, including seven duplicated genes, which are a distinctive characteristic of this group. Phylogenetic analysis confirms the close evolutionary relationship between the dwarf and medium-sized forms of S. oualaniensis, suggesting they share a recent common ancestor. The observed genetic divergence of 12% between these forms indicates the potential for cryptic speciation, implying that the two forms may represent distinct evolutionary units despite their morphological similarities. These findings underscore the value of mitochondrial genome analysis in resolving evolutionary relationships and speciation processes, particularly within the complex S. oualaniensis species complex. Further research, incorporating genetic, ecological, and morphological data, will be crucial for elucidating the mechanisms driving their divergence and for informing conservation strategies that ensure the sustainable management of their genetic diversity and ecological roles.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes16020226/s1, Table S1. PCR primers were used in this research. Table S2. The details of deposited mitochondrial genomes in Genbank used in this research.

Author Contributions

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

Funding

This research was supported by Hainan Provincial Natural Science Foundation of China (324MS131) and The National Key Research and Development Program of China (2024YFD2400602).

Institutional Review Board Statement

All procedures involving the handling and treatment of animals in this study were performed in strict compliance with the Guide for Animal Care.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genome sequence data supporting the findings on the dwarf form of S. oualaniensis are publicly accessible in GenBank (NCBI) at [https://www.ncbi.nlm.nih.gov] under accession number MW542205. The authors confirm that the data supporting the findings of this study are available at the following link: https://doi.org/10.5281/zenodo.4489989 (accessed on 2 February 2021).

Acknowledgments

We thank Xin Liang, Yuezhong Wang for field work and Yubin Zhou from Jierui Biotech Company for his invaluable assistance with data analysis. We thank all colleagues and students for their help with sampling.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Dunning, M.C. A review of the systematics, distribution, and biology of arrow squids genera Ommastrephes Orbigny, 1835, Sthenoteuthis Verrill, 1880, and Ornithoteuthis Okada, 1927 (Cephalopoda: Ommastrephidae). In Systematics and Biogeography of Cephalopods. Smithsonian Contributions to Zoology 586: Volume 2; Voss, N.A., Vecchione, M., Toll, R.B., Sweeney, M.J., Eds.; Smithsonian Institution Press: Washington, DC, USA, 1998; pp. 425–433. [Google Scholar]
  2. Jereb, P.; Roper, C.F.E. Cephalopod of the World. An Annotated and Illustrated Catalogue of Cephalopod Species Know to Date. Volume 2. Myopsid and Oegopsid Squids; FAO Species Catalogue for Fishery Purposes: Rome, Italy, 2010; Volume 4, p. 649. [Google Scholar]
  3. Markaida, U.; Gilly, W.F. Cephalopods of Pacific Latin America. Fish. Res. 2016, 173, 113–121. [Google Scholar] [CrossRef]
  4. Ward, P.; Barord, G.J.; Schauer, A.; Veloso, J. Comparative Trophic Levels of Phragmocone-Bearing Cephalopods (Nautiloids, Ammonoids, and Sepiids). Integr. Comp. Biol. 2023, 63, 1285–1297. [Google Scholar] [CrossRef]
  5. Merten, V.; Christiansen, B.; Javidpour, J.; Piatkowski, U.; Puebla, O.; Gasca, R.; Hoving, H.-J.T. Diet and stable isotope analyses reveal the feeding ecology of the orangeback squid Sthenoteuthis pteropus (Steenstrup 1855) (Mollusca, Ommastrephidae) in the eastern tropical Atlantic. PLoS ONE 2017, 12, e0189691. [Google Scholar] [CrossRef] [PubMed]
  6. Camarillo-Coop, S.; Salinas-Zavala, C.A.; Lavaniegos, B.E.; Markaida, U. Food in early life stages of Dosidicus gigas (Cephalopoda: Ommastrephidae) from the Gulf of California, Mexico. J. Mar. Biol. Assoc. U. K. 2013, 93, 1903–1910. [Google Scholar] [CrossRef]
  7. Moltschaniwskyj, N.A. Understanding the process of growth in cephalopods. Mar. Freshwat. Res. 2004, 55, 379–386. [Google Scholar] [CrossRef]
  8. Leporati, S.C.; Pecl, G.T.; Semmens, J.M. Cephalopod hatchling growth: The effects of initial size and seasonal temperatures. Mar. Biol. 2007, 151, 1375–1383. [Google Scholar] [CrossRef]
  9. Pierce, G.J.; Valavanis, V.D.; Guerra, A.; Jereb, P.; Orsi-Relini, L.; Bellido, J.M.; Katara, I.; Piatkowski, U.; Pereira, J.; Balguerias, E.; et al. A review of cephalopod–environment interactions in European Seas. Hydrobiologia 2008, 612, 49–70. [Google Scholar] [CrossRef]
  10. Ji, F.; Guo, X. A new way to understand migration routes of oceanic squid (Ommastrephidae) from satellite data. Remote Sens. Ecol. Conserv. 2024, 10, 248–263. [Google Scholar] [CrossRef]
  11. Yu, W.; Chen, X. Habitat suitability response to sea-level height changes: Implications for Ommastrephid squid conservation and management. Aquac. Fish. 2021, 6, 309–320. [Google Scholar] [CrossRef]
  12. Arkhipkin, A.I.; Hendrickson, L.C.; Payá, I.; Pierce, G.J.; Roa-Ureta, R.H.; Robin, J.-P.; Winter, A. Stock assessment and management of cephalopods: Advances and challenges for short-lived fishery resources. ICES J. Mar. Sci. 2021, 78, 714–730. [Google Scholar] [CrossRef]
  13. Hunsicker, M.E.; Essington, T.E.; Watson, R.; Sumaila, U.R. The contribution of cephalopods to global marine fisheries: Can we have our squid and eat them too? Fish Fish. 2010, 11, 421–438. [Google Scholar] [CrossRef]
  14. González, Á.F.; Pierce, G.J. Advances in the study of cephalopod fisheries and ecosystems. Fish. Res. 2021, 242, 105975. [Google Scholar] [CrossRef]
  15. Piatkowski, U.; Pierce, G.J.; Morais da Cunha, M. Impact of cephalopods in the food chain and their interaction with the environment and fisheries: An overview. Fish. Res. 2001, 52, 5–10. [Google Scholar] [CrossRef]
  16. Anderson, C.I.H.; Rodhouse, P.G. Life cycles, oceanography and variability: Ommastrephid squid in variable oceanographic environments. Fish. Res. 2001, 54, 133–143. [Google Scholar] [CrossRef]
  17. Oesterwind, D.; Piatkowski, U.; Brendelberger, H. On distribution, size and maturity of shortfin squids (Cephalopoda, Ommastrephidae) in the North Sea. Mar. Biol. Res. 2015, 11, 188–196. [Google Scholar] [CrossRef]
  18. Pang, Y.; Tian, Y.; Fu, C.; Wang, B.; Li, J.; Ren, Y.; Wan, R. Variability of coastal cephalopods in overexploited China Seas under climate change with implications on fisheries management. Fish. Res. 2018, 208, 22–33. [Google Scholar] [CrossRef]
  19. Ibáñez, C.M.; Argüelles, J.; Yamashiro, C.; Sepúlveda, R.D.; Pardo-Gandarillas, M.C.; Keyl, F. Population dynamics of the squids Dosidicus gigas (Oegopsida: Ommastrephidae) and Doryteuthis gahi (Myopsida: Loliginidae) in Northern Peru. Fish. Res. 2016, 173, 151–158. [Google Scholar] [CrossRef]
  20. Xavier, J.C.; Cherel, Y.; Allcock, L.; Rosa, R.; Sabirov, R.M.; Blicher, M.E.; Golikov, A.V. A review on the biodiversity, distribution and trophic role of cephalopods in the Arctic and Antarctic marine ecosystems under a changing ocean. Mar. Biol. 2018, 165, 93. [Google Scholar] [CrossRef]
  21. Peng, D.; Liu, H.; Zhang, W.; Xu, L.; Jiang, R.; Zhu, Y.; García-Lorenzo, I.; Chu, J.; Sumaila, U.R. Global sustainability assessment of cephalopod fisheries based on pressure-state-response framework. iScience 2024, 27, 110986. [Google Scholar] [CrossRef] [PubMed]
  22. Lishchenko, F.; Perales-Raya, C.; Barrett, C.; Oesterwind, D.; Power, A.M.; Larivain, A.; Laptikhovsky, V.; Karatza, A.; Badouvas, N.; Lishchenko, A.; et al. A review of recent studies on the life history and ecology of European cephalopods with emphasis on species with the greatest commercial fishery and culture potential. Fish. Res. 2021, 236, 105847. [Google Scholar] [CrossRef]
  23. Pardo-Gandarillas, M.C.; Torres, F.I.; Fuchs, D.; Ibáñez, C.M. Updated molecular phylogeny of the squid family Ommastrephidae: Insights into the evolution of spawning strategies. Mol. Phylogen. Evol. 2018, 120, 212–217. [Google Scholar] [CrossRef] [PubMed]
  24. Fernández-Álvarez, F.Á.; Taite, M.; Vecchione, M.; Villanueva, R.; Allcock, A.L. A phylogenomic look into the systematics of oceanic squids (order Oegopsida). Zool. J. Linn. Soc. 2022, 194, 1212–1235. [Google Scholar] [CrossRef]
  25. Pei, L.; Jiang, L.; Chen, C.; Ye, Y.; Jing, F.; Liu, Y.; Liu, B. Gene rearrangements in the mitochondrial genome of Eucleoteuthis luminosa (Ommastrephidae, Cephalopoda) and phylogenetic implications for Coleoidea. Hydrobiologia 2023, 850, 3611–3626. [Google Scholar] [CrossRef]
  26. Fernández-Álvarez, F.Á.; Braid, H.E.; Nigmatullin, C.M.; Bolstad, K.S.R.; Haimovici, M.; Sánchez, P.; Sajikumar, K.K.; Ragesh, N.; Villanueva, R. Global biodiversity of the genus Ommastrephes (Ommastrephidae: Cephalopoda): An allopatric cryptic species complex. Zool. J. Linn. Soc. 2020, 190, 460–482. [Google Scholar] [CrossRef]
  27. Murata, M. Oceanic resources of squids. Mar. Behav. Physiol. 1990, 18, 19–71. [Google Scholar] [CrossRef]
  28. Nesis, K.N. Population structure in the squid Sthenoteuthis oulaniensis (Lesson, 1830) (Ommastrephidae) in the Western tropical Pacific. Tr. Instituta Okeanol. 1977, 107, 15–29. [Google Scholar]
  29. Zuyev, G.; Nigmatullin, C.; Chesalin, M.; Nesis, K. Main results of long-term worldwide studies on tropical nektonic oceanic squid genus Sthenoteuthis: An overview of the Soviet investigations. Bull. Mar. Sci. 2002, 71, 1019–1060. [Google Scholar]
  30. Nesis, K.N. Population structure of oceanic ommastrephids, with particular reference to S. oualaniensis: A review. In Recent Advances in Fisheries Biology; Okutani, T., O’Dor, R.K., Kubodera, T., Eds.; Tokai University Press: Tokyo, Japan, 1993; pp. 375–383. [Google Scholar]
  31. Jeena, N.S.; Sajikumar, K.K.; Rahuman, S.; Ragesh, N.; Koya, K.P.S.; Chinnadurai, S.; Sasikumar, G.; Mohamed, K.S. Insights into the divergent evolution of the oceanic squid S. oualaniensis (Cephalopoda: Ommastrephidae) from the Indian Ocean. Integr. Zool. 2023, 18, 924–948. [Google Scholar] [CrossRef] [PubMed]
  32. Xu, L.; Liu, P.; Wang, X.; Van Damme, K.; Du, F. Phylogenetic relationships and cryptic species in the genus Sthenoteuthis (Cephalopoda: Ommastrephidae) in the South China Sea. Mol. Phylogen. Evol. 2020, 149, 106846. [Google Scholar] [CrossRef]
  33. White, D.J.; Wolff, J.N.; Pierson, M.; Gemmell, N.J. Revealing the hidden complexities of mtDNA inheritance. Mol. Ecol. 2008, 17, 4925–4942. [Google Scholar] [CrossRef]
  34. Harrison, R.G. Animal mitochondrial DNA as a genetic marker in population and evolutionary biology. Trends Ecol. Evol. 1989, 4, 6–11. [Google Scholar] [CrossRef] [PubMed]
  35. Ballard, J.W.O.; Whitlock, M.C. The incomplete natural history of mitochondria. Mol. Ecol. 2004, 13, 729–744. [Google Scholar] [CrossRef]
  36. Bernt, M.; Braband, A.; Schierwater, B.; Stadler, P.F. Genetic aspects of mitochondrial genome evolution. Mol. Phylogen. Evol. 2013, 69, 328–338. [Google Scholar] [CrossRef] [PubMed]
  37. Barratt, I.; Allcock, L.S. oualaniensis. The IUCN Red List of Threatened Species 2014, e.T163152A977501. Available online: https://www.iucnredlist.org/species/163152/977501 (accessed on 10 May 2010).
  38. Snyder, R. Aspects of the biology of the giant form of S. oualaniensis (Cephalopoda ommastrephidae) from the Arabian Sea. J. Molluscan Stud. 1998, 64, 21–34. [Google Scholar] [CrossRef]
  39. Yokobori, S.-i.; Fukuda, N.; Nakamura, M.; Aoyama, T.; Oshima, T. Long-Term Conservation of Six Duplicated Structural Genes in Cephalopod Mitochondrial Genomes. Mol. Biol. Evol. 2004, 21, 2034–2046. [Google Scholar] [CrossRef]
  40. Hahn, C.; Bachmann, L.; Chevreux, B. Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads A baiting and iterative mapping approach. Nucleic Acids Res. 2013, 41, e129. [Google Scholar] [CrossRef] [PubMed]
  41. Swindell, S.; Plasterer, T. Seqman, contig assembly. In Sequence Data Analysis Guidebook; Swindell, S., Ed.; Humana Press: Totowa, NJ, USA, 1997; pp. 75–89. [Google Scholar]
  42. Lowe, T.M.; Eddy, S.R. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Res. 1997, 25, 955–964. [Google Scholar] [CrossRef] [PubMed]
  43. Grant, J.R.; Stothard, P. The CGView Server: A comparative genomics tool for circular genomes. Nucleic Acids Res. 2008, 36, W181–W184. [Google Scholar] [CrossRef] [PubMed]
  44. Perna, N.T.; Kocher, T.D. Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. J. Mol. Evol. 1995, 41, 353–358. [Google Scholar] [CrossRef] [PubMed]
  45. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar] [CrossRef]
  46. Xu, L.; Wang, X.; Huang, D.; Li, Y.; Wang, L.; Ning, J.; Du, F. The complete mitochondrial genome of middle-sized form of S. oualaniensis (Cephalopoda: Ommastrephidae) from the South China Sea. Mitochondrial DNA B Resour. 2020, 5, 3030–3032. [Google Scholar] [CrossRef] [PubMed]
  47. Rozas, J.; Ferrer-Mata, A.; Sánchez-DelBarrio, J.C.; Guirao-Rico, S.; Librado, P.; Ramos-Onsins, S.E.; Sánchez-Gracia, A. DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Mol. Biol. Evol. 2017, 34, 3299–3302. [Google Scholar] [CrossRef] [PubMed]
  48. Hall, T.A. BioEdit: A user-friendly biological sequence algnment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 1999, 41, 95–98. [Google Scholar]
  49. Zhang, D.; Gao, F.; Jakovlić, I.; Zou, H.; Zhang, J.; Li, W.X.; Wang, G.T. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol. Ecol. Resour. 2020, 20, 348–355. [Google Scholar] [CrossRef] [PubMed]
  50. Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.F.; von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 2017, 14, 587–589. [Google Scholar] [CrossRef] [PubMed]
  51. Nguyen, L.-T.; Schmidt, H.A.; von Haeseler, A.; Minh, B.Q. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol. Biol. Evol. 2015, 32, 268–274. [Google Scholar] [CrossRef]
  52. Li, X.-R.; Sun, C.-H.; Zhan, Y.-J.; Jia, S.-X.; Lu, C.-H. Complete mitochondrial genome sequence of Nannostomus eques and comparative analysis with Nannostomus beckfordi. Mol. Genet. Genomics 2024, 300, 3. [Google Scholar] [CrossRef] [PubMed]
  53. Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M.A.; Huelsenbeck, J.P. MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Syst. Biol. 2012, 61, 539–542. [Google Scholar] [CrossRef]
  54. Gissi, C.; Iannelli, F.; Pesole, G. Evolution of the mitochondrial genome of Metazoa as exemplified by comparison of congeneric species. Heredity 2008, 101, 301–320. [Google Scholar] [CrossRef]
  55. Duchene, S.; Frey, A.; Alfaro-Núñez, A.; Dutton, P.H.; Thomas, P.; Gilbert, M.; Morin, P.A. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogen. Evol. 2012, 65, 241–250. [Google Scholar] [CrossRef]
  56. Lee, Y.; Kwak, H.; Shin, J.; Kim, S.-C.; Kim, T.; Park, J.-K. A mitochondrial genome phylogeny of Mytilidae (Bivalvia: Mytilida). Mol. Phylogen. Evol. 2019, 139, 106533. [Google Scholar] [CrossRef] [PubMed]
  57. Zhu, Y.; Yan, S.; Ma, P.; Zhang, Y.; Zuo, C.; Ma, X.; Zhang, Z. Comparative mitochondrial genome analysis provides new insights into the classification of Modiolinae. Mol. Biol. Rep. 2024, 51, 823. [Google Scholar] [CrossRef]
  58. Zhong, J.; Zhang, Q.; Xu, Q.; Schubert, M.; Laudet, V.; Wang, Y. Complete mitochondrial genomes defining two distinct lancelet species in the West Pacific Ocean. Mar. Biol. Res. 2009, 5, 278–285. [Google Scholar] [CrossRef]
  59. Sun, C.-H.; Lu, C.-H.; Wang, Z.-J. Comparison and phylogenetic analysis of the mitochondrial genomes of Synodontis eupterus and Synodontis polli. Sci. Rep. 2024, 14, 15393. [Google Scholar] [CrossRef] [PubMed]
  60. Sun, C.-H.; Gu, Y.-L.; Liu, D.-W.; Du, H.-W.; Lu, C.-H. Sequencing and Analysis of the Complete Mitochondrial Genome of Lentipes ikeae. Animals 2024, 14, 943. [Google Scholar] [CrossRef] [PubMed]
  61. Sun, C.-H.; Lu, C.-H. Comparative Analysis and Phylogenetic Study of Dawkinsia filamentosa and Pethia nigrofasciata Mitochondrial Genomes. Int. J. Mol. Sci. 2024, 25, 3004. [Google Scholar] [CrossRef] [PubMed]
  62. Uribe, J.E.; Zardoya, R. Revisiting the phylogeny of Cephalopoda using complete mitochondrial genomes. J. Molluscan Stud. 2017, 83, 133–144. [Google Scholar] [CrossRef]
  63. Jiang, L.; Liu, W.; Zhang, J.; Zhu, A.; Wu, C. Complete mitochondrial genome of Argentine shortfin squid (Illex argentines). Mitochondrial DNA Part A 2016, 27, 3335–3336. [Google Scholar] [CrossRef] [PubMed]
  64. Winkelmann, I.; Campos, P.F.; Strugnell, J.; Cherel, Y.; Smith, P.J.; Kubodera, T.; Allcock, L.; Kampmann, M.-L.; Schroeder, H.; Guerra, A.; et al. Mitochondrial genome diversity and population structure of the giant squid Architeuthis: Genetics sheds new light on one of the most enigmatic marine species. Proc. R. Soc. B. 2013, 280, 20130273. [Google Scholar] [CrossRef] [PubMed]
  65. Tomita, K.; Yokobori, S.-i.; Oshima, T.; Ueda, T.; Watanabe, K. The Cephalopod Loligo bleekeri Mitochondrial Genome: Multiplied Noncoding Regions and Transposition of tRNA Genes. J. Mol. Evol. 2002, 54, 486–500. [Google Scholar] [CrossRef] [PubMed]
  66. Fan, F.; Pei, L.; Jiang, L.; Ye, Y.; Liu, Y.; Liu, B. Gene Rearrangements in the Mitochondrial Genome of Gonatopsis borealis and Onychoteuthis compacta Reveal Their Phylogenetic Implications for Oegopsida. Biochem. Genet. 2024. online ahead of print. [Google Scholar] [CrossRef]
  67. Tang, Y.; Zhang, X.; Ma, Y.; Zheng, X. Descriptive study of the mitogenome of the diamondback squid (Thysanoteuthis rhombus Troschel, 1857) and the evolution of mitogenome arrangement in oceanic squids. J. Zool. Syst. Evol. Res. 2021, 59, 981–991. [Google Scholar] [CrossRef]
  68. Misuzu, A.; Hideyuki, I.; Tohru, N.; Yuzuru, I. Low Genetic Diversity of Oval Squid, Sepioteuthis cf. lessoniana (Cephalopoda: Loliginidae), in Japanese Waters Inferred from a Mitochondrial DNA Non-coding Region. Pac. Sci. 2008, 62, 403–411. [Google Scholar] [CrossRef]
  69. Staaf, D.J.; Ruiz-Cooley, R.I.; Elliger, C.; Lebaric, Z.; Campos, B.; Markaida, U.; Gilly, W.F. Ommastrephid squids S. oualaniensis and Dosidicus gigas in the eastern Pacific show convergent biogeographic breaks but contrasting population structures. Mar. Ecol. Prog. Ser. 2010, 418, 165–178. [Google Scholar] [CrossRef]
  70. Strugnell, J.; Norman, M.; Jackson, J.; Drummond, A.J.; Cooper, A. Molecular phylogeny of coleoid cephalopods (Mollusca: Cephalopoda) using a multigene approach; the effect of data partitioning on resolving phylogenies in a Bayesian framework. Mol. Phylogen. Evol. 2005, 37, 426–441. [Google Scholar] [CrossRef] [PubMed]
  71. Strugnell, J.M.; Lindgren, A.R. A barcode of life database for the Cephalopoda? Considerations and concerns. Rev. Fish Biol. Fish. 2007, 17, 337–344. [Google Scholar] [CrossRef]
  72. Kawashima, Y.; Nishihara, H.; Akasaki, T.; Nikaido, M.; Tsuchiya, K.; Segawa, S.; Okada, N. The complete mitochondrial genomes of deep-sea squid (Bathyteuthis abyssicola), bob-tail squid (Semirossia patagonica) and four giant cuttlefish (Sepia apama, S. latimanus, S. lycidas and S. pharaonis), and their application to the phylogenetic analysis of Decapodiformes. Mol. Phylogen. Evol. 2013, 69, 980–993. [Google Scholar] [CrossRef]
  73. Hurst, L.D. The Ka/Ks ratio: Diagnosing the form of sequence evolution. Trends Genet. 2002, 18, 486–487. [Google Scholar] [CrossRef]
  74. Yang, Z.; Bielawski, J.P. Statistical methods for detecting molecular adaptation. Trends Ecol. Evol. 2000, 15, 496–503. [Google Scholar] [CrossRef]
  75. Wakabayashi, T.; Suzuki, N.; Sakai, M.; Ichii, T.; Chow, S. Phylogenetic relationships among the family Ommastrephidae (Mollusca: Cephalopoda) inferred from two mitochondrial DNA gene sequences. Mar. Genom. 2012, 7, 11–16. [Google Scholar] [CrossRef] [PubMed]
  76. Wakabayashi, T.; Suzuki, N.; Sakai, M.; Ichii, T.; Chow, S. Identification of ommastrephid squid paralarvae collected in northern Hawaiian waters and phylogenetic implications for the family Ommastrephidae using mtDNA analysis. Fish. Sci. 2006, 72, 494–502. [Google Scholar] [CrossRef]
  77. Carvalho, G.R.; Thompson, A.; Stoner, A.L. Genetic diversity and population differentiation of the shortfin squid Illex argentinus in the south-west Atlantic. J. Exp. Mar. Biol. Ecol. 1992, 158, 105–121. [Google Scholar] [CrossRef]
  78. Carlini, D.B.; Kunkle, L.K.; Vecchione, M. A molecular systematic evaluation of the squid genus Illex (Cephalopoda: Ommastrephidae) in the North Atlantic Ocean and Mediterranean Sea. Mol. Phylogen. Evol. 2006, 41, 496–502. [Google Scholar] [CrossRef] [PubMed]
Genes 16 00226 g001
Figure 1. Annotated mitochondrial genome of dwarf form of S. oualaniensis. Blue bars denote protein-coding genes, virescent bars represent rRNA genes, and lavender bars indicate tRNA genes. The direction of transcription is shown by the orientation of gene arrows: arrows pointing to the right indicate the heavy strand, while those pointing to the left denote the light strand. The black circle represents GC content, with outward projections indicating GC content above the average level and inward projections indicating below-average content. The GC skew is depicted using purple and green circles, where green represents negative GC skew and deep purple indicates positive GC skew.
Figure 1. Annotated mitochondrial genome of dwarf form of S. oualaniensis. Blue bars denote protein-coding genes, virescent bars represent rRNA genes, and lavender bars indicate tRNA genes. The direction of transcription is shown by the orientation of gene arrows: arrows pointing to the right indicate the heavy strand, while those pointing to the left denote the light strand. The black circle represents GC content, with outward projections indicating GC content above the average level and inward projections indicating below-average content. The GC skew is depicted using purple and green circles, where green represents negative GC skew and deep purple indicates positive GC skew.
Genes 16 00226 g001
Genes 16 00226 g002
Figure 2. Relative synonymous codon usage (RSCU) patterns in the medium-sized and dwarf forms of S. oualaniensis.
Figure 2. Relative synonymous codon usage (RSCU) patterns in the medium-sized and dwarf forms of S. oualaniensis.
Genes 16 00226 g002
Genes 16 00226 g003
Figure 3. The ratio of nonsynonymous to synonymous substitutions (Ka/Ks) across 13 protein coding genes in two forms of S. oualaniensis.
Figure 3. The ratio of nonsynonymous to synonymous substitutions (Ka/Ks) across 13 protein coding genes in two forms of S. oualaniensis.
Genes 16 00226 g003
Genes 16 00226 g004
Figure 4. Bayesian inference (BI) (A) and maximum likelihood (ML) (B) phylogenetic trees illustrating the evolutionary relationships among cephalopod species based on mitochondrial genome sequences. The tree is rooted with A. dux as the outgroup. Posterior probabilities and support value are displayed at the nodes. Notable clades include multiple mitochondrial haplotypes of S. oualaniensis forming a well-supported cluster, and a close relationship between D. gigas and Eucleoteuthis luminosa. The longer branch lengths of T. pacificus and I. argentinus indicate greater genetic divergence compared to other taxa. The scale bar represents genetic distance.
Figure 4. Bayesian inference (BI) (A) and maximum likelihood (ML) (B) phylogenetic trees illustrating the evolutionary relationships among cephalopod species based on mitochondrial genome sequences. The tree is rooted with A. dux as the outgroup. Posterior probabilities and support value are displayed at the nodes. Notable clades include multiple mitochondrial haplotypes of S. oualaniensis forming a well-supported cluster, and a close relationship between D. gigas and Eucleoteuthis luminosa. The longer branch lengths of T. pacificus and I. argentinus indicate greater genetic divergence compared to other taxa. The scale bar represents genetic distance.
Genes 16 00226 g004
Table 1. Characteristic constituents of the mitochondrial genome of two forms of S. oualaniensis.
Table 1. Characteristic constituents of the mitochondrial genome of two forms of S. oualaniensis.
Dwarf Form of S. oualaniensisMedium-Sized Form of S. oualaniensis
FeatureStrandPositionLengthInitiation/
Stop Codon		StrandPositionLengthInitiation/
Stop Codon
COX3H1-780780ATG/TAAH1-780780ATG/TAA
tRNA-AlaH814-88168 H811-87868
tRNA-AsnH890-95869 H888-95669
tRNA-IleH961-102666 H959-102365
ND3H1027-1380354ATG/TAAH1024-1377354ATG/TAA
COX1H1434-29661533ATG/TAAH1421-29531533ATG/TAG
COX2H2968-3655688ATG/AATH2955-3642688ATG/TAA
tRNA-AspH3656-372267 H3643-370967
ATP8H3724-3879156ATG/TAAH3711-3866156ATG/TAA
ATP6H3881-4573693ATG/TAGH3868-4560693ATG/TAG
tRNA-PheL4666-460067 L4652-458766
tRNA-ValL4733-466569 L4719-465169
12S rRNAL5723-4734990 L5704-4720985
tRNA-CysL5788-572465 L5770-570566
tRNA-GlnL5858-579267 L5839-577367
long non-coding regionH5859-6419561 H5843-6403561
COX3H6420-7199780ATG/TAAH6404-7183780ATG/TAA
tRNA-LysH7206-727368 H7189-725668
tRNA-ArgH7274-734067 H7257-732367
tRNA-SerH7381-744868 H7365-743268
ND2H7449-84891041ATG/TAAH7433-84731041ATT/TAA
COX1H8461-99931533ATG/TAAH8445-99771533ATG/TAG
COX2H9995-10682688ATG/AATH9979-10666688ATG/TAA
tRNA-AspH10683-1074967 H10667-1073367
ATP8H10751-10906156ATG/TAAH10735-10890156ATG/TAA
ATP6H10908-11600693ATG/TAGH10892-11584693ATG/TAG
ND5L13322-116251698ATG/TAAL13306-116091698ATT/TAA
tRNA-HisL13390-1332368 L13373-1330767
ND4L14750-133911360ATA/TAGL14734-133741361ATA/TAG
ND4LL15043-14747297ATG/TAGL15027-14731297ATG/TAG
tRNA-ThrL15116-1505265 L15036-1510166
tRNA-SerL15182-1511865 L15167-1510365
CytbL16326-151831144ATA/TAGL16311-151681144ATA/TAG
ND6L16829-16323507ATG/TAGL16814-16308507ATG/TAG
tRNA-ProL16897-1683167 L16882-1681667
ND1L17833-16898936ATG/TAAL17818-16883936ATA/TAG
tRNA-LeuL17903-1783470 L17888-1781970
tRNA-LeuL17975-1790967 L17960-1789467
16S rRNAL19400-179761425 L19391-179611431
tRNA-MetL19471-1940171 L19462-1939271
tRNA-TyrL19541-1947666 L19531-1946765
tRNA-TrpL19608-1954267 L19598-1953267
tRNA-GlyL19681-1961765 L19668-1960465
tRNA-GluL19758-1968574 L19744-1967273
long non-coding regionH19759-20320562 H19747-20308562
H and L refer to the heavy and light strand, respectively.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Duo, W.; Xu, L.; Mohd Yusof, M.J.; Wang, Y.; Ng, S.B.; Du, F. Characterization of the Complete Mitochondrial Genome of Dwarf Form of Purpleback Flying Squid (Sthenoteuthis oualaniensis) and Phylogenetic Analysis of the Family Ommastrephidae. Genes 2025, 16, 226. https://doi.org/10.3390/genes16020226

AMA Style

Duo W, Xu L, Mohd Yusof MJ, Wang Y, Ng SB, Du F. Characterization of the Complete Mitochondrial Genome of Dwarf Form of Purpleback Flying Squid (Sthenoteuthis oualaniensis) and Phylogenetic Analysis of the Family Ommastrephidae. Genes. 2025; 16(2):226. https://doi.org/10.3390/genes16020226

Chicago/Turabian Style

Duo, Wenjuan, Lei Xu, Mohd Johari Mohd Yusof, Yingmin Wang, Seng Beng Ng, and Feiyan Du. 2025. "Characterization of the Complete Mitochondrial Genome of Dwarf Form of Purpleback Flying Squid (Sthenoteuthis oualaniensis) and Phylogenetic Analysis of the Family Ommastrephidae" Genes 16, no. 2: 226. https://doi.org/10.3390/genes16020226

APA Style

Duo, W., Xu, L., Mohd Yusof, M. J., Wang, Y., Ng, S. B., & Du, F. (2025). Characterization of the Complete Mitochondrial Genome of Dwarf Form of Purpleback Flying Squid (Sthenoteuthis oualaniensis) and Phylogenetic Analysis of the Family Ommastrephidae. Genes, 16(2), 226. https://doi.org/10.3390/genes16020226

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop