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Article

The Complete Mitochondrial Genome of the Deep-Dwelling Goby Suruga fundicola (Teleostei, Gobiidae) Reveals Evidence of Recombination in the Control Region

by
Changting An
1,2,
Ang Li
1,2,
Huan Wang
1,2,
Shuai Che
1,2,
Richard van der Laan
3,
Shufang Liu
1,2,* and
Zhimeng Zhuang
1
1
State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
2
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266237, China
3
Independent Researcher, Grasmeent 80, 1357JJ Almere, The Netherlands
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(17), 8317; https://doi.org/10.3390/ijms26178317
Submission received: 12 July 2025 / Revised: 19 August 2025 / Accepted: 20 August 2025 / Published: 27 August 2025
(This article belongs to the Section Molecular Biophysics)
Browse Figures
Review Reports Versions Notes

Abstract

Suruga fundicola, one of the few known deep-dwelling gobies, is found in Japan, South Korea, and China. Owing to the limited availability of specimens, little is known about its mitogenome characterization, phylogenetic relationship, and adaptive evolution. In this study, we sequenced four complete mitogenomes using the DNBSEQ platform and Sanger sequencing. The mitogenomes in length ranged from 17,138 to 17,352 bp, primarily due to the variation in the number of long tandem repeat (LTR) sequences within variable region 3 (VR3). Although the gene composition and arrangement of the S. fundicola mitogenome are largely consistent with those of other gobies, we identified an expansion of the ND2 gene (78 bp), and an unexpected noncoding region (NC, 35 bp) located between the ND2 and tRNAtrp genes. To further investigate the variation in VR3, we sequenced this region in all nineteen individuals with the Sanger sequencing method. We detected eight distinct LTR types, containing one–three mutation sites, which formed ten different VR3 patterns. Most VR3 patterns (14/19) consisted of a single type of pure LTR, while the remaining five exhibited heterogeneous patterns composed of two different LTRs. Notably, in LTR types T1 and T3, which co-occur in heterogeneous patterns P1 and P9, we found their respective pure patterns (P2–3 and P7). Recombination provides a better, more plausible mechanism for generating the heterogeneity patterns than slipped-strand mispairing, which better explains the homogeneous LTR expansions. These findings provide evidence of recombination in the control region of a vertebrate mitogenome. A phylogenetic analysis confirmed that S. fundicola has a close relationship with Am. hexanema and C. stigmatias. Compared to five shallow-water species of the AcanthogobiusGroup, the deep-dwelling goby S. fundicola was found to be under stronger purifying selection. Within its mitochondrial protein-coding genes (PCGs), ND2 and ND6 genes were subject to stronger purifying selection than the others. Additionally, four genes showed signs of selection sites with high credibility (one in ATP6, ND3, and ND4; eight in ND2). This study provides valuable genomic resources for S. fundicola and enhances our understanding of the phylogenetic relationship, mitogenome recombination, and adaptive evolution of the goby.

1. Introduction

Mitochondria are essential cellular organelles for life found in almost all eukaryotes, and they contain their own genetic information, the mitogenome [1]. It has generally been assumed that this mitogenome does not undergo recombination, but this assumption has been challenged by a few cases of heteroplasmy and mtDNA recombination, as already reported for mussels [2,3], fishes [4,5], birds [6], cats [7], and humans [8,9]. Genes typically encoded in the vertebrate mitogenome contain 13 proteins, 2 ribosomal RNAs (rRNAs), and 22 transfer RNAs (tRNAs) [10,11,12]. Two noncoding regions (NC), the control region (CR), and the origin of L-strand replication (OL), were identified in almost all vertebrate mitogenomes [13]. The CR contains regulatory elements for controlling the transcription and replication of the mtDNA molecule, such as the termination-associated site (TAS), conserved sequence blocks (CSBs I to III), and variable regions (VR1, VR2, and VR3); see Figure 1 [13]. The length of the CR is highly variable due to the appearance or absence of variable numbers of tandem repeats (VNTRs). Tandem repeats have been widely reported in the mitochondrial CR of animal species, and variations in the number of repeat units have been found in birds [6], owls [14], cats [7], rabbits [15], and fishes [4,16,17]. Moreover, an extreme variation in the number of repeats has been found in interspecific [16], intraspecific [7,15], and intra-individual levels [14,18].
Based principally on surveys of the axial skeletal features of gobiid fishes, Suruga fundicola Jordan & Snyder, 1901, was placed into a putative monophyletic group (the Acanthogobius Group) with seven other genera [19,20]. However, due to the characteristics of gobiid fishes—exceptional species diversity (more than 2400 species), broad biogeographic distribution, diminutive body sizes, and heterogeneous morphological specialization/degeneration—significant challenges arise in their traditional morphological classification [21,22,23]. With the help of molecular evidence, the monophyly of the Acanthogobius Group was verified by recent studies (although based on a few species of the group), and the group was confirmed to be part of a larger clade, the Acanthogobius lineage (a monophyletic lineage within the family Gobiidae). This clade comprises about 29 genera, mainly living in the Northern Pacific [21,22,23]. McCraney, Thacker, and Alfaro further confirmed the monophyly of this group based on more taxa [23]. The monophyly of the Acanthogobius Group was recently challenged, although this study was based on only a few mitogenomes [24]. In our prior study, a preliminary phylogenetic analysis of concatenated COX1 and 12S sequences (704 bp) incorporating S. fundicola failed to recover a monophyletic Acanthogobius Group. This likely resulted from an insufficient number of phylogenetically informative sites due to the limited sequence length [25]. Thus, it is a better choice to use more mitogenomes to investigate both the phylogenetic status of S. fundicola and the monophyly of the Acanthogobius Group.
Suruga fundicola inhabits temperate regions of the Northwest Pacific, including Japan, South Korea, and China. This species has been occasionally recorded in marine surveys as one of the deepest-dwelling gobies [25,26,27,28]. Compared to other shallow-water species of the Acanthogobius Group, S. fundicola occupies significantly deeper habitats (depths of about 40–400 m) [20,28], enduring high hydrostatic pressure, low temperatures (mean: 8 °C), and permanent low-light conditions [25,29]. Down to the limits of daylight (1000 m) in the sea, the eyes of fishes generally increase in size relative to body length [29]. The eyes of S. fundicola are notably larger than those of other fishes in this group [19,28], clearly an adaptation to this deepwater environment. The harsh deepwater conditions are expected to result not only in the morphological traits but also in the adaptive evolution of the protein-coding genes that are vital for the oxidative phosphorylation pathway [7]. Intriguingly, energy-intensive species like bats exhibit much higher selection pressures on mitochondrial-encoded OXPHOS genes relative to their nuclear counterparts [30]. The positive selection sites identified in Triplophysa cavefishes indicated adaptive evolution in mitochondrial PCGs in response to extreme subterranean conditions [31]. However, whether mitochondrial proteins in this deep-water goby have undergone adaptive evolution remains unknown.
To date, only five mitogenomes (a total of ~150) of gobies within the Acanthogobius Group have been deposited in the MitoFish database (https://mitofish.aori.u-tokyo.ac.jp/species/search/ accessed on 20 May 2024). Few published studies have mainly involved the mitogenome architecture and phylogenetic relationships of only a few species [24,32,33], and have also addressed the interspecific variations between Chaeturichthys stigmatias and Amblychaeturichthys hexanema [24]. Due to the absence of mitogenomic data for the deep-dwelling goby, its mitochondrial architecture, phylogenetic relationships, and deepwater adaptive evolution remain poorly characterized. Thus, we sequenced four complete mitogenomes of S. fundicola and the VR3 part of the mitogenomes of nineteen individuals, based on next-generation sequencing (NGS) and/or the Sanger sequencing method. Firstly, we characterized the basic mitogenomic structure of this species and the arrangement of the various mt-genes. Secondly, we performed a comparative analysis with other goby fishes, and we analyzed the phylogenetic relationship of the group within the Gobiiformes. Finally, we analyzed the evolutionary selection pressure on 13 proteins coded by the mitogenome of S. fundicola.

2. Results and Discussion

2.1. General Features of S. fundicola Mitogenome

The ND2 gene and tandem repeats of the four mitogenomes were once more determined by the Sanger sequencing method. The four complete mitogenomes of S. fundicola and four sequences of the ND2 expansion were submitted to GenBank with the accession numbers PV067722-25 and PV067729-32. The four circular mitogenomes ranged from 17,138 to 17,352 bp in length, the sizes of which were longer than most mitogenomes of known goby fishes. All the typical 37 genes, including 13 PCGs (ND1–6, ND4L, COX1–3, CYTB, ATP6, and ATP8), 22 tRNA genes, and 2 rRNA genes (12S rRNA and 16S rRNA) were identified. Most of the genes were encoded on the H-strand; only ND6 and 8 tRNAs were on the L-strand (Figure 2, Table 1). The identified 37 genes of the four mitogenomes had all the same length and relative position. An unexpected NC (36 bp length in PV067722 and 35 bp length in PV067723-25) was identified between ND2 and tRNTrp, which has not been found in other fishes (as far as we know).
The base composition of the complete mitogenome (PV067722) was as follows: A% = 28.6, T% = 27.6, C% = 27.0, G% = 16.8 (Table 2). The overall A + T content was 56.4%, which was slightly richer than the G + C content (43.6%). The overall GC- and AT-skews of the mitogenome were −0.23 and 0.02, respectively; thus, indicating a strand compositional bias characterized by a substantial excess of over G nucleotides and a slight excess of over T nucleotides, similar to other fishes [34,35].
The PCGs accounted for 66.7% (11, 506 bp) of the S. fundicola mitogenome, with nucleotide composition of the PCGs: T% (30.4%), C% (28.5%), A% (24.8%), and G% (16.3%), with an AT content of 55.2%. The start codon ATG was found in 12 of the 13 PCGs, only COX1 was initiated with the codon GTG. The stop codons of the 13 PCGs are mostly TAA (ND2, COX1, ATP8, ND4L, ND5) and include TAG (ND1, ND6), TA (ATP8), and T (COX2, COX3, ND3, ND4, CYTB).
The 13 protein-coding genes have 3839 codons. Leu, Ala, Thr, and Ile were the most abundant amino acids in the mitogenome PCGs (Figure 3). Codon frequencies were calculated by the occurrence frequency of codons in all PCGs. Leu (CUU, CUA), Ala (GCC), Arg (CGA), Gly (GGC), Phe (UUU), and Thr (ACC) were the most abundant codons; meanwhile, stop codons UAG and UGU were the least frequent. The relative synonymous codon usage (RSCU) is a widely used measurement for the codon bias of each amino acid. The RSCU value for a codon close to 1 means that there is no preference for that codon, and an RSCU value >1 indicates that there is a strong bias for that codon. The RSCU values of the S. fundicola genome are displayed in Figure 4. Pro (CCC), Arg (CGA), Ala (GCC), Thr (ACC), Ser (UCC), and Leu (CUC and CUA) were the most frequently used codons, while Ser (UCG and AGU), Pro (CCG), Ala (GCG), Thr (ACG), and Leu (UUG) were the least.
Two common noncoding regions, the OL and the CR, were similar to those reported in other fish species [13]. The OL was 33 bp in length, located in the WANCY cluster between tRNAAsn and tRNACys. The CR was 1495 bp in length, only slightly less than the CR of Am. hexanema, but longer than any other gobies of the Acanthogobius Group. Three termination-associated sequences (TAS), two conserved sequence blocks (CSB II to III), and one T-homopolymer were identified, but we could not identify the CSB-D block [13,16]. The LTR was 107 bp in length, consisting of an upstream part (52 bp) and a downstream part (55 bp), as shown in Figure 5. The homolog of the upstream part can be found in the CR of other species, and the homolog of the downstream part is its tRNA Phe. In addition, a 5′ imperfect copy was found in all individuals downstream of the CR, which includes the complete upstream part and the first five bases of the downstream part. In addition, the LTR can form a second structure with a higher free energy dG = −11.8 (Supplementary Figure S1a), and complex structures with multiple stem-loops were found between two LTRs with a higher free energy dG = −21.57 (Supplementary Figure S1b). The complex structures can help to form the VRNT [6].

2.2. Variation in the Long Terminal Repeat

The nineteen LTR sequences in the VR3 with lengths ranging from 582 to 930 bp were submitted to GenBank with the accession numbers PV077033-51 (Supplementary Table S1). The number of LTRs in the sequenced nineteen individuals was very variable: three (once), four (12 times), 5 (5 times), and six (once), see Figure 6b. In the phylogenetic tree, the individuals with the same LTRs were distributed in different clades, the changes in the LTRs did happen much faster than changes in the nuclear base composition. At the same time, the individuals from the same location were clustered in different clades. This phenomenon is also observed with yellow-browed tits [6] and domestic cats [7], but these studies differ from ours: these variable repeats are related to pedigree and geographic groups [36,37,38,39]. In our study, the observed variation in the LTRs was not a suitable marker for discussing lineage divergence. The LTRs have been observed in several vertebrates, and extreme variation in the number of repeats has been found in many intraspecific [6,7] and intra-individual studies [14,18,40]. The structure and functions of the coding regions are well studied, but the CR’s function is not entirely clear. It has been demonstrated that the CR region holds the promoters for the replication; however, the function of a series of tandem repeats is still unknown [15].
There were eight types (T1–T8) of LTRs found in the VR3 (Figure 6a), six with a single variation point compared to T1, one (T6) with 2, and one (T4) with 3 points. The eight types formed ten different patterns (P1–P10), five of which have only one type of LTR, and the other five have two heteroplasmic types of LTRs. The T1 LTR can be found in six different patterns (P1–3, P5–6, and P9), and the T3 LTR can be found in three different patterns (P1, P7, and P9). Most duplication events in mtDNAs can be explained by slipped-strand mispairing [6,41,42]. Considering that two LTRs can form complex structures, slipped-strand mispairing would be the best explanation for the patterns with one type of LTR. Slipped-strand mispairing was not a good explanation for the occurrence of the heteroplasmic patterns, especially considering that the LTRs occupied by P1, P5, P6, and P9 can be identified in other patterns (individuals) as well. That is to say, the “P2” and “P7” type mitochondria must be present in the same cell, and their DNA must be coupled. This implies paternal leakage followed by fusion of the mitochondria [3]. Insertion via recombination is probably a better explanation for the origin of these heteroplasmic LTRs. This heteroplasmy in the LTRs at the intraspecific level was also found in a variety of species [3,4,5,6,7,8,9], and direct evidence for recombination has already been discovered in two fishes, the Atlantic Cod (Gadus morhua) [4] and the European Flounder (Platichthys flesus) [5]. Here, we present another case for recombination in the mtDNA of a vertebrate.

2.3. Comparative Analysis

2.3.1. A + T% and AT-Skew of the Acanthogobius Group Mitogenomes PCGs

The A + T content and AT-skew of the 13 mitogenome PCGs of the Acanthogobius Group were calculated (six gobioid species; see Figure 7a). The six species all exhibit AT bias. The A + T content of S. fundicola (55.21%) was significantly higher than that of the other species (p < 0.01), with a 95% CI of [53.43, 55.08]. The value of A. flavimanus was the lowest. The AT-skew of S. fundicola was significantly lower than that of the other five species (p < 0.01), with a 95% CI of [−0.11, −0.04], see Figure 7b.

2.3.2. Transfer RNA of the Acanthogobius Group

The typical 22 tRNAs were identified in the Acanthogobius Group mitogenomes, as in other fishes. Taking S. fundicola as an example, the secondary structure of tRNAs generally contains four domains and a short variable loop: the AA (amino acid acceptor) stem, the dihydrouridine arm (D stem and loop, D), the thymidine arm (T stem and loop, T), the anticodon arm (AC stem and loop, AC), and the variable loop (V; Figure 8). Among the tRNAs, tRNASer1 (AGC) is the only one that is not folded into the typical clover-leaf secondary structure, due to the absence of the DHU arm, which is similar to the structure found in other fish [13,43]. In addition, tRNACys is supposed to have lost the D-loop [44]. Unmatched base pairs in the stem are widespread among tRNAs; only three (tRNAAla, tRNAGln, and tRNATyr) have a fully paired stem in the investigated mitogenomes. Compared to the standard tRNAPhe, the putative “tRNAPhe” identified in the lower region of the CR does not have the AA stem, and the code “GAA” is changed into “AAA”.

2.3.3. ND2 Gene of the Acanthogobius Group Mitogenomes

Compared to the other five species of the Acanthogobius Group, the length of the ND2 gene of the S. fundicola is 1122 bp, obviously longer than the length of 1044 bp of the other five members of the Acanthogobius Group. The ND2 gene of S. fundicola has a unique expanded 78 bp sequence at the end of this gene, the homologous sequence of which was not found by the BLASTn tool in GenBank.
Most motifs of the ND2 gene are similar, except for Nos. 20, 22, 24, and 25 (Figure 9). The No. 24 motif cannot be identified in S. fundicola, Am. hexanema, and C. stigmatias. The No. 20 motif in Lophiogobius ocellicauda was transferred to the upper side of the No. 17 motif. The No. 22 motif was not found in S. fundicola, and No. 25 was found in the expanded region of the ND2 gene of S. fundicola.

2.3.4. Gene Arrangement Analysis of Goby Fish Mitogenomes

The typical 37 genes (13 PCGs, 22 tRNAs, and 2 rRNA genes) and the two noncoding regions (CR and OL) were identified in 150 gobies’ mitogenomes, only the OL of E. klunzingerii, was not identified (Figure 10 type VII). The 150 goby fishes mitogenomes display nine types of gene arrangement (Figure 10). Most species (n = 138, 92.0%) of the goby fishes compared in the present study had the typical gene order, widely shared among vertebrate mitogenomes. However, deviations from the conserved gene order were found in twelve species from four families (12/150 species = 8.0%). All of the gene rearrangements mainly involve local position changes (reshuffling) or displacement to another location (translocation). Switching of the encoded strand (inversion) was not found in this study [13].
The local reshuffling was identified within three tRNA gene clusters (IQM, WANCY, and HS2L2). The tRNA gene order IQM was mutated to QIM in types V, VI, and IX. The relative location of gene WANCY was changed to NC and OL in types II and IX, respectively. The gene order HS2L2 in type VIII was changed into S2L2H. The translocation was found in type IX. The tRNA gene F was duplicated in types II, IV, VI, and IX.

2.3.5. Start and Stop Codons of the Mitogenomes of Gobies

The 13 protein-coding genes of 150 goby fishes were found to possess eight types of start codons (Figure 11; Supplementary Table S2). Among these eight codons, ATG was predominant (90.7%) as already found in other fishes [13], and it was found exclusively in the COX2, COX3, ND3, and CYTB genes. GTG was only found in the COX1 gene as a start codon. Of the eight codons, CTA was identified as a start codon in the ND1 gene of the Rhinogobius leavelli (MH729000, Oxudercidae), which was the first example reported in a goby [13,43]. The code ATT was only found in the ND1 gene of Brachygobius doriae (KU674800, Oxudercidae), and ATC was only found in the ND5 gene of Tridentiger trigonocephalus (KT282115, Oxudercidae).
The stop codons TAA (41.3%) and T-- (39.2%) were most common in gobies, a result usually encountered in other fishes [13,41]. AG- was found once in the ND4 gene by Odontobutis haifengensis (MF383619, Odontobutidae). The TA-, T-, and AG- stop codons were found mostly when the 3′-end of the protein-coding genes (i.e., COX2, ATP6, COX3, ND3, ND4, and CYTB) was followed by a tRNA gene on the same strand. The complete stop codons were found in the ND5, ND6, and COX1 genes. The most common stop codon of ND2 is TAA (56.0%), which differs from other fishes (91.2%) [13]. Only one stop codon, TAA, was found in ND4L, and seven stop codons (T--, TA-, TAA, AGA, AG-, AGG, and TAG) were found in the other genes (Figure 11, Supplementary Table S3).

2.4. Phylogenetic Inferences of Gobies

Molecular phylogenetic analyses were performed using 13 PCGs from 150 mitogenomes of Gobiiformes. The ML and IQ analyses generated similar topologies (Supplementary Figure S2 and Figure 12). In the ML tree, the species of gobies were divided into five clades (Gobiidae, Eleotridae, Odontobutidae, Rhyacichthyidae, and Trichonotidae), which was similar to recent studies [10,23]. The species of Eleotridae did not cluster into a monophyletic group but were divided into two clades: Eleotrinae (I) and Butinae (II). The latter clade was sister to the clade Gobiidae [10,45]. The species of Gobiidae formed a monophyletic group and clustered into two main clades: Gobiine-like (III) and Gobionelline-like (IV).
The Acanthogobius lineage is closely related to a clade consisting of the species of Gymnogobius, Luciogobius, Chaenogobius, Eucyclogobius, and Gillichthys, a result similar to recent studies [16,23,45]. Relationships within the Acanthogobius lineage recovered by our analysis conflicted with results from molecular phylogenies incorporating nuclear genes [21,22,23] or single-copy nuclear coding markers [45] but were consistent with the study based on mitogenome PCG sequence [10]. The species of the Acanthogobius Group formed a monophyletic group, containing A. hasta, A. flavimanus, L. ocellicauda, S. fundicola, Am. Hexanema, and C. stigmatias, as found in recent studies [16,21,23,45], with the exception of two other studies [24,27]. S. fundicola was sister to the clade formed by Am. hexanema and C. stigmatias (BP 100%), a result already found by An et al. [25].

2.5. Selective Pressure Analysis

The Ka/Ks ratio values were calculated to represent the selective pressure magnitude and direction of the PCGs. The average Ka/Ks values of 13 PCGs of the Acanthogobius Group were <1, suggesting a purifying selection of functional genes. Among the six species, S. fundicola has a lower value than other species of this group (Figure 13), indicating a stronger purifying selection, resulting in the accumulation of less nonsynonymous mutations [46,47]. Our results of the Ka/Ks comparison between S. fundicola and the other Acanthogobius Group species showed that ATP8 (Ka/Ks < 0.35; p < 0.05), ND2 (Ka/Ks < 0.20; p < 0.05), and ND6 (Ka/Ks < 0.20; p < 0.05) were the genes with the highest Ka/Ks values (Figure 14). This result indicates less intense purifying selection compared to the remaining mitochondrial PCGs [48]. In turn, COX1 possesses the lowest Ka/Ks values, indicating the strongest purifying selection, resulting in the accumulation of less nonsynonymous mutations. According to the literature, purifying selection is the predominant force acting during the molecular evolution of mitogenomes [47,48,49].
The result of signatures of positive selection in all 13 mitochondrial PCGs based on BM in EasyCodeML is presented in Table 3. All mitochondrial PCGs have values of ω < 1, with Model 0. Two genes, ND2 (ω = 0.182; p = 0.0489) and ND4 (ω = 0.071; p = 0.026), provided a statistically significant better fit than the one-ratio model. According to this result, the two genes (ND2 and ND6) experienced stronger purifying selection than the other mitochondrial PCGs.
The BSM analysis in EasyCodeML v.1.41 was used to detect signatures of positive selection in S. fundicola (as foreground branch) compared to the other five Acanthogobius Group species (background branches). Four genes were found to have positive selection sites (one in ATP6, ND3, and ND4; eight in ND2), based on the MA, although this result did not receive the support of the p-value of the LRTs (>0.05) (Table 3). These sites were mapped onto the 3D structures of corresponding homologous proteins (marked with red color in Supplementary Figure S3). Most sites were located within the functional domains of α-helices, particularly near or on the junction sites of the α-helices and loop areas, which were probably crucial for the conformational stability of the relevant proteins. The mitochondrial respiratory chain generates 95% of the adenosine triphosphate (ATP) required for cellular activities. All 13 mitochondrially encoded proteins participate irreplaceably in electron transport and aerobic respiration [50]. Studies have demonstrated that mitochondrial OXPHOS genes in bats undergo significantly stronger selective pressure than nuclear-encoded counterparts (23.08% vs. 4.9%) [30]. In this study, signals of positive selection were detected in four mitochondrial OXPHOS genes. ATP6 encodes subunit a of ATP synthase Fo, directly driving ATP synthesis, and ND2–ND4 constitute core functional units of the NADH dehydrogenase complex (Complex I), pivotal for energy transduction. Compared to shallow-water gobies of this Acanthogobius Group, S. fundicola and cavefishes (e.g., Triplophysa) face analogous extreme environmental pressures (high hydrostatic pressure, darkness, extreme temperature, and food scarcity) [25,31]. Notably, all four positively selected genes identified here also exhibit adaptive signatures in cavefishes. ND2—the gene with the highest number of positively selected sites in S. fundicola (eight sites)—also shows multi-site adaptive evolution in Triplophysa (six sites). We thus propose that in S. fundicola, the ND2 gene likely experiences dual selective pressures (purifying selection coupled with positive selection) to adapt to extreme habitats. Furthermore, accumulating evidence also reveals positive selection in mitochondrial genes under extreme environments, including deep-sea fishes [51], deep-sea mussels [52], deep-sea crab [53], and plateau mammals [54].

3. Materials and Methods

3.1. Sample Collection and DNA Extraction

During the 2021–2023 R/V cruises in the Yellow Sea, a small Agassiz trawl was employed to catch the samples, at an average speed of 3 n mile/h (=5556 m/h) for 20 min. A total of nineteen specimens of S. fundicola were collected from the South Yellow Sea, during the 2021–2023 R/V cruises in the Yellow Sea (Supplementary Table S1). The muscle of the right lateral dorsal was sampled and stored in anhydrous alcohol for subsequent DNA extraction. The voucher specimens attached with a unique number were fixed in 10% formalin preservative for morphological examination and also for permanent curation and were deposited in the National Marine Fishery Biological Germplasm Resource Bank, China.
Genomic DNA was extracted using TIANamp Genomic DNA Kit (Tiangen DP324, Biotech, Beijing, China), according to the manufacturer’s protocol. The quality was estimated at wavelength 260/280 nm by a Nano-300 micro-spectrophotometer (Allsheng, Hangzhou, China).

3.2. Library Construction and DNA Sequencing

The total mitogenome of S. fundicola was sequenced on the DNBSEQ platform by MGI Tech Co., Ltd. (Hongshan District, Wuhan, Hubei, China). To confirm the expansion of the ND2 gene, one pair of primers [4707 (S) F: 5′-CCTGGCCAAGTCATGAACCA-3′, 5583 (S) R: 5′-CAACGTGTGATTGCCACAGG-3′] was designed for the Sanger sequencing method, with the Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, accessed on 1 May 2024). The PCR product can cover the region of the ND2 expansion. PCR was conducted in 25 μL volumes, including 12.5 μL Master mix Taq, 1 μL of each primer, 1 μL template DNA, adding double distilled water to adjust the volume (Table 4). Thermocycling conditions were as follows: initial denaturation for 4 min at 94 °C, denaturation for 50 s at 94 °C, annealing for 50 s at 55 °C, and extension 50 s at 72 °C. After 35 cycles, the final extension was performed at 72 °C for 10 min. The PCR products were checked by the uncropped gel photograph method. The PCR products were bidirectionally sequenced by BGI Genomics Co., Ltd (West Coast New Area, Qingdao, Shandong, China).
The existence of LRT in the VR3 region can cause errors in mitochondrial genome assembly, manifested as varying VR3 lengths in repeated assembly runs using NOVOPlasty under identical parameters. The primers were designed similarly to ND2, the sequences are labelled as follows: 16068TR(S) F: 5′-TTCAATCGCATCTCAGAGT-3′, 75TR (S) R: 5′-CGGGAAGGAATATGTAGGG-3′. To investigate the VR3 region across different individuals, the region of all nineteen individuals was sequenced using this pair of primers. The PCR reaction conditions are detailed in Table 4 and all amplified sequences were submitted to GenBank, with the help of BankIt (https://www.ncbi.nlm.nih.gov/WebSub/, accessed on 30 January 2025).

3.3. Sequence Assembly, Annotation, and Analysis of S. fundicola

NOVOPlasty 4.3.5 was used for the assembly of the four mitogenomes in Linux, with the template (seed) of the COX1 sequence [55], and the SeqMan of Lasergene. v. 7.1 (DNASTAR software package) was used to assemble the sequences generated from the Sanger sequencing method. The ND2 and VR3 sequences of mitogenomes were confirmed in MEGA X, based on ClustalW. The mitogenomes were annotated with Mito Annotator [56]. The identification and secondary structure of tRNA were conducted in tRNAscan-SE version 1.21 [57], and the tRNA secondary structures were visualized using the Forna web server [58]. The circular mitogenome map was visualized using Proksee [59]. Base composition and amino acid distributions were calculated with MEGA X [60]. The mitochondrial genes’ skewness was calculated using the following formulas: GC-skew = (G − C)/(G + C) and AT-skew = (A − T)/(A + T).
The CR sequences were separately extracted, based on the annotation with Mito Annotator [56]. The length, A + T content, and AT-skew were calculated with MEGA X [60]. The number of repeats of CR was investigated with the web server Tandem Repeat Finder v. 4.09 (http://tandem.bu.edu/trf/trf.html, accessed on 10 October 2023) [61]. The Mfold web server (http://mfold.rna.albany.edu/?q=mfold, accessed on 15 October 2023) was employed to predict its secondary structures. Based on the aligned sequences with ClustalW in MEGA X [60], we identified the termination-associated site (TAS), conserved sequence blocks (CSB I to III), and T-homopolymer with Jalview 2.9.0b2 [13,62,63,64]. Here, we identified TAS motifs using the query ATGN (8–9) CAT/TAT and searched for CSB-like motifs based on the following queries: CSB-D: TTC/A (11)T/CTC, CSB-I: ATA (15) ATA; CSB-II: AAACCCCNNNNCCCCC; CSB-III: AAACCCCC [13,16,62]. The T-homopolymer was also recognized based on a continuous “TTTTTT” base. The Mfold web server (http://mfold.rna.albany.edu/?q=mfold, accessed on 25 October 2023) was employed to predict L-strand origin (OL), secondary structures [65], and the conserved teleost motif “5′-GCGGG-3′” was identified [11,13]. The holotype of the LTR in the 3′ terminal of the CR was identified in DnaSP 6 [66].

3.4. Comparative Analysis of the Goby Mitogenomes

For the Acanthogobius Group, we downloaded the five available goby fish mitogenomes of this group from Mitofish (http://mitofish.aori.u-tokyo.ac.jp/species/search/, accessed 20 May 2024). The base compositions of the PCGS of the Acanthogobius Group mitogenomes were calculated as in the S. fundicola (the PV067722 mitogenome was chosen as an example). To further examine whether these two parameters exhibit significant differences between S. fundicola and other species of the Acanthogobius Group, we performed one-sample t-tests using SPSS 27. The motifs of the ND2 gene of this group were identified by MEME-SUITE 5.5 (https://meme-suite.org/meme/tools/meme, accessed on 22 August 2024), based on the maximum number of 25 motifs.
We downloaded the other 149 Gobiiformes genomes from Mitofish and annotated the genes with the annotation function in the same database. We analyzed the gene arrangement of these Gobiiformes fishes. In addition, the start and stop codons of 13 PCGS were calculated with MEGA X [60].

3.5. Molecular Phylogenetic Analysis

The sequences of the 3′ terminal CR of nineteen individuals were aligned and trimmed in MEGA X. These sequences of the partial CR were 571 to 898 bp in length, which consisted of one section before LTR (with a length of 256 bp) and the LTRs (with a length from 321 bp to 642 bp). The NJ method was chosen to construct the phylogenetic tree with the same software, based on the Kimura 2-parameter [60].
The taxonomic position of S. fundicola was assessed using concatenated sequences of 13 PCGs (without stop codon) of 150 Gobiiformes mitogenomes, which represented 74 genera from 6 families of Gobiiformes. Kurtus gulliveri was chosen as an out-group to the Gobioidei (Supplementary Table S4). The taxonomy of Eschmeyer’s Catalog of Fishes was followed [67]. We performed sequence alignment using the MAFFT program (alignment mode: code, code: vertebrate mitochondrial code, strategy: auto [68] and multi-gene datasets concatenated by SequenceMatrix [69]. The aligned DNA sequences were used for the phylogenetic analysis. Maximum likelihood trees (ML trees) were constructed using concatenated data (12,276 bp) based on the GTRGAMMA model in RaxML v8.2.8 [70] with 1000 bootstrap replicates. Data were partitioned by codon position and by a partitioning scheme identified in PartitionFinder v2.1.1 [71]. Initial data blocks of codons were analyzed under the models (GTR, GTR + G, GTR + G + I) and with the clustering algorithm in PartitionFinder v2.1.1 [72]. In addition, IQ-TREE multicore version 2.1.4-beta was employed to infer an ML tree for comparison, with the best-fit model GTR + F + R10 as suggested by ModelFinder [73], based on the BIC value with 1000 SH-aLRT (Shimodaira–Hasegawa-like approximate likelihood ratio test) [74]. The resulting ML and IQ trees were visualized using Figtree v.1.4.3 [75].

3.6. Selective Pressure Analyses

The selective pressure magnitudes and directions of 13 concatenated PCGS sequences of the six Acanthogobius Group species, the number of non-synonymous substitutions per non-synonymous site (Ka), the synonymous substitutions per synonymous site (Ks), and the Ka/Ks ratio were determined [76]. For this analysis, Ka and Ks values were calculated between each Acanthogobius Group species and the other 149 goby species, based on the 13 concatenated PCGS sequences (without stop codon). To investigate the pressures acting on each of these PCGs of the Acanthogobius Group. The Ka/Ks ratio of each PCG between S. fundicola and other Acanthogobius Group species was determined with the same method [76].
The EasyCodeML v.1.41 was employed to examine for signatures of positive selection in all 13 mitochondrial PCGs that could be altered by the colonization of deep-water environments in S. fundicola [77]. We used the phylogeny obtained with MEGA X as input files based on each gene sequence of the PCGs.
We first used Branch Models (BMs) to test for statistically significant differences in ω among branches of the phylogenetic tree. Foreground branches correspond to S. fundicola living in deep-water habitats. In turn, background branches are those leading to the five other species inhabiting the bottom depth less than 90 m. The one-ratio model (M0) assumes that ω is constant for all branches. The free-ratio model (M1) allows each branch to have an independent ω, and the two-ratio model (M2) assumes that the branches of interest (i.e., the foreground lineages) have different ω values than the background lineages. Here, M1 and M0 were compared to test whether different lineages in the tree had different ω values, and M2 was compared with M0 to determine whether S. fundicola was subjected to more selection pressure than the other Acanthogobius Group species. Pairwise models were compared using likelihood ratio tests (LRTs) to test whether M1 or M2 fit the data significantly better than M0 [78]. Considering that positive selection often occurs over a short period of evolutionary time and/or at a few sites, we chose to use branch-site models (BSM) in EasyCodeML to investigate the case of positive selection for the deep-dwelling species S. fundicola with the help of two models, model A and model Anull [77,79]. The three-dimensional structure of the proteins was predicted with Phyre2 [80], and the positive sites were marked with PyMOL.

4. Conclusions

This study sequenced and assembled four complete mitogenomes of this deepwater goby and the variable region 3 (VR3) in the 3′ terminal control region (CR) of nineteen specimens. The lengths of the four mitogenomes of S. fundicola ranged from 17,138 to 17,352 bp, with size variation attributed primarily to differences in the number of long tandem repeats (LTRs) within the CR. The gene composition and arrangement of the S. fundicola mitogenome are not significantly different from those of other gobies, but an expansion of the ND2 gene was identified. Meanwhile, an unexpected NC was also identified between the ND2 and tRNATrp coding regions. Using species-specific primer/probe design targeting unique extended regions (e.g., ND2 and the NC), facilitating definitive species identification, quick species identification can be achieved without sequencing. Among gobiid mitogenomes, nine distinct gene arrangements were observed, mainly involving local position changes (reshuffling) or displacement to another location (translocation). Analysis of VR3 showed that insertion via recombination provides a better explanation for the heterogeneity patterns LTR T1 and T3, compared with the explanation of slipped-strand mispairing for pure LTR. The analysis also reveals evidence of recombination in the vertebrate mitochondrial control region. Phylogenetic reconstruction based on 13 protein-coding genes (PCGs) from 150 gobioid mitogenomes confirmed the monophyly of the Acanthogobius Group, S. fundicola formed a sister group relationship to the clade consisting of two other gobies, Am. hexanema and C. stigmatias. These results contribute to the systematic classification of these gobies. Selection pressure analysis indicated that ND2 and ND6 underwent stronger purifying selection than the other mitochondrial PCGs. Four genes harbored high-confidence positive selection sites (one site each in ATP6, ND3, and ND4; eight sites in ND2). These candidate mutations can provide prioritized targets for future mitochondrial proteomics.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26178317/s1.

Author Contributions

Formal analysis, funding acquisition, writing—original draft, and writing—review and editing, C.A.; methodology, writing—review and editing, A.L.; visualization and writing—review and editing, H.W.; methodology and writing—review and editing, S.C.; writing—review and editing, R.v.d.L.; conceptualization, funding acquisition, and writing—review and editing, S.L.; conceptualization, supervision, and writing—review and editing Z.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key R&D Program of China (2024YFD2400401), and the Central Public-interest Scientific Institution Basal Research Fund, YSFRI, CAFS (NO.20603022024023). Data and samples were collected onboard R/V “Lanhai 101” implementing the open research cruise NORC2022-01 supported by NSFC Shiptime Sharing Project (Project number: 42149901).

Institutional Review Board Statement

All specimens in this study were collected in strict compliance with Chinese laws and regulations. This study was approved by the Institutional Animal Care and Use Committee of the Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences (Approval code: YSFRI-2025067 on 7 July 2025).

Informed Consent Statement

Not applicable.

Data Availability Statement

The four complete mitogenomes of S. fundicola and four sequences of the ND2 expansion were submitted to GenBank with the accession numbers PV067722-25 and PV067729-32. The nineteen VR3 sequences were submitted to GenBank with the accession number PV077033-51. Other supporting results are included within this article and its supplementary files.

Acknowledgments

Our sincere thanks are given to Fang-Qun Dai and other staff (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences) who shared the specimens with us, and the whole staff of the Lanhai 101 for their help. We are especially grateful to Chen-Hong Li of Shanghai Ocean University, who provided the high-performance computing environment to construct the ML and IQ trees.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Hampl, V.; Epika, I.; Eliá, M. Was the mitochondrion necessary to start Eukaryogenesis? Trends Microbiol. 2019, 27, 96–104. [Google Scholar] [CrossRef]
  2. Burzyński, A.; Zbawicka, M.; Skibinski, D.O.F.; Wenne, R. Evidence for Recombination of mtDNA in the marine mussel Mytilus trossulus from the Baltic. Mol. Biol. Evol. 2003, 20, 388–392. [Google Scholar] [CrossRef]
  3. Ladoukakis, E.D.; Eleftherios, Z. Direct Evidence for Homologous Recombination in Mussel (Mytilus galloprovincialis) Mitochondrial DNA. Mol. Biol. Evol. 2001, 18, 1168–1175. [Google Scholar] [CrossRef] [PubMed]
  4. Arnason, E.; Rand, D.M. Heteroplasmy of short tandem repeats in mitochondrial DNA of Atlantic cod, Gadus morhua. Genetics 1992, 132, 211–220. [Google Scholar] [CrossRef]
  5. Hoarau, G.; Holla, S.; Lescasse, R.; Stam, W.T.; Olsen, J.L. Heteroplasmy and Evidence for Recombination in the Mitochondrial Control Region of the Flatfish Platichthys flesus. Mol. Biol. Evol. 2002, 19, 2261–2264. [Google Scholar] [CrossRef] [PubMed]
  6. Wang, X.-Y.; Liu, N.; Zhang, H.-L.; Yang, X.-J.; Huang, Y.; Lei, F. Extreme variation in patterns of tandem repeats in mitochondrial control region of yellow-browed tits (Sylviparus modestus, Paridae). Sci. Rep. 2015, 5, 13227. [Google Scholar] [CrossRef]
  7. Patterson, E.C.; Lall, G.M.; Neumann, R.; Ottolini, B.; Batini, C.; Sacchini, F.; Foster, A.P.; Wetton, J.H.; Jobling, M.A. Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite. BMC Genom. 2023, 24, 690. [Google Scholar] [CrossRef]
  8. Marilena, D.A.; Gajewski, C.D.; Lin, M.-T.; Mauck, W.M.; Shao, L.-Z.; Giorgio, L.; Moraes, C.T.; Giovanni, M. Heterologous mitochondrial DNA recombination in human cells. Hum. Mol. Genet. 2004, 13, 3171–3179. [Google Scholar] [CrossRef]
  9. Strakova, A.; Leathlobhair, M.N.; Wang, G.D.; Yin, T.-T.; Airikkalaotter, I.; Allen, J.L.; Allum, K.M.; Bansseissa, L.; Bisson, J.L.; Castillo, D.A. Mitochondrial genetic diversity, selection and recombination in a canine transmissible cancer. eLife 2016, 5, e14552. [Google Scholar] [CrossRef] [PubMed]
  10. Li, J.-Y.; Cui, X.-F.; Chen, S.-H.; Li, Y.; Zhang, S.-Y.; Yang, Y.-F.; Li, Y.-Y.; Guo, Y.-S.; Wang, Z.-D.; Liao, J. The First Complete Mitogenome Characterization and Phylogenetic Implications of Elops machnata (Teleostei: Elopiformes: Elopidae). Biology 2025, 14, 739. [Google Scholar] [CrossRef]
  11. Bibb, M.J.; Van Etten, R.A.; Wright, C.T.; Walberg, M.W.; Clayton, D.A. Sequence and gene organization of mouse mitochondrial DNA. Cell 1981, 26, 167–180. [Google Scholar] [CrossRef]
  12. Bohnsack, J.A.; Sutherland, D.L. Artificial Reef Research: A Review with Recommendations for Future Priorities. Bull. Mar. Sci. -Miami-. 1985, 37, 11–39. [Google Scholar]
  13. Satoh, T.P.; Miya, M.; Mabuchi, K.; Nishida, M. Structure and variation of the mitochondrial genome of fishes. BMC Genom. 2016, 17, 719. [Google Scholar] [CrossRef]
  14. Serrano, M.J.; Goudet, J.; Cumer, T. Characterization of the diversity of barn owl’s mitochondrial genome reveals high copy number variations in the control region. PLoS ONE 2024, 19, e0295595. [Google Scholar] [CrossRef] [PubMed]
  15. Tapanainen, R.; Aasumets, K.; Fekete, Z.; Goffart, S.; Dufour, E.; Pohjoismäki, J.L.O. Species-specific variation in mitochondrial genome tandem repeat polymorphisms in hares (Lepus spp., Lagomorpha, Leporidae) provides insight into their evolution. Gene 2024, 926, 148644. [Google Scholar] [CrossRef]
  16. Adrian-Kalchhauser, I.; Svensson, O.; Kutschera, V.E.; Alm Rosenblad, M.; Pippel, M.; Winkler, S.; Schloissnig, S.; Blomberg, A.; Burkhardt-Holm, P. The mitochondrial genome sequences of the round goby and the sand goby reveal patterns of recent evolution in gobiid fish. BMC Genom. 2017, 18, 177. [Google Scholar] [CrossRef] [PubMed]
  17. Dadkhah, K.; Mianji, G.R.; Barzegar, A.; Farhadi, A. Characterization of the mitochondrial Huso huso genome and new aspects of its organization in the presence of tandem repeats in 12S rRNA. BMC Ecol. Evol. 2023, 23, 55. [Google Scholar] [CrossRef]
  18. Wilkinson, G.S.; Mayer, F.; Kerth, G.; Petri, B. Evolution of repeated sequence arrays in the D-loop region of bat mitochondrial DNA. Genetics 1997, 146, 1035–1048. [Google Scholar] [CrossRef]
  19. Birdsong, R.S.; Murdy, E.O.; Pezold, F.L. A Study of the Vertebral Column and Median Fin Osteology in Gobioid Fishes with Comments on Gobioid Relationships. Bull. Mar. Sci. 1988, 42, 174–214. [Google Scholar]
  20. Shibukawa, K.; Iwata, A. Review of the East Asian gobiid genus Chaeturichthys (Teleostei: Perciformes: Gobioidei), with description of a new species. Bull. Natl. Mus. Nat. Sci. 2013, 4, 31–51. [Google Scholar]
  21. Agorreta, A.; San Mauro, D.; Schliewen, U.; Van Tassell, J.L.; Kovačić, M.; Zardoya, R.; Rüber, L. Molecular phylogenetics of Gobioidei and phylogenetic placement of European gobies. Mol. Phylogenetics Evol. 2013, 69, 619–633. [Google Scholar] [CrossRef] [PubMed]
  22. Thacker, C.E. Phylogenetic placement of the European sand gobies in Gobionellidae and characterization of gobionellid lineages (Gobiiformes: Gobioidei). Zootaxa 2013, 3619, 369–382. [Google Scholar] [CrossRef] [PubMed]
  23. McCraney, W.T.; Thacker, C.E.; Alfaro, M.E. Supermatrix phylogeny resolves goby lineages and reveals unstable root of Gobiaria. Mol. Phylogenetics Evol. 2020, 151, 106862. [Google Scholar] [CrossRef]
  24. Zheng, J.; Chen, B.-J.; Gao, T.-X.; Song, N. The mitochondrial genome of Chaeturichthys stigmatias provides novel insight into the interspecific difference with Amblychaeturichthys hexanema. Acta Oceanol. Sin. 2021, 40, 74–81. [Google Scholar] [CrossRef]
  25. An, C.-T.; Li, A.; Wang, H.; Li, B.-S.; Liu, K.-Y.; Sun, H.-Y.; Liu, S.; Zhuang, Z.-M.; van der Laan, R. Identification of the rare deep-dwelling goby Suruga fundicola Jordan & Snyder, 1901 (Gobiiformes, Gobiidae) from the Yellow Sea. Zoosyst. Evol. 2023, 99, 489–501. [Google Scholar]
  26. Akihito; Ikeda, Y.; Aizawa, M. Suborder Gobioidei. In Fishes of Japan with Pictorial Keys to the Species, 3rd ed.; Nakabo, T., Ed.; Tokai University Press: Tokyo, Japan, 2013; pp. 1347–1608 + 2109–2211. ISBN 978-4-4860-1804-9. [Google Scholar]
  27. Akihito; Hayashi, M.; Yoshino, T.; Shimada, K.; Yamamoto, T. Suborder Gobioidei. In The fishes of the Japanese Archipelago; Masuda, H., Amaoka, K., Araga, C., Uyeno, T., Yoshino, T., Eds.; Tokai University Press: Tokyo, Japan, 1984; pp. 236–289, pls. 235–258, 353–355. [Google Scholar]
  28. Choi, Y.; Lee, H.-H. First Record of the Goby, Suruga fundicola (Perciformes: Gobiidae) from Tongyeong, Korea. Korean J. Ichthyol. 2019, 31, 255–258. [Google Scholar] [CrossRef]
  29. Warrant, E. The eyes of deep-sea fishes and the changing nature of visual scenes with depth. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2000, 355, 1155–1159. [Google Scholar] [CrossRef]
  30. Shen, Y.-Y.; Liang, L.; Zhu, Z.-H.; Zhou, W.-P.; Irwin, D.M.; Zhang, Y.-P. Adaptive evolution of energy metabolism genes and the origin of flight in bats. Proc. Natl. Acad. Sci. USA 2010, 107, 8666–8671. [Google Scholar] [CrossRef] [PubMed]
  31. Song, S.; Cao, J.-H.; Xiang, H.-M.; Liu, Z.-X.; Jiang, W.-S. Comparative mitogenomic analysis of Chinese cavefish Triplophysa (Cypriniformes: Nemacheilidae): Novel gene tandem duplication and evolutionary implications. BMC Genom. 2025, 26, 293. [Google Scholar] [CrossRef]
  32. Kim, I.C.; Kweon, H.S.; Kim, Y.J.; Kim, C.B.; Gye, M.C.; Lee, W.O.; Lee, Y.S.; Lee, J.S. The complete mitochondrial genome of the javeline goby Acanthogobius hasta (Perciformes, Gobiidae) and phylogenetic considerations. Gene 2004, 336, 147–153. [Google Scholar] [CrossRef] [PubMed]
  33. Quan, X.-Q.; Jin, X.-X.; Sun, Y.-N. The complete mitochondrial genome of Lophiogobius ocellicauda (Perciformes, Gobiidae). Mitochondrial DNA 2014, 25, 95–97. [Google Scholar] [CrossRef] [PubMed]
  34. Li, B.; Wang, H.; Yang, L.; Liu, S.; Zhuang, Z. Complete Mitochondrial Genome of Pseudocaranx dentex (Carangidae, Perciformes) Provides Insight into Phylogenetic and Evolutionary Relationship among Carangidae Family. Genes 2021, 12, 1234. [Google Scholar] [CrossRef] [PubMed]
  35. Zhong, L.-Q.; Wang, M.-H.; Li, D.-M.; Tang, S.-K.; Zhang, T.-Q.; Bian, W.-J.; Chen, X.-H. Complete mitochondrial genome of Odontobutis haifengensis (Perciformes, Odontobutiae): A unique rearrangement of tRNAs and additional non-coding regions identified in the genus. Odontobutis Genom. 2018, 110, 382–388. [Google Scholar]
  36. Hernández, M.Á.; Campos, F.; Gutiérrez-corchero, F.; Amezcua, A. Identification of Lanius species and subspecies using tandem repeats in the mitochondrial DNA control region. IBIS 2003, 146, 227–230. [Google Scholar] [CrossRef]
  37. Keogh, S.M.; Johnson, N.A.; Smith, C.H.; Sietman, B.E.; Garner, J.T.; Randklev, C.R.; Simons, A.M. Secondary contact erodes Pleistocene diversification in a wide-ranging freshwater mussel (Quadrula). Mol. Ecol. 2025, 34, e17572. [Google Scholar] [CrossRef]
  38. Padhi, A. Geographic variation within a tandemly repeated mitochondrial DNA D-loop region of a North American freshwater fish, Pylodictis olivaris. Gene 2014, 538, 63–68. [Google Scholar] [CrossRef]
  39. Mundy, N.I.; Winchell, C.S.; Woodruff, D.S. Tandem repeats and heteroplasmy in the mitochondrial DNA control region of the loggerhead shrike (Lanius ludovicianus). J. Hered. 1996, 87, 21–26. [Google Scholar] [CrossRef]
  40. Françoso, E.; Zuntini, A.R.; Ricardo, P.C.; Araujo, N.S.; Silva, J.P.N.; Brown, M.J.F.; Arias, M.C. The complete mitochondrial genome of Trigonisca nataliae (Hymenoptera, Apidae) assemblage reveals heteroplasmy in the control region. Gene 2023, 881, 147621. [Google Scholar] [CrossRef]
  41. Cerasale, D.J.; Dor, R.; Winkler, D.W.; Lovette, I.J. Phylogeny of the Tachycineta genus of New World swallows: Insights from complete mitochondrial genomes. Mol. Phylogenetics Evol. 2012, 63, 64–71. [Google Scholar] [CrossRef]
  42. Slack, K.E.; Delsuc, F.; McLenachan, P.A.; Arnason, U.; Penny, D. Resolving the root of the avian mitogenomic tree by breaking up long branches. Mol. Phylogenet. Evol. 2007, 42, 1–13. [Google Scholar] [CrossRef] [PubMed]
  43. Yu, P.; Zhou, L.; Yang, W.T.; Miao, L.J.; Li, Z.; Zhang, X.J.; Wang, Y.; Gui, J.F. Comparative mitogenome analyses uncover mitogenome features and phylogenetic implications of the subfamily Cobitinae. BMC Genom. 2021, 22, 50. [Google Scholar] [CrossRef]
  44. Tang, Q.; Liu, Y.; Li, C.H.; Zhao, J.F.; Wang, T. Comparative Mitogenome Analyses Uncover Mitogenome Features and Phylogenetic Implications of the Reef Fish Family Holocentridae (Holocentriformes). Biology 2023, 12, 1273. [Google Scholar] [CrossRef]
  45. Ting, K.; Luke, T.; Jing Yan, L.; Jia Mei, J.; Prosanta, C.; Sparks, J.S.; Naylor, G.J.P.; Chen Hong, L. Phylogenomic analysis on the exceptionally diverse fish clade Gobioidei (Actinopterygii: Gobiiformes) and data-filtering based on molecular clocklikeness. Mol. Phylogenet. Evol. 2018, 128, 192–202. [Google Scholar]
  46. Sha, Z.; Xiao, N. The first two complete mitogenomes of the order Apodida from deep-sea chemoautotrophic environments: New insights into the gene rearrangement, origin and evolution of the deep-sea sea cucumbers. Comp. Biochem. Physiol. Part. D Genom. Proteom. 2021, 39, 100839. [Google Scholar]
  47. Zhao, B.; Gao, S.; Zhao, M.; Lv, H.; Song, J.; Wang, H.; Zeng, Q.; Liu, J. Mitochondrial genomic analyses provide new insights into the “missing” atp8 and adaptive evolution of Mytilidae. BMC Genom. 2022, 23, 738. [Google Scholar] [CrossRef] [PubMed]
  48. Stewart, J.B.; Freyer, C.; Elson, J.L.; Larsson, N.G.R. Purifying selection of mtDNA and its implications for understanding evolution and mitochondrial disease. Nat. Rev. Genet. 2008, 9, 657–662. [Google Scholar] [CrossRef] [PubMed]
  49. Kuhle, B.; Chihade, J.; Schimmel, P. Relaxed sequence constraints favor mutational freedom in idiosyncratic metazoan mitochondrial tRNAs. Nat. Commun. 2020, 11, 969. [Google Scholar] [CrossRef]
  50. Wallace, D.C. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: A dawn for evolutionary medicine. Annu. Rev. Genet. 2005, 39, 359–407. [Google Scholar] [CrossRef]
  51. Shen, X.-J.; Pu, Z.-Q.; Chen, X.; Murphy, R.W.; Shen, Y.-Y. Convergent Evolution of Mitochondrial Genes in Deep-Sea Fishes. Front. Genet. 2019, 10, 925. [Google Scholar] [CrossRef]
  52. Zhang, K.; Sun, J.; Xu, T.; Qiu, J.-W.; Qian, P.-Y. Phylogenetic Relationships and Adaptation in Deep-Sea Mussels: Insights from Mitochondrial Genomes. Int. J. Mol. Sci. 2021, 22, 1900. [Google Scholar] [CrossRef]
  53. Zhang, B.; Wu, Y.-Y.; Wang, X.; Jiang, W.; Yin, J.-P.; Lin, Q. Comparative analysis of mitochondrial genome of a deep-sea crab Chaceon granulates reveals positive selection and novel genetic features. J. Ocean. Limnol. 2020, 38, 427–437. [Google Scholar] [CrossRef]
  54. Gutiérrez, E.G.; Ortega, J.; Savoie, A.; Baeza, J.A. The mitochondrial genome of the mountain wooly tapir, Tapirus pinchaque and a formal test of the effect of altitude on the adaptive evolution of mitochondrial protein coding genes in odd-toed ungulates. BMC Genom. 2023, 24, 527. [Google Scholar] [CrossRef] [PubMed]
  55. Dierckxsens, N.; Mardulyn, P.; Smits, G. NOVOPlasty: De novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 2017, 45, e18. [Google Scholar]
  56. Iwasaki, W.; Fukunaga, T.; Isagozawa, R.; Yamada, K.; Maeda, Y.; Satoh, T.P.; Sado, T.; Mabuchi, K.; Takeshima, H.; Miya, M.; et al. MitoFish and MitoAnnotator: A mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol. Biol. Evol. 2013, 30, 2531–2540. [Google Scholar] [CrossRef] [PubMed]
  57. Chan, P.-P.; Lin, B.-Y.; Mak, A.J.; Lowe, T.M. tRNAscan-SE 2.0: Improved Detection and Functional Classification of Transfer RNA Genes. Nucleic Acids Res. 2021, 49, 9077–9096. [Google Scholar] [CrossRef]
  58. Kerpedjiev, P.; Hammer, S.; Hofacker, I.L. Forna (force-directed RNA): Simple and effective online RNA secondary structure diagrams. Bioinformatics 2015, 31, 3374–3376. [Google Scholar] [CrossRef]
  59. Grant, J.R.; Enns, E.; Marinier, E.; Mandal, A.; Herman, E.K.; Chen, C.-Y.; Graham, M.; Van Domselaar, G.; Stothard, P. Proksee: In-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 2023, 5, 484–489. [Google Scholar] [CrossRef]
  60. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547. [Google Scholar] [CrossRef]
  61. Benson, G. Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res. 1999, 27, 573–580. [Google Scholar] [CrossRef]
  62. Jemt, E.; Persson, Ö.; Shi, Y.; Mehmedovic, M.; Uhler, J.P.; López, M.D.; Freyer, C.; Gustafsson, C.M.; Samuelsson, T.; Falkenberg, M. Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element. Nucleic Acids Res. 2015, 43, 9262–9275. [Google Scholar] [CrossRef]
  63. Nicholls, T.J.; Minczuk, M. In D-loop: 40 years of mitochondrial 7S DNA. Exp. Gerontol. 2014, 56, 175–181. [Google Scholar] [CrossRef] [PubMed]
  64. Sbisà, E.; Tanzariello, F.; Reyes, A.; Pesole, G.; Saccone, C. Mammalian mitochondrial D-loop region structural analysis: Identification of new conserved sequences and their functional and evolutionary implications. Gene 1997, 205, 125–140. [Google Scholar] [CrossRef] [PubMed]
  65. Zuker, M. Mfold Web Server for Nucleic Acid Folding and Hybridization Prediction. Nucleic Acids Res. 2003, 31, 3406–3415. [Google Scholar] [CrossRef]
  66. 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]
  67. Fricke, R.; Eschmeyer, W.; Van der Laan, R. Eschmeyer’s Catalog of Fishes: Genera, Species, References; California Academy of Sciences: San Francisco, CA, USA; Available online: http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp (accessed on 10 August 2025).
  68. Katoh, K.; Standley, D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013, 30, 772–780. [Google Scholar] [CrossRef]
  69. Vaidya, G.; Lohman, D.J.; Meier, R. SequenceMatrix: Concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 2011, 27, 171–180. [Google Scholar] [CrossRef] [PubMed]
  70. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef]
  71. Lanfear, R.; Frandsen, P.B.; Wright, A.M.; Senfeld, T.; Calcott, B. PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol. Biol. Evol. 2017, 34, 772–773. [Google Scholar] [CrossRef]
  72. Lanfear, R.; Calcott, B.; Kainer, D.; Mayer, C.; Stamatakis, A. Selecting optimal partitioning schemes for phylogenomic datasets. BMC Evol. Biol. 2014, 14, 82. [Google Scholar] [CrossRef]
  73. Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.; Von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 2017, 14, 587–589. [Google Scholar] [CrossRef]
  74. Anisimova, M.; Gil, M.; Dufayard, J.-F.; Dessimoz, C.; Gascuel, O. Survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihood-based approximation schemes. Syst. Biol. 2011, 60, 685–699. [Google Scholar] [CrossRef] [PubMed]
  75. Rambaut, A. FigTree-Version 1.4.3, A Graphical Viewer of Phylogenetic Trees. Computer Program Distributed by the Author. 2017. Available online: http://tree.bio.ed.ac.uk/software/figtree (accessed on 5 June 2024).
  76. Wang, D.-P.; Zhang, Y.-B.; Zhang, Z.; Zhu, J.; Yu, J. KaKs_Calculator 2.0: A Toolkit Incorporating Gamma-Series Methods and Sliding Window Strategies. Genom. Proteom. Bioinform. 2010, 8, 77–80. [Google Scholar] [CrossRef]
  77. Gao, F.-L.; Chen, C.-J.; Arab, D.A.; Du, Z.-G.; He, Y.-H.; Ho, S.Y.W. EasyCodeML: A visual tool for analysis of selection using CodeML. Ecol. Evol. 2019, 9, 3891–3898. [Google Scholar] [CrossRef] [PubMed]
  78. Anisimova, M.; Bielawski, J.P.; Yang, Z. Accuracy and power of the likelihood ratio test in detecting adaptive molecular evolution. Mol. Biol. Evol. 2001, 18, 1585–1592. [Google Scholar] [CrossRef] [PubMed]
  79. Zhang, J.-Z.; Nielsen, R.; Yang, Z.-H. Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol. Biol. Evol. 2005, 22, 2472–2479. [Google Scholar] [CrossRef] [PubMed]
  80. Kelley, L.A.; Mezulis, S.; Yates, C.M.; Wass, M.N.; Sternberg, M.J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 2015, 10, 845–858. [Google Scholar] [CrossRef]
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Figure 1. A schematic diagram of the control region (CR) of the fishes’ mitogenome, following Satoh et al. (2016) [13]. The locations of conserved sequence blocks (CSBs) and variable regions (VRs) are mapped. The T-homopolymer region is represented by a black bar.
Figure 1. A schematic diagram of the control region (CR) of the fishes’ mitogenome, following Satoh et al. (2016) [13]. The locations of conserved sequence blocks (CSBs) and variable regions (VRs) are mapped. The T-homopolymer region is represented by a black bar.
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Figure 2. The circular mitogenome map of S. fundicola (PV067722). Genes encoded by the H-strand and L-strand are displayed in the outer and inner rings, respectively.
Figure 2. The circular mitogenome map of S. fundicola (PV067722). Genes encoded by the H-strand and L-strand are displayed in the outer and inner rings, respectively.
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Ijms 26 08317 g003
Figure 3. The amino acid distribution of the S. fundicola mitogenome. On the horizontal axis and vertical axis are the number and types of amino acids, respectively.
Figure 3. The amino acid distribution of the S. fundicola mitogenome. On the horizontal axis and vertical axis are the number and types of amino acids, respectively.
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Figure 4. The relative synonymous codon usage (RSCU) of all 13 mitochondrial protein-coding genes of S. fundicola.
Figure 4. The relative synonymous codon usage (RSCU) of all 13 mitochondrial protein-coding genes of S. fundicola.
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Figure 5. The sequence of the mitochondrial control region of S. fundicola PV067722. TAS, T-homopolymer, CSB elements, and VR3 consist of two regions: the upstream and downstream parts.
Figure 5. The sequence of the mitochondrial control region of S. fundicola PV067722. TAS, T-homopolymer, CSB elements, and VR3 consist of two regions: the upstream and downstream parts.
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Figure 6. (a) The eight types of LTRs in the VR3 of S. fundicola; (b) the NJ tree constructed based on the VR3 of the LTR. The ten patterns of the LTRs found in the CR of the S. fundicola mitogenome are marked on the tree; the sampling location is listed in square brackets after the voucher number. The “*” represents multiplication and is used to indicate the repetition count of different types of LTRs.
Figure 6. (a) The eight types of LTRs in the VR3 of S. fundicola; (b) the NJ tree constructed based on the VR3 of the LTR. The ten patterns of the LTRs found in the CR of the S. fundicola mitogenome are marked on the tree; the sampling location is listed in square brackets after the voucher number. The “*” represents multiplication and is used to indicate the repetition count of different types of LTRs.
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Figure 7. (a) The A + T content of species from the Acanthogobius Group; (b) the AT-skew of the same species. p-values and 95% confidence intervals (CIs) are noted.
Figure 7. (a) The A + T content of species from the Acanthogobius Group; (b) the AT-skew of the same species. p-values and 95% confidence intervals (CIs) are noted.
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Figure 8. (a) The secondary structure of the 22 tRNA genes present in the mitogenome of S. fundicola; (b) the secondary structure of the putative tRNAPhe homologous sequence by tRNAscan-SE 2.0 in the CR.
Figure 8. (a) The secondary structure of the 22 tRNA genes present in the mitogenome of S. fundicola; (b) the secondary structure of the putative tRNAPhe homologous sequence by tRNAscan-SE 2.0 in the CR.
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Figure 9. The motifs identified by MEME-SUITE in the ND2 sequence of Acanthogobius Group mitogenomes.
Figure 9. The motifs identified by MEME-SUITE in the ND2 sequence of Acanthogobius Group mitogenomes.
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Figure 10. The linearized representation of the mitogenome of S. fundicola compared to other gobies. The mitochondrial genes, including 22~27 tRNAs (represented by a single uppercase italic letter), 2 mitochondrial rRNAs (12S and 16S), and 13 protein-coding genes, were labeled in red, light green, and purple, respectively. NC (noncoding sequence of ≥30 bp) was marked in green. “F”: the tRNAPhe homologous sequence identified by tRNAscan-SE 2.0. CR: control region. The variant regions are marked with black underlines.
Figure 10. The linearized representation of the mitogenome of S. fundicola compared to other gobies. The mitochondrial genes, including 22~27 tRNAs (represented by a single uppercase italic letter), 2 mitochondrial rRNAs (12S and 16S), and 13 protein-coding genes, were labeled in red, light green, and purple, respectively. NC (noncoding sequence of ≥30 bp) was marked in green. “F”: the tRNAPhe homologous sequence identified by tRNAscan-SE 2.0. CR: control region. The variant regions are marked with black underlines.
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Figure 11. The usage bias of start and stop codons of 13 PCGs in 150 goby mitogenomes. Pie graphs show the frequency of the occurrence of the start and stop codons. Start codons (upper section) and stop codons (lower section) are represented by distinct colors, with the proportion of each color indicating the probability of occurrence of each respective codon across the 13 proteins.
Figure 11. The usage bias of start and stop codons of 13 PCGs in 150 goby mitogenomes. Pie graphs show the frequency of the occurrence of the start and stop codons. Start codons (upper section) and stop codons (lower section) are represented by distinct colors, with the proportion of each color indicating the probability of occurrence of each respective codon across the 13 proteins.
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Figure 12. The ML tree was constructed based on concatenated sequences of 13 PCGs (without stop codon) of 151 mitogenomes. Bootstrap support values based on maximum likelihood analysis are shown (with 1000 replicates). The clade represented by S. fundicola is highlighted in red.
Figure 12. The ML tree was constructed based on concatenated sequences of 13 PCGs (without stop codon) of 151 mitogenomes. Bootstrap support values based on maximum likelihood analysis are shown (with 1000 replicates). The clade represented by S. fundicola is highlighted in red.
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Figure 13. Individual Ka/Ks ratio values of the species of the Acanthogobius Group.
Figure 13. Individual Ka/Ks ratio values of the species of the Acanthogobius Group.
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Figure 14. Analysis of selective pressure in the PCGs of S. fundicola vs. the other species of the Acanthogobius Group; the vertical axis shows the KA/KS ratio for each of the 13 protein-coding genes.
Figure 14. Analysis of selective pressure in the PCGs of S. fundicola vs. the other species of the Acanthogobius Group; the vertical axis shows the KA/KS ratio for each of the 13 protein-coding genes.
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Table 1. The mitogenome features of S. fundicola (PV067722).
Table 1. The mitogenome features of S. fundicola (PV067722).
GeneStrandLocationSize
(bp)		AnticodonStart
Codon		Stop
Codon		Intergenics
Length
tRNAPhe (S)+1–6868GAA 0
12srRNA+69–1012944 0
tRNAVal (V)+1013–108371TAC 0
16srRNA+1084–27611678 0
tRNALeu(UUR) (L1)+2762–283675TAA 0
ND1+2837–3811975 ATGTAG3
tRNAIle (I)+3815–388470GAT −1
tRNAGln (Q)−3884–395471TTG −1
tRNAMet (M)+3954–402370CAT 1
ND2+4025–51461122 ATGTAA0
NC+5147–518135
tRNATrp (W)+5183–525371TCA 2
tRNAAla (A)−5256–532469TGC 1
tRNAAsn (N)−5326–539873GTT 33
OL−5399–5431
tRNACys (C)−5432–549665GCA 0
tRNATyr (Y)−5496–556671GTA 1
COX1+5569–71221554 GTGTAA0
tRNASer (S2)−7123–719371TGA 3
tRNAAsp (D)+7197–726872GTC 3
COX2+7272–7962691 ATGT–0
tRNALys (K)+7963–803674TTT 1
ATP8+8038–8202165 ATGTAA−5
ATP6+8196–8878683 ATGTA–0
COX3+8879–9662784 ATGT–0
tRNAGly (G)+9663–973472TCC 0
ND3+9735–10,083349 ATGT–0
tRNAArg (R)+10,084–10,15269TCG 0
ND4L+10,153–10,449297 ATGTAA−5
ND4+10,443–11,8231381 ATGT–0
tRNAHis (H)+11,824–11,89168GTG 0
tRNASerr(AGY) (S1)+11,892–11,95968GCT 3
tRNALeu(CUN) (L2)+11,963–12,03573TAG 0
ND5+12,036–13,8741839 ATGTAA−2
ND6−13,871–14,392522 ATGTAG0
tRNAGlu (E)−14,393–14,46169TTC 5
CYTB+14,467–15,6101144 ATGT–0
tRNAThr (T)+15,611–15,68272TGT −1
tRNAPro (P)−15,682–15,75170TGG 0
Control region+15,752–17,2461495 –
Table 2. The nucleotide composition and strand asymmetry of the S. fundicola mitogenome.
Table 2. The nucleotide composition and strand asymmetry of the S. fundicola mitogenome.
LengthT%C%A%G%AT%AT-
Skew%		GC-
Skew%
PCGs11,22330.428.524.816.355.2−0.10−0.27
tRNA155227.020.928.623.555.60.030.06
rRNA262220.925.333.220.654.10.23−0.10
CR149528.819.337.714.266.50.13−0.15
Genome17,24627.627.028.616.856.20.02−0.23
Table 3. Positive selection in all 13 mitochondrial PCGs based on BM in EasyCodeML. The number with “*” indicates that the positive selection site has high confidence (p > 0.95).
Table 3. Positive selection in all 13 mitochondrial PCGs based on BM in EasyCodeML. The number with “*” indicates that the positive selection site has high confidence (p > 0.95).
ModelLn LModel ComparedLRT p-ValuePositive Sites
ATP6MA−2587.829MA vs. MA 00.2667 D 0.960 *
MA 0−2588.449Not Allowed
ATP8MA−726.169MA vs. MA 01.000
MA 0−726.169Not Allowed
COX1MA−4856.932MA vs. MA 01.000
MA 0−4856.932Not Allowed
COX2MA−2035.809MA vs. MA 00.670
MA 0−2035.718Not Allowed
COX3MA−2355.767MA vs. MA 01.000
MA 0−2355.767Not Allowed
CYTBMA−3942.845MA vs. MA 01.000
MA 0−3942.845Not Allowed
ND1MA−3522.005MA vs. MA 01.000
MA 0−3522.005Not Allowed
ND2MA−4337.265MA vs. MA 00.3852 N 0.987 *, 18 G 0.980 *, 21 A 0.980 *, 62 T 0.976 *, 240 T 0.967 *, 329 T 0.953 *, 345 L 0.982 *
MA 0−4337.643Not Allowed
ND3MA−1202.254MA vs. MA 00.34688 T 0.952 *
MA 0−1202.698Not Allowed
ND4MA−5157.592MA vs. MA 01.000351 S 0.957 *
MA 0−5157.592 Not Allowed
ND4LMA−1051.586MA vs. MA 00.804
MA 0−1051.617Not Allowed
ND5MA−6756.556MA vs. MA 00.472
MA 0−6756.815Not Allowed
ND6MA−2199.261MA vs. MA 01.000
MA 0−2199.261Not Allowed
Table 4. PCR conditions for the two pairs of primers used in this study.
Table 4. PCR conditions for the two pairs of primers used in this study.
RegionInitial DenaturationDenaturationAnnealingExtensionCyclesVolume
ND294 °C, 4 min94 °C, 50 s57 °C, 30 S50 S3525 μL
VR350 °C, 30 S
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An, C.; Li, A.; Wang, H.; Che, S.; van der Laan, R.; Liu, S.; Zhuang, Z. The Complete Mitochondrial Genome of the Deep-Dwelling Goby Suruga fundicola (Teleostei, Gobiidae) Reveals Evidence of Recombination in the Control Region. Int. J. Mol. Sci. 2025, 26, 8317. https://doi.org/10.3390/ijms26178317

AMA Style

An C, Li A, Wang H, Che S, van der Laan R, Liu S, Zhuang Z. The Complete Mitochondrial Genome of the Deep-Dwelling Goby Suruga fundicola (Teleostei, Gobiidae) Reveals Evidence of Recombination in the Control Region. International Journal of Molecular Sciences. 2025; 26(17):8317. https://doi.org/10.3390/ijms26178317

Chicago/Turabian Style

An, Changting, Ang Li, Huan Wang, Shuai Che, Richard van der Laan, Shufang Liu, and Zhimeng Zhuang. 2025. "The Complete Mitochondrial Genome of the Deep-Dwelling Goby Suruga fundicola (Teleostei, Gobiidae) Reveals Evidence of Recombination in the Control Region" International Journal of Molecular Sciences 26, no. 17: 8317. https://doi.org/10.3390/ijms26178317

APA Style

An, C., Li, A., Wang, H., Che, S., van der Laan, R., Liu, S., & Zhuang, Z. (2025). The Complete Mitochondrial Genome of the Deep-Dwelling Goby Suruga fundicola (Teleostei, Gobiidae) Reveals Evidence of Recombination in the Control Region. International Journal of Molecular Sciences, 26(17), 8317. https://doi.org/10.3390/ijms26178317

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