Comparative Analysis of Complete Mitogenomes of Two Oxyurichthys Gobies and Their Phylogenetic Implication

© 2022 Academia Sinica, Taiwan Open Access Comparative Analysis of Complete Mitogenomes of Two Oxyurichthys Gobies and Their Phylogenetic Implication Quan Van Nguyen1, Chien Van Pham1, Thanh Xuan Nguyen1, Jung-Il Kim2, Frank L. Pezold3,4, The Duc Nguyen1, Hiep Minh Le1, Chang-Bae Kim2, Viet Do Hung Dang1,*, and Thinh Dinh Do1,* 1Institute of Marine Environment and Resources, Vietnam Academy of Science and Technology, Haiphong, Vietnam. *Correspondence: E-mail: [email protected] (Dang); [email protected] (Do). E-mail: [email protected] (Nguyen); [email protected] (Pham); [email protected] (TX Nguyen); [email protected] (TD Nguyen); [email protected] (Le) 2Department of Biotechnology, Sangmyung University, Seoul 03016, South Korea. E-mail: [email protected] (JI Kim); [email protected] (CB Kim) 3Department of Life Sciences, Texas A&M University-Corpus Christi, Corpus Christi, TX 78412-5806, USA. E-mail: [email protected] (Pezold) 4Florida Museum of Natural History, University of Florida, Gainesville, FL 32611-7800, USA Received 20 July 2021 / Accepted 17 October 2022 / Published 26 December 2022 Communicated by Ka Hou Chu Oxyurichthys is a genus of goby that is widespread in the tropical Indo-West Pacific region. Oxyurichthys species are usually found in estuarine and coastal marine habitats. In Southeast Asia, they are commercial fishes and often collected by trawling to serve the market’s demand. The mitogenome serves as a good marker for investigating the systematics and evolution of fishes, but the mitogenome of Oxyurichthys species remains unknown. In this study, mitogenomes of two Oxyurichthys gobies, O. ophthalmonema and O. microlepis, were characterized and compared. The sizes of the mitogenomes were 16,504 bp and 16,506 bp for O. ophthalmonema and O. microlepis, respectively. Mitogenomes of these two species were similar in gene content and structure. Both included 37 genes and a control region. The two Oxyurichthys mitogenomes shared similar gene features and base composition with other recorded gobies. Typical conserved blocks (CSB-1, CSB-2, CSB-3 and CSB-D) were found in the control region of both species. Phylogenetic analyses based on concatenation of 13 protein-coding genes and 2 rRNAs revealed that the two Oxyurichthys species clustered together and were sister to species of the genera Sicydium, Sicyopterus and Stiphodon. The findings of the present study support previous evolutionary studies of gobies using other molecular markers. Key words: Gobies, Mitogenome, Gene structure, Control region, Phylogeny. BACKGROUND Gobies are relatively small fishes with a typical length of less than 10 cm that are ubiquitous in most seas and estuaries of the world (Patzner et al. 2011). The majority of gobies belong to two families, Gobiidae and Oxudercidae, comprising roughly 2,000 species in about 200 genera (Thacker 2015; Betancur et al. 2017). Gobies are not only important for aquatic ecosystems, but also for humans. Gobies play a key role in the ecosystem as prey for larger, more commercially important fishes (Yokoo et al. 2012). Some gobies are also important as direct sources of food for humans (e.g., Bell 1999; Zarev et al. 2013). Gobies are also popular in the aquarium fish trade, particularly species of the genus Brachygobius (Larson et al. 2008). Despite their ubiquity and economic and ecological significance, phylogenetic studies of goby evolution have been hampered by the simplification and Citation: Nguyen QV, Pham CV, Nguyen TX, Kim JI, Pezold FL, Nguyen TD, Le HM, Kim CB, Dang VDH, Do TD. 2022. Comparative analysis of complete mitogenomes of two Oxyurichthys gobies and their phylogenetic implication. Zool Stud 61:88. doi:10.6620/ZS.2022.61-88. Zoological Studies 61:88 (2022) doi:10.6620/ZS.2022.61-88 1 © 2022 Academia Sinica, Taiwan loss of many morphological characteristics (Thacker 2003; Jin et al. 2015). Therefore, analyses of molecular data have been considered attractive tools for resolving goby relationships (Akihito et al. 2000; Jin et al. 2015; Adrian-Kalchhauser et al. 2017). Oxyurichthys species are large gobies found in the tropical Indo-west Pacific region (Pezold and Larson 2015). Species of this genus are usually distributed in shallow estuarine and coastal marine habitats. Oxyurichthys species are commercially important for fishermen in Southeast Asia (Pezold and Larson 2015). Two species, O. ophthalmonema and O. microlepis are common in mangroves, estuaries and lagoons. Both species are important sources of livelihood for the people living around the Tam Giang–Cau Hai lagoon in Vietnam. Considerable progress has been made in diagnosing Oxyurichthys over the last half century (Gilbert and Randall 1979; Hoese 1986; Pezold 1991 1998), but the genus had never been adequately revised until the comprehensive review of Pezold and Larson (2015). Oxyurichthys is distinguished from other gobioid genera in having a transversely broad third neural spine, an A’BCDFH’ pattern of oculoscapular canal pores and no preopercular canal. All but one species have a single row of teeth in the upper jaw. The genus is a member of the Stenogobius lineage in the family Oxudercidae (Agorreta et al. 2013; Thacker 2015) and twenty species are currently recognized (Pezold and Larson 2015). While more morphological and biological data of Oxyurichthys has been accumulated, little is known about the genetic characteristics of species in the genus. The mitogenome has been used as an effective marker for identification, phylogeny and population genetics in vertebrates. The vertebrate mitogenome is circular and compact, generally spanning 16–17 kb in size (Satoh et al. 2016). Typically, it contains 37 genes, including 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes. In addition, two non-coding regions, origin of L-strand replication (OL) and control region (CR), are found in the vertebrate mitogenome. CR contains regulatory elements that control the transcription and replication of the mitogenome (Yu et al. 2021). With the development of sequencing technology, the number of mitogenomes of fishes, including gobies, has rapidly increased. Based on available sequences, some studies have been performed to investigate the interrelationships among species and genera of gobies (Jin et al. 2015; Adrian-Kalchhauser et al. 2017). Nevertheless, many genera, including Oxyurichthys, have never been sampled for these analyses. This lack of data has limited the investigation of goby phylogeny based on mitogenomes. This study was performed to sequence and analyze the mitogenomes of O. ophthalmonema and O. microlepis from Tam Giang–Cau Hai lagoon, Vietnam. Content and structure of the mitogenomes were investigated and compared with available mitogenomes of other gobies. In addition, phylogenetic trees were reconstructed to determine the position of Oxyurichthys in relation to other gobies. MATERIALS AND METHODS Fish sampling and genomic DNA extraction The specimens of Oxyurichthys species were collected from Tam Giang–Cau Hai lagoon, Thua Thien Hue province, Vietnam (16°36'38.49"N–107°31'40.95"E) in April 2021. Taxonomic identification of the collected specimens was performed based on morphological characteristics guided by Pezold and Larson (2015) together with cox1 sequences (data not shown). Whole fin clips of fishes were removed and preserved in 95% ethanol. Total genomic DNA was extracted from fin clips using DNeasy Blood & Tissue Kit following manufacturer’s instructions (Qiagen, Germany). PCR amplification and sequencing PCR amplification was conducted using different sets of primers shown in table S1. Universal primers for the control region (Cheng et al. 2012) and fragments of cox1 (Ward et al. 2005) were adopted from previous studies. Additional primers for overlapping sequences were designed with Primer3 ver. 4.1.0 program based on available goby mitogenomes (Untergasser et al. 2012). The mixture of 20 µl reaction volume contained 10 μL of 2X TOPsimpleTM DyeMIX-Tenuto (Enzynomics, South Korea), 1 μL of each primer (10 pmoles/μL), 100 ng of extracted DNA, and distilled water. PCR reaction was performed under the following conditions: initial denaturation at 95°C for 5 min, followed by 35 cycles at 95°C for 45 s, annealing at 50°C for 1 min, and extension at 72°C for 1 min, with a final extension at 72°C for 5 min. PCR products were visualized on 1% agarose gel under a UV transilluminator. Sequencing of the products was conducted with an ABI 3730 DNA Analyzer (Applied Biosystems, USA). Sequence alignment and mitogenome annotation The overlapping fragments of mitogenome sequences were aligned and analyzed using Geneious Prime ver. 2022.1 (Kearse et al. 2012). Mitogenome annotation was conducted using MitoFish ver. 3.72 page 2 of 14Zoological Studies 61:88 (2022) © 2022 Academia Sinica, Taiwan (Iwasaki et al. 2013) and MITOS web server (Bernt et al. 2013). The secondary structure of tRNAs was predicted with tRNAscan-SE ver. 2.0 (Chan and Lowe 2019). The complete mitogenome map of the two species was generated using Geneious Prime ver. 2022.1 (Kearse et al. 2012). The nucleotide compositions and genetic code were estimated in MEGA X ver. 10.2.4 (Kumar et al. 2018). Circular maps of complete mitogenomes were generated and annotated using Geneious Prime ver. 2022.1 (Kearse et al. 2012). The secondary structure of the putative origin of L-strand replication was predicted using Mfold web server (Zuker 2003). Skewness was assessed as follows: AT skew = [A − T] / [A + T]; GC skew = [G − C] / [G + C] (Perna and Kocher 1995). RSCU values were calculated using MEGA X ver. 10.2.4 to evaluate the level of nucleotide bias in each codon (Kumar et al. 2018). Phylogenetic analyses The mitogenomes of Oxyurichthys species from the present study and mitogenomes from different genera of Gobiidae obtained from GenBank were used for phylogenetic investigation (Table S2). Gobiomorus maculatus, Eleotris fusca and Odontobutis platycephala were used as outgroups. Phylogenetic trees were reconstructed for different concatenated datasets: 13 PCGs and 13 PCGs + 2 rRNAs. First, each sequence was extracted and aligned using MAFFT ver. 7 (Katoh and Standley 2013) in Geneious Prime ver. 2022.1 (Kearse et al. 2012). Subsequently, GBlocks 0.91b was applied to exclude poorly aligned regions (Castresana 2000). PartitionFinder 2 was used to identify the best partition scheme and the best fit model (Lanfear et al. 2017). The phylogenetic trees were constructed using Maximum Likelihood (ML) and Bayesian Inference (BI) approaches. Maximum Likelihood (ML) trees were constructed using IQ-tree ver. 2.1.2 with 1,000 bootstrap replicates (Minh et al. 2020). Meanwhile, Bayesian Inference (BI) trees were constructed using MrBayes ver. 3.2.7 with four chains, 10,000,000 generations and sampling every 100 generations (Ronquist et al. 2012). The first 25,000 trees before stationarity were excluded as burn-in, and the remaining trees were used to generate consensus trees. Effective sample size (ESS) values for the convergence of MCMC runs were assessed in Tracer ver. 1.7 (Rambaut et al. 2018). The convergence was accepted with ESS values above 200. The generated tree was visualized using FigTree ver. 1.4.4 (Rambaut 2018). RESULTS General features of Oxyurichthys mitogenome The mitogenomes of Oxyurichthys ophthalmonema and O. microlepis (GenBank accession numbers: ON755178 and ON755179) were 16,504 bp and 16,506 bp in length, respectively. Gene order and direction of the two mitogenomes are shown in figures 1–2. Both mitogenomes contained 13 PCGs (nd1–6, nd4l, cox1–3, cytb, atp6 and atp8), two rRNA genes (12S rRNA and 16S rRNA) and 22 tRNA genes (Tables 1–2). In addition, two non-coding regions (OL and CR) that are important for replication and transcription were also found in the two mitogenomes. While nd6 and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNACys, tRNA-Tyr, tRNA-Ser, tRNA-Glu and tRNAPro) were encoded on the L-strand, remaining genes of O. ophthalmonema and O. microlepis mitogenomes were encoded on the H-strand (Tables 1–2). The base composition of O. ophthalmonema was 28.7% A, 26.1% C, 16.7% G and 28.5% T, while the base composition of O. microlepis was 28% A, 25.9% C, 17.2% G and 28.9% T. Overlapping regions in O. ophthalmonema and O. microlepis mitogenomes ranged from 1 to 7 bp, with the longest overlapping region (7 bp) located between atp8 and atp6 as well as between nd4l and nd4 (Tables 1–2). Meanwhile, intergenic regions ranged from 1 to 34 bp, and the largest spacer was located between tRNA-Asn and tRNA-Cys (Tables 1–2). The analysis of nucleotide frequencies revealed a slight bias for A and T. A-T content accounted for 57.2% in O. ophthalmonema and 56.9% in O. microlepis. The control region was the main A+T-rich region with 63.8% in O. ophthalmonema and 63.1% in O. microlepis. A-T skew was 0.003 and -0.016 in O. ophthalmonema and O. microlepis, respectively. This indicates that O. ophthalmonema had A > T, while O. microlepis had A < T. In contrast, both species had negative G-C skew, -0.220 in O. ophthalmonema and -0.202 in O. microlepis, indicating that G < C. There were 3,799 amino acids of PCGs encoded in the mitogenomes of O. ophthalmonema and O. microlepis. The amino acids Leu (16.46%), Ala (8.75%), and Thr (7.96%) were most abundant in the two mitogenomes. Among these, the frequencies of Leu (CUC, CUA), Ala (GCC), and Thr (ACC) were highest. The frequencies of Cys (UGU), and Arg (CGG) in PCGs were lowest. Analysis of relative synonymous codon usage (RSCU) showed that the codons UCC for Ser, CUC and CUA for Leu and GCC for Ala occurred most frequently, while UUG for Leu, AGU for Ser and GCG for Ala were rare in the mitogenome of Oxyurichthys (Figs. 3–4). page 3 of 14Zoological Studies 61:88 (2022) © 2022 Academia Sinica, Taiwan Protein-coding genes (PCGs) The total length of 13 protein-coding genes was 11,426 bp for both O. ophthalmonema and O. microlepis, ranging from atp8 165 (bp) to nd5 (1,839 bp). Most of 13 PCGs were encoded on the H-strand, except for nd6, which was encoded on the L-strand. Twelve PCGs initiated with a typical ATG codon, except for atp6 gene, which was GTG. The two species had almost similar stop codons, except for the nd5 gene. For this gene, O. ophthalmonema had TAG for termination while O. microlepis had TAA. For the remaining genes, TAA was the common termination codon, while incomplete T-- was the termination for cox2, cox3, cytb, nd3 and nd4. Ribosomal RNA and transfer RNA genes In total, the tRNA genes were 1,556 bp in length, varying from 66 bp (tRNA-Cys) to 76 bp (tRNA-Leu) in size. Of 22 tRNA genes, 8 genes were encoded on the L-strand and 14 genes were encoded on the H-strand. The prediction for the secondary structure of tRNA genes is presented in figures S1 and S2. The prediction result revealed that all tRNAs could be folded into a typical cloverleaf secondary structure except for tRNASer (GCT). There was no recognizable dihydrouridine (DHU) stem found for tRNA-Ser. In vertebrate mitogenomes, the lack of a DHU stem in tRNA-Ser is commonly observed (Lee and Kocher 1995). The three tRNA clusters (IQM, WANCY and HSL) showed Fig. 1. Gene map of Oxyurichthys ophthalmonema mitogenome. page 4 of 14Zoological Studies 61:88 (2022) © 2022 Academia Sinica, Taiwan conservative features in the two Oxyurichthys species typical of vertebrate mitogenomes. 12S rRNA was located between tRNA-Phe and tRNA-Val, with a length of 952 bp. Meanwhile, 16S rRNA was located between tRNA-Val and tRNA-Leu, with a length of 1,675 bp. The G-C content of rRNA genes was 44.8% and 45.4% for O. ophthalmonema and O. microlepis, respectively. Non-coding region The putative origin of L-strand replication (OL) was determined in the mitogenomes of the two Oxyurichthys species (Fig. 5). OL was found between tRNA-Asn and tRNA-Cys in the WANCY region that includes a block of five tRNA genes (Trp, Ala, Asp, Cys, and Tyr). OL could fold into the secondary structure as a stable stem-loop, with 13 bp in the stem and 11 bp in the loop. The control region of both species was found between tRNA-Pro and tRNA-Phe. Control region sizes of O. ophthalmonema and O. microlepis were 847 bp and 848 bp, respectively. The region showed high A-T content with A-T = 63.8% for O. ophthalmonema and 63.1% for O. microlepis, which was higher than the A-T content of the whole mitogenome (57.2% and 56.9%). Conservative elements, including Terminationassociated sequence (TAS), the conserved sequence block domain (CSB-1, CSB-2 and CSB-3), the central conserved sequences (CSB-D) and A GTGGG box were Fig. 2. Gene map of Oxyurichthys microlepis mitogenome. page 5 of 14Zoological Studies 61:88 (2022) © 2022 Academia Sinica, Taiwan also observed in the mitogenomes of O. ophthalmonema and O. microlepis (Fig. 6). Phylogenetic analyses For investigation of the phylogenetic position of Oxyurichthys, the concatenated sets of nucleotide sequences of available mitogenomes were used for phylogenetic analyses. Bayesian Inference and Maximum Likelihood approaches were performed for tree reconstruction. The phylogenetic position of Oxyurichthys presented in figures 7–8 and figures S3–4 was relative to goby species based on mitogenome sequences. The ML and BI analyses indicated a slightly different topology, but all lineages as recognized by Agorreta et al. (2013) and Thacker (2015) were recovered as monophyletic. The two species of Oxyurichthys from our samples were clustered together with high values of posterior probability (PP = 1) and ultrafast bootstrap (UFBoot = 100) and were sister to a clade containing the genera Sicydium, Sicyopterus and Stiphodon (PP = 1, UFBoot = 100). Monophyly for the clade of sicydiines was well-supported with high strong posterior probability and ultrafast bootstrap support Table 1. Gene organization of Oxyurichthys ophthalmonema mitogenome Gene Position Size Codon Intergenic nucleotide Strand From To Start Stop tRNA-Phe 1 68 68 0 H 12S rRNA 69 1,021 953 0 H tRNA-Val 1,022 1,093 72 0 H 16S rRNA 1,094 2,780 1,687 0 H tRNA-Leu 2,781 2,856 76 0 H nd1 2,857 3,831 975 ATG TAG 0 H tRNA-Ile 3,835 3,904 70 3 L tRNA-Gln 3,904 3,974 71 -1 H tRNA-Met 3,974 4,042 69 -1 H nd2 4,043 5,089 1,047 ATG TAA 0 H tRNA-Trp 5,091 5,161 71 1 H tRNA-Ala 5,164 5,232 69 2 L tRNA-Asn 5,234 5,306 73 1 L tRNA-Cys 5,341 5,406 66 34 L tRNA-Tyr 5,407 5,477 71 0 L cox1 5,479 7,032 1,554 GTG TAA 1 H tRNA-Ser 7,033 7,103 71 0 L tRNA-Asp 7,107 7,178 72 3 H cox2 7,181 7,871 691 ATG T-- 2 H tRNA-Lys 7,872 7,946 75 0 H atp8 7,948 8,112 165 ATG TAA 1 H atp6 8,106 8,789 684 ATG TAA -7 H cox3 8,789 9,572 784 ATG T-- -1 H tRNA-Gly 9,573 9,644 72 0 H nd3 9,645 9,993 349 ATG T-- 0 H tRNA-Arg 9,994 10,062 69 0 H nd4l 10,063 10,359 297 ATG TAA 0 H nd4 10,353 11,733 1,381 ATG T-- -7 H tRNA-His 11,734 11,802 69 0 H tRNA-Ser 11,803 11,870 68 0 H tRNA-Leu 11,875 11,947 73 4 H nd5 11,948 13,786 1,839 ATG TAG 0 H nd6 13,783 14,301 519 ATG TAA -4 L tRNA-Glu 14,302 14,370 69 0 L cytb 14,376 15,516 1,141 ATG T-- 5 H tRNA-Thr 15,517 15,588 72 0 H tRNA-Pro 15,588 15,657 70 -1 L D-loop 15,658 16,504 847 0 H page 6 of 14Zoological Studies 61:88 (2022) © 2022 Academia Sinica, Taiwan values (Figs. 7–8). Oxyurichthys formosanus however was revealed to be sister to a clade of Gobiopsis lineage species of the family Gobiidae. DISCUSSION The mitogenomes of the two Oxyurichthys species sequenced here contain 37 genes typical of the metazoan mitogenome. The total length between the two sequences is comparable, differing only by 2 bp. The length of goby mitogenomes range from 16,396 bp (Pomatoschistus minutus) to 18,999 bp (Neogobius melanostomus) (Adrian-Kalchhauser et al. 2017). The mitogenome lengths for O. ophthalmonema (16,504 bp) and O. microlepis (16,506 bp) fall within this range. Gene arrangement of the two Oxyurichthys species examined in this study is similar to the majority of recorded goby mitogenomes. In general, gene arrangement in goby mitogenomes is pretty conservative with few exceptions. For example, in the case of the gene arrangement of O. ophthalmonema, O. microlepis and most goby species, the order is Ile/Gln/ Met, while it is Gln/Ile/Met in Neogobius melanostomus Table 2. Gene organization of Oxyurichthys microlepis mitogenome Gene Position Size Codon Intergenic nucleotide Strand From To Start Stop tRNA-Phe 1 68 68 0 H 12S rRNA 69 1,021 953 0 H tRNA-Val 1,022 1,093 72 0 H 16S rRNA 1,094 2,780 1,687 0 H tRNA-Leu 2,781 2,856 76 0 H nd1 2,857 3,831 975 ATG TAG 0 H tRNA-Ile 3,835 3,904 70 3 L tRNA-Gln 3,904 3,974 71 -1 H tRNA-Met 3,974 4,042 69 -1 H nd2 4,043 5,089 1,047 ATG TAA 0 H tRNA-Trp 5,091 5,161 71 1 H tRNA-Ala 5,164 5,232 69 2 L tRNA-Asn 5,234 5,306 73 1 L tRNA-Cys 5,341 5,406 66 34 L tRNA-Tyr 5,407 5,477 71 0 L cox1 5,479 7,032 1,554 GTG TAA 1 H tRNA-Ser 7,033 7,103 71 0 L tRNA-Asp 7,107 7,178 72 3 H cox2 7,182 7,872 691 ATG T-- 3 H tRNA-Lys 7,873 7,947 75 0 H atp8 7,949 8,113 165 ATG TAA 1 H atp6 8,107 8,790 684 ATG TAA -7 H cox3 8,790 9,573 784 ATG T-- -1 H tRNA-Gly 9,574 9,645 72 0 H nd3 9,646 9,994 349 ATG T-- 0 H tRNA-Arg 9,995 10,063 69 0 H nd4l 10,064 10,360 297 ATG TAA 0 H nd4 10,354 11,734 1,381 ATG T-- -7 H tRNA-His 11,735 11,803 69 0 H tRNA-Ser 11,804 11,871 68 0 H tRNA-Leu 11,876 11,948 73 4 H nd5 11,949 13,787 1,839 ATG TAA 0 H nd6 13,784 14,302 519 ATG TAA -4 L tRNA-Glu 14,303 14,371 69 0 L cytb 14,377 15,517 1,141 ATG T-- 5 H tRNA-Thr 15,518 15,589 72 0 H tRNA-Pro 15,589 15,658 70 -1 L D-loop 15,659 16,506 848 0 H page 7 of 14Zoological Studies 61:88 (2022) © 2022 Academia Sinica, Taiwan and Ponticola kessleri (Adrian-Kalchhauser et al. 2017). The putative origin of L-strand replication and control region of O. ophthalmonema and O. microlepis identified here were similar to those found for other goby mitogenomes. The control region showed rich A-T content with 63.8% for O. ophthalmonema and 63.1% for O. microlepis. These values are comparable to previous records such as 64.2 % for Tridentiger bifasciatus and 64.5% for T. barbatus (Jin et al. 2015). Conservative elements in the control region are expected as they play an important role in duplication and transcription of mitogenome. A number of conservative elements were identified in the two Oxyurichthys mitogenomes, consistent with previously recorded goby mitogenomes. The termination-associated sequence (TAS) is reported to be a recognition site for termination of heavy strand synthesis (Jin et al. 2015). Meanwhile, the conserved sequence block domains (CSB-1, CSB2 and CSB-3) may function in positioning RNA polymerase for transcription as well as for priming replication (Clayton 1991; Shadel and Clayton 1997). In addition, the central conserved sequence (CSB-D) was recorded in O. ophthalmonema and O. microlepis just as it had been in mitogenomes of other gobies (Jin et al. 2015; Adrian-Kalchhauser et al. 2017). CSB-D is well-known as a universally conserved block in the mitogenomes of teleost fishes (Lee et al. 1995). For phylogenetic analyses, 87 mitogenome sequences belonging to the Gobiidae and Oxudercidae and relatives were included (Table S1). The BI and ML Fig. 3. Relative synonymous codon usage of Protein-coding genes in Oxyurichthys ophthalmonema. Fig. 4. Relative synonymous codon usage of Protein-coding genes in Oxyurichthys microlepis. page 8 of 14Zoological Studies 61:88 (2022) © 2022 Academia Sinica, Taiwan Fig. 5. The putative origin of L-strand replication (OL) of Oxyurichthys ophthalmonema (a) and Oxyurichthys microlepis (b). Fig. 6. Termination-associated sequences (TAS), conserved sequence blocks (CSB-1, CSB-2, and CSB-3) and central conserved sequences (CSB-D) and GTGGG box in control region of two Oxyurichthys species mitogenomes. page 9 of 14 Zoological Studies 61:88 (2022)