First records of Bathytoshia brevicaudata (Hutton, 1875) (Myliobatiformes, Dasyatidae) from China with mitogenomic evidence

First records of Bathytoshia brevicaudata (Hutton, 1875) (Myliobatiformes, Dasyatidae) from China with mitogenomic evidence Jia-Jie Chen1,2*, Jiang-Yuan Chen1,3*, Xing-Le Guo4, Sheng Zeng5, De-Yuan Yang5,6, Jun-Sheng Zhong2, Shen-Long Zhao7, Wen-Hong Fang1*, Jian Chen7*, Pan Liu8* 1 East China Sea Fisheries Research Institute, Fisheries Science of Chinese Academy, 200090, Shanghai, China 2 Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, Shanghai Ocean University, 201306, Shanghai, China 3 Fisheries College, Ocean University of China, Qingdao 266003, China 4 Laboratory of Fisheries Ecology and Biodiversity, College of Fisheries, Zhejiang Ocean University, Zhoushan 316022, China 5 College of the Environment and Ecology, Xiamen University, 361102, Xiamen, China 6 National Taiwan Ocean University, Keelung, 202301, Taiwan, China 7 Marine Science and Technology College, National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang Ocean University, Zhoushan 316022, China 8 Conversation and Research Center for Collections, Shanghai Natural History Museum, Branch of Shanghai Science and Technology Museum, 200041, Shanghai, China https://zoobank.org/05A33FE9-5BF1-4C9F-8432-92E172DF5DEA Corresponding authors: Wen-Hong Fang ([email protected]); Jian Chen ([email protected]) Pan Liu ([email protected]g.cn) * These authors contributed equally to this work. Academic editor: Nalani Schnell ♦ Received 4 July 2025 ♦ Accepted 16 October 2025 ♦ Published 17 November 2025 Abstract This study presents the first records of the Broad-tail Stingray, Bathytoshia brevicaudata (Hutton, 1875), in the East China Sea, significantly extending its known distribution in the Northwest Pacific. Through comprehensive morphological examination of male and female specimens, we identified several sexually dimorphic traits, including the number of oral papillae rows, dorsal tubercle spines, and tooth cusp morphology. Stomach content analysis revealed predation on teleosts exceeding 30 cm total length, challenging previous dietary descriptions. Furthermore, the co-occurrence of distinct fishing hook types (coastal J-hook and pelagic circle hook) within one specimen provides empirical evidence of transboundary movements between neritic and oceanic zones. We also sequenced and characterized the complete mitogenomes of both sexes (17,640 bp; GenBank: PV699610–PV699611), which exhibited conserved dasyatid genomic features, including pronounced AT bias (59.2%), purifying selection across protein-coding genes (strongest in cox2), and structural traits such as an incomplete stop codon in ND4 and a DHU-arm-deficient trnS1. These integrative findings enhance the ecological and molecular understanding of B. brevicaudata and provide critical baseline data for its conservation in poorly studied regions of the Indo-Pacific. Key Words mitochondrial genome, phylogenetic analysis, East China Sea, taxonomic key Zoosyst. Evol. 101 (4) 2025, 2191–2205|DOI 10.3897/zse.101.164102 Copyright Chen, J.-J. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. zse.pensoft.net Chen, J.-J. et al.: First records of Bathytoshia brevicauda (Hutton, 1875) from China2192 Introduction Bathytoshia Whitley, 1933, is a widely distributed genus in temperate Atlantic and Indo-West Pacific waters that has long been taxonomically disputed regarding its species composition. Current consensus recognizes three valid species: B. centroura (Mitchill, 1815) in the Atlantic, and Bathytoshia brevicaudata (Hutton, 1875) and B. lata (Garman, 1880) occurring in the northwestern Pacific, among other regions (Last et al. 2016a, 2016b). The type locality of Bathytoshia brevicaudata is in New Zealand (Hutton 1875) and the species exhibits a disjunct distribution, divided by tropical seas into three isolated regions: the coastal waters of southern Africa, southern Australia-New Zealand, and the temperate northwestern Pacific (Last et al. 2016b). Due to the vast geographical separation between the northern and southern populations, the northwestern Pacific population was historically regarded as a distinct species, Dasyatis matsubarai (Miyoshi, 1939) (Miyosi 1939). However, subsequent morphological and molecular studies have relegated D. matsubarai to a junior synonym of B.brevicaudata (Le Port et al. 2013). Recent records include its first occurrence in southern Japan (Takushi et al. 2025) and South Korea (Kim et al. 2020). These recent records reported its complete mitochondrial genome sequence without detailed characterizing. Biological and ecological knowledge of this species remains limited. Studies on Bathytoshia spp. remain limited in China, with only B. lata having been historically recorded from Chinese waters (Zhu and Meng 2001; Wu and Zhong 2021). B. brevicaudata distribution was considered restricted to East coast waters of Taiwan Island, recorded as a specimen (FRIP20268; catalog 0268; 22.06°N, 121.51°E; http://catalog.digitalarchives.tw/item/00/26/49/f9.html) caught near Lanyu Island in 1987, originally identified as Dasyatis matsubarai (793 mm TL). However, this specimen likely represents Pteroplatytrygon violacea (Bonaparte, 1832) based on photographic evidence showing a purple-black dorsum and absent pectoral fin spots. No other Chinese records of B. brevicaudata exist, yet field surveys document abundant B. lata (1.2–1.5 m disc width, 200–300 kg) numbers in trawl catches at Taizhou, Zhejiang (Suppl. material 1: fig. S1) and Xiamen, Fujian of East China Sea and the Taiwan Strait (Suppl. material 1: fig. S2). We hypothesize that unreported B. brevicaudata populations may exist due to confusion between these large, broad stingrays. In this study, we report the first formal records of B. brevicaudata from the open water of northern Zhejiang, East China Sea, based on two voucher specimens (♂ ZJOUMBM F00035; ♀ ECSFRI 28263), and provide detailed morphological descriptions. We conducted dissection and qualitative stomach content analysis of the female specimen. We also sequenced, annotated, and characterized complete mitogenomes of the specimens with comparative analysis against Korean conspecifics (Kim et al. 2020), and reconstructed a myliobatiform phylogeny using mitogenomes from 52 species to elucidate their evolutionary relationships. Materials and methods Sample collection, morphological examination, and DNA extraction Two specimens of Bathytoshia brevicaudata, comprising a mature male (voucher ZJOUMBM F00035, 42 kg, Fig. 1, Suppl. material 1: fig. S3) and a mature female (voucher ECSFRI 28263, 60 kg, Fig. 2, Suppl. material 1: fig. S4), were collected as bycatch during bottom trawling in the East China Sea in March 2025. The specimens were landed at Songmen Port, Zhejiang Province, China. Subsequently, our team performed morphological measurements and tissue sampling on the specimens. Methods for measurements followed Compagno and Roberts (1982). Radial cartilage counts were obtained through dissection during the preparation of stripped specimens. An approximately 5 × 5 mm2 block of muscle tissue, which had been preserved in 100% ethanol, was clipped below the ventral dorsal fin of the specimens for DNA extraction. Whole genomic DNA was extracted using the TIANamp Genomic DNA kit (TIANGEN, Beijing, China). The two specimens are deposited at the Zhejiang Ocean University Museum of Marine Biodiversity (ZJOUMBM) and East China Sea Fisheries Research Institute (ECSFRI), respectively. The male individual was preserved in 10% neutral buffered formalin, while the female specimen underwent systematic biological dissection followed by taxidermy preparation. The stomach contents of the dissected female specimen (ECSFRI 28263) are deposited at the Shanghai Natural History Museum (SHNHM, voucher SNHM-Hfi13442, 95% ethanol), while its stomach and liver remain preserved at ECSFRI (voucher 28366~28367, in 10% neutral buffered formalin). Measurement of fishing hooks in stomach contents followed the standard protocols of Alós (2008) and Swimmer et al. (2010). Mitogenome sequencing and assembly The mitochondrial genomes were sequenced and assembled following established protocols (Chen et al. 2024; Xu et al. 2024; Yang et al. 2024). DNA libraries were prepared with the Illumina TruSeqTM DNA Sample Preparation Kit (Illumina, USA) following manufacturer guidelines. Novogene Bioinformatics Technology Co., Ltd. (China) conducted sequencing on a DNBSEQ-T7 platform, generating 150 bp paired-end reads yielding ~5 Gb raw data per sample. Data preprocessing involved Fastp v0.23.2 (Chen et al. 2018) with default parameters and quality assessment using FastQC v0.12.1 (http://www.bioinformatics.babraham. ac.uk/projects/fastqc/). Mitogenome assembly employed the FastMitoAssembler pipeline, augmented by GetOrganelle v1.7.6.1 (Jin et al. 2020) and NovoPlasty v4.3.1 (Dierckxsens et al. 2017). Zoosyst. Evol. 101 (4) 2025, 2191–2205 zse.pensoft.net 2193 Mitogenome annotation and sequence analyses The complete mitochondrial genome was annotated using MITOS2 (Donath et al. 2019) and Mitoz v3.6 (Meng et al. 2019), with manual validation in Geneious v2021.0.3. PhyloSuite v1.2.3 (Zhang et al. 2020) was employed to quantify base composition, codon usage, and relative synonymous codon usage (RSCU) of protein-coding genes (PCGs). Nucleotide skewness (A+T skew = [A%−T%]/ [A%+T%]; G+C skew = [G%−C%]/[G%+C%]) was calculated following Perna and Kocher (1995). Selective pressure analysis through Ka/Ks ratios was conducted for Dasyatinae species mitogenomes using DnaSP 6.0 (Rozas et al. 2017). Transfer RNA secondary structures predicted by tRNAscan-SE 2.0 (Lowe and Chan 2016; Chan et al. 2021) and MITOS (Bernt et al. 2013) were visualized Figure 1. Dorsal (A) and ventral (B) view of Bathytoshia brevicaudata, ZJOUMBM F00035, male, photo by Rui-Lin Huang. zse.pensoft.net Chen, J.-J. et al.: First records of Bathytoshia brevicauda (Hutton, 1875) from China2194 through ViennaRNA Web Services (Kerpedjiev et al. 2015; http://rna.tbi.univie.ac.at/forna/). The complete mitogenome sequences of the two specimens were ultimately determined and deposited in NCBI under accession numbers PV699610 (voucher ECSFRI 28263, female) and PV699611 (voucher ZJOUMBM F00035, male). Figure 2. Dorsal (A) and ventral (B) view of Bathytoshia brevicaudata, ECSFRI 28263, female, photo by Rui-Lin Huang. Zoosyst. Evol. 101 (4) 2025, 2191–2205 zse.pensoft.net 2195 Phylogenetic analysis We reconstructed the myliobatiform phylogeny using mitogenomes from 52 species, with Narcine timlei (Bloch & Schneider, 1801) and Rhinobatos schlegelii Müller & Henle, 1841 designated as outgroups. Mitogenome sequences retrieved from GenBank (Suppl. material 1: table S1) were processed in PhyloSuite 1.2.3 (Zhang et al. 2020). Thirteen protein-coding genes (PCGs) were aligned codon-wise using MAFFT v7.313 (Katoh and Standley 2013), and the resulting alignments were trimmed to the length of the shortest sequence to ensure consistent data length. Based on these alignments, pairwise genetic distances (p-distance) were calculated in MEGA X (Kumar et al. 2018), reporting the number of comparisons, mean, and standard deviation for each group. The pairwise p-distance matrices for the genus Bathytoshia are provided in Suppl. material 1: table S6. Subsequently, ambiguous regions were further trimmed using Gblocks v0.91 (Castresana 2000). ModelFinder v.2.2.0 (Kalyaanamoorthy et al. 2017) was used to select the best substitution models under the Bayesian Information Criterion (BIC) for maximum likelihood (ML) analysis and the corrected Akaike Information Criterion (AICc) for Bayesian inference (BI) analysis (Suppl. material 1: table S2). Maximum likelihood analysis used IQ-TREE v2.2.2 with edge-linked partitioning (5,000 ultrafast bootstrap replicates), while Bayesian inference was done using MrBayes v3.2.7a (two parallel runs; 2 × 106 generations) (Ronquist et al. 2012). Final topologies were visualized using iTOL v6 (Letunic and Bork 2016). Results Species description Bathytoshia brevicaudata (Hutton, 1875) Diagnosis. Large, plain-colored stingray with broad rhombic disc, short snout; dorsal surface greyish-brown, ventral surface whitish with grey margins; oblique white spots and paired anterolaterally-directed white pores at pectoral-fin bases dorsally; pelvic fins small; tail dusky, midline preceding caudal sting bearing row of spearshaped or star-based thorns and tubercles, post-sting portion blackish distally and covered with sharp thornlets. Description. Based on 2 specimens, 1,810–1,955 mm TL. Proportional measurements and counts are given in Table 1. Body large and flattened but thick, with a broad rhombic disc (Figs 1, 2). Disc width 1.2 (female)–1.4 (male) times its length (Table 1). Back smooth with large backward-pointing tubercles (9 in male, 3 in female); tail midline with similar tubercles extending anterior to caudal sting (Figs 1, 2). Snout short and blunt, length 0.22–0.23 times disc length, with irregularly distributed upright tubercles bearing blunt stellate bases, clustered discontinuously between snout tip and anterior orbital margins (Fig. 3A, B). Eyes small, length of orbit and spiracle 2.2 times (male) and 2.7/1.7 times (female) of snout length; interorbital space broad, up to 6 times orbit length (Table 1). Pectoral fins broad; anterior margins nearly straight; distal apex narrowly rounded; posterior margins smoothly convex; posterior insertion deeply notched; posterior angle bluntly pointed. Pectoral insertions slightly exceeding interorbital width. Pelvic fin origin slightly anterior to pectoral insertion; extending posteriorly beyond pectorals; anterior margin and lateral margins nearly straight, lateral and posterior angles smoothly rounded; pelvic insertion deeply notched (Figs 1, 2). Claspers (male) moderately elongate, length 0.24 times tail length; tips not reaching caudal sting (Table 1). Tail stout and tapered (not whip-like), short to moderately elongate, length 0.8–1.0 times disc width (males 0.8, females 1.0, estimated); base broad and depressed, width 1.4–1.6 times depth (Table 1); single caudal sting positioned midway, immediately posterior to dorsal thorn row comprising 15 densely arranged enlarged thorns with sharp-tipped projections from base (male) or 7 sparsely spaced thorns (female) (Fig. 3C, D); lateral serrated thorns upright, blunt, stellate-based, numerous, extending to tip; minute thorns densely cover dorsal surface from posterior to caudal sting, extending to terminus; ventral fold distinct (Fig. 3E, F), moderately long (0.3 times tail length) (Table 1), originating slightly posterior to caudal sting origin, extending toward tip but falls far of terminus; dorsal fold degenerated (Fig. 3E, F). Labial furrows deep; lower jaw weakly convex. Nasal curtain square-shaped, very broad, with margin fringed; nostrils oval, oblique (Fig. 4A, B). Mouth small (Figs 1, 2); floor of mouth with transverse series of 5 (female) or 7 (male) oral papillae; midregion with 3 (male) or 4 (female) prominent elongated papillae, lateral papillae shorter, 1–2 per side (Fig. 4C, D). Teeth small, granular, with 31 (male) and 33 (female) rows on upper jaws, and 34 (male) and 37 (female) rows on lower jaws, arranged in an interlocking pavement pattern. Upper and lower teeth similar, with sharp cusps in male while rounded in female (Fig. 4C, D). The male specimen ZJOUMBM F00035 possesses claspers measuring 257 mm in length, situated on the posteromedial aspect of the pelvic fins (Suppl. material 1: fig. S5). The dorsal surface of the claspers exhibit a slate-gray coloration, while the ventral surface appears pinkish due to subcutaneous hemorrhaging, with a seminal groove on the dorsal side. The claspers are robust, rigid, and exhibit a fully calcified structure, confirming the adult status of the specimen. Color. Dorsal surface pale grey, darkest above eyes and on tail tip, with 26–30 (female) or 21–24 (male) white pores arranged diagonally from pectoral fin bases toward pectoral fin leading edges; spiracles white internally; lateral head-encircling pore rows white; disc margin grey; ventral surface white (Figs 1, 2). To aid in the morphological identification of genus Bathytoshia, we propose the following diagnostic key, building upon the taxonomic framework of Last et al. (2016b): zse.pensoft.net Chen, J.-J. et al.: First records of Bathytoshia brevicauda (Hutton, 1875) from China2196 1 Labial furrow deep; pores around side of head and anterior disc white; tail short, usually shorter than disc width ........ ...................................................................................B. brevicaudata (Japan, Australia, New Zealand, southern Africa) – Labial furrow weak; pores around side of head and anterior disc without prominent color; tail long, longer than disc width ........................................................................................................................................................................ 2 2 Mouth usually with 6 oral papillae; tail length 1.6–1.8 times disc width ........................... B. centroura (western Atlantic) – Mouth with 3–5 oral papillae; tail length about 2 times disc width ........... B. lata (Indo-western Pacific, eastern Atlantic) Feeding habits. Dissection of the female specimen revealed stomach contents (Fig. 5) comprising fragmentary prey remains, specifically two Branchiostegus japonicus (Houttuyn, 1782) (Horsehead Tilefish), one Trachurus japonicus (Temminck & Schlegel, 1844) (Japanese Jack Mackerel), one Neobythites Goode & Bean, 1885 sp., and three longline fishhooks. The hooks included one circle hook (front length (FL) 20.2 mm, width (WD) 25.0 mm, gap (GP) 12.4 mm, shaft length (SL) 38.7 mm, offset 10°) piercing the gastric wall into the liver (Suppl. material 1: figs S6, S7) and two J hooks (FL 17.8 mm, WD 17.8 mm, GP 15.2 mm, SL 42.6 mm) lodged within the stomach lumen. Mitochondrial genomic structure and base composition The complete mitogenome sequences of two B. brevicaudata specimens (PV699610 and PV699611) were both 17,640 bp long, consistent with known Dasyatinae and Table 1. Measurements and counts data for Bathytoshia brevicaudata from China. Proportional measurements ZJOUMBM F00035 (male) ECSFRI 28263 (female) Absolute measurements (mm) Percentages of disc width (%DW) Absolute measurements (mm) Percentages of disc width (%DW) Total length 1,810 170.75 1,955 150.97 Disc length 868 81.89 1,070 82.63 Head length 428 40.38 525 40.54 Disc width 1,060 100.00 1,295 100.00 Disc depth 145 13.68 176 13.59 Eyeball 26 2.45 29 2.24 Cornea 19 1.79 13 1.00 Interorbital width 160 15.09 180 13.90 Spiracle 61 5.75 63 4.86 lnterspiracular width 158 14.91 210 16.22 Nasal curtain 53 5.00 59 4.56 lnternarial width 122 11.51 150 11.58 Mouth width 100 9.43 114 8.80 1st gill slit 35 3.30 43 3.32 5th gill slit 22 2.08 31 2.39 Width between 1st gill slits 232 21.89 280 21.62 Width between 5th gill slits 160 15.09 190 14.67 Snout tip to eye 195 18.40 270 20.85 Snout tip to nostril 148 13.96 188 14.52 Snout tip to mouth 195 18.40 240 18.53 Snout tip to 1st gill slit 297 28.02 374 28.88 Snout tip to 5th gill slit 423 39.91 502 38.76 Snout tip to pelvic fin 768 72.45 955 73.75 Snout tip to vent 806 76.04 990 76.45 Pectoral fin inner margin 142 13.40 145 11.20 Pelvic fin anterior margin 192 18.11 180 13.90 Pelvic fin posterior margin 68 6.42 85 6.56 Pelvic fin base 122 11.51 99 7.64 Span of pelvic fins 131 12.36 200 15.44 Clasper length 257 24.25 —— —— Tail length 1,077 101.60 910 (broken) 70.27 (broken) Tail base width 116 10.94 98 7.57 Tail base depth 71 6.70 72 5.56 Tail fold length 356 33.58 380 29.34 Counts: Oral papillae 7 5 Tooth rows, upper jaw 31 33 Tooth rows, lower jaw 34 37 Total pectoral radials —— 123 Zoosyst. Evol. 101 (4) 2025, 2191–2205 zse.pensoft.net 2197 Myliobatiform species (Fig. 6, Suppl. material 1: table S1). Each contained 37 mitochondrial genes (13 PCGs, 22 tRNAs, 2 rRNAs) and one control region, with all genes encoded on the H-chain except ND6 and eight tRNAs (trnS2, trnE, trnP, trnQ, trnA, trnN, trnC, trnY) on the L-chain (Fig. 6, Suppl. material 1: table S3). Three (PV699610) and four (PV699611) overlapping regions (1–10 bp) were identified, with the longest overlap (10 bp) at trnK-ATP8 in both mitogenomes. Fifteen gene spacers (1–36 bp) were present in each assembly, and the largest gap (1,896 bp) occurred between the control region and trnF (Suppl. material 1: table S3). The overall base compositions of two B. brevicaudata mitogenomes (PV699610 and PV699611) were biased toward A and T at 59.2% (A = 30.8%, T = 28.4%, G = 13.5%, and C = 27.3%) with a positive A + T skew (PV699610: 0.041; PV699611: 0.040) and a negative G + C skew (PV699610: -0.340; PV699611: -0.339) (Suppl. material 1: table S4). Both mitogenomes exhibited a clear A + T preference, similar to other Dasyatinae fish (Suppl. material 1: table S1). The ATP8 gene had the highest A + T content (PV699610: 63.7%; PV699611: 63.1%), while the first codon position of the PCGs had the lowest (PV699610: 52.2%; PV699611: 52.1%) (Suppl. material 1: table S4). Protein-coding mitochondrial genes and codon usage The combined length of all protein-coding genes (PCGs) was 11,463 bp, with individual gene lengths ranging from 168 bp (ATP8) to 1,845 bp (ND5) (Suppl. material 1: table S4). All PCGs initiated with the canonical start codon ATG. Twelve PCGs possessed complete stop codons, while ND4 terminated with an incomplete T stop codon (Suppl. material 1: table S3). AT-skew and GC-skew values for PCGs were consistently positive and negative, respectively (PV699610: AT-skew = 0.041, GC-skew = -0.340; PV699611: Figure 3. Morphological features of Bathytoshia brevicaudata: A, B. Ventral view of rostral region; C, D. Dorsal view of caudal peduncle base, with a pair of claspers on male; E, F. Lateral view of tail. Male (ZJOUMBM F00035: A, C, E); female (ECSFRI 28263: B, D, F). Scale bars: 2 cm (A, B); 5 cm (C–F), photo by Pan Liu. zse.pensoft.net Chen, J.-J. et al.: First records of Bathytoshia brevicauda (Hutton, 1875) from China2198 Figure 4. Dentition and oral papillae of Bathytoshia brevicaudata. A, C. Male specimen (ZJOUMBM F00035); B, D. Female specimen (ECSFRI 28263), photo by Pan Liu. Figure 5. Stomach contents of Bathytoshia brevicaudata (female, ECSFRI 28263) and associated fishhooks. These contents are deposited at the SHNHM, preserved in 95% ethanol (SNHM-Hfi13442), photo by Pan Liu. Zoosyst. Evol. 101 (4) 2025, 2191–2205 zse.pensoft.net 2199 AT-skew = 0.040, GC-skew = -0.339; Suppl. material 1: table S4), indicating higher genomic abundances of adenine and cytosine relative to their complementary bases. The amino acid usage and RSCU values in the PCGs of B. brevicaudata are summarized in Suppl. material 1: table S5, fig. S8. The mitogenome encoded a total of 3,821 amino acids, among which leucine (17.51%) and cysteine (0.60%) were the most and the least frequently used amino acids, respectively. The six most frequently used codons in B. brevicaudata were CUA(Leu), AUU(Ile), CUC(Leu), AUC(Ile), UUC(Phe), ACA(Thr). Selection pressure analysis To investigate the selective pressure on 13 PGCs of 17 Dasyatinae species, we calculated the non-synonymous substitutions rate (Ka) to the synonymous substitutions rate (Ks) ratio (Ka/Ks). The Ka/Ks ratios of all PCGs were far lower than one (Suppl. material 1: fig. S9). The atp8 gene exhibited the highest ratio (Ka/Ks = 0.1903) of all the PCGs, whereas the COX2 gene had the lowest ratio (Ka/Ks = 0.0302). Transfer RNA and ribosomal RNA genes The mitogenome of B. brevicaudata consisted of 22 tRNA genes individually ranging in size from 68 bp (trnS1) to 75 bp (trnL2) (Suppl. material 1: table S3), representing 8.83% (1557 bp) of the entire mitogenome (Suppl. material 1: table S4). Among the 22 tRNA genes, 14 tRNA genes were encoded on the H strand, while 8 tRNA genes were encoded on the L strand (Suppl. material 1: table S3). All tRNA genes were predicted to fold into the typical cloverleaf secondary structures except that the trnS1 lacked the dihydrouridine (DHU) arm (Suppl. material 1: fig. S10). The 22 tRNA genes exhibited high A+T content in both strains (PV69960: 62.0%; PV69961: 61.7%). Both strains showed positive skew values: PV69960 had an A+T skew of +0.039 and a G+C skew of +0.069; PV69961 had an A+T skew of +0.047 and a G+C skew of +0.057 (Suppl. material 1: table S4). The two rRNA genes, 12S and 16S rRNA genes, were 964 bp and 1,696 bp in length, respectively (Suppl. material 1: table S4). The rRNA genes were located between trnF(gaa) and trnL(uaa), separated from each other by trnV(uac) (Fig. 6, Suppl. material 1: table S3), as observed in other vertebrates. The two rRNA genes exhibited an A+T content of 56.4% and G+C content of 60.2% in PV69960 (G+C: 60.3% in PV69961). The 12S rRNA gene showed consistent positive A+T skew (+0.165) and negative G+C skew (-0.167) in both strains. For the 16S rRNA gene, PV69960 had an A+T skew of +0.162 and G+C skew of -0.108, while PV69961 showed values of +0.160 and -0.107, respectively (Suppl. material 1: table S4). These values indicate a clear bias in favor of the utilization of A and C nucleotides. Genetic divergence among Bathytoshia species The pairwise genetic p-distances within and among the three Bathytoshia species are summarized in Table 2. The analysis of mitochondrial p-distances revealed distinct patterns of genetic divergence within and among the three Bathytoshia species (Table 2). For the COI gene, intraspecific distances were lowest, averaging 0.0021 for B. brevicaudata (Bb), 0.0081 for B. centroura (Bc), and 0.0831 for B. lata (Bl). Interspecific distances were markedly higher, with the Bb-Bc comparison (0.0375) showing a clear separation, while distances involving B. lata were the greatest (Bb-Bl: 0.0797; Bc-Bl: 0.0620). A similar trend was observed for the ND2 gene, where intraspecific distances for Bb and Bc were minimal (0.0037 and 0.0008, respectively). The interspecific Table 2. Genetic p-distances (Mean ± SD) among Bathytoshia species. Comparison COI (n; Mean ± SD) ND2 (n; Mean ± SD) Bb vs Bb 36; 0.0021 ± 0.0018 6; 0.0037 ± 0.0034 Bl vs Bl 225; 0.0831 ± 0.0870 - Bc vs Bc 400; 0.0081 ± 0.0067 36; 0.0008 ± 0.0006 Bb vs Bc 147; 0.0375 ± 0.0026 28; 0.0522 ± 0.0008 Bb vs Bl 112; 0.0797 ± 0.0700 4; 0.0540 ± 0.0006 Bc vs Bl 336; 0.0620 ± 0.0788 7; 0.0059 ± 0.0007 Note: Species abbreviations: Bb, Bathytoshia brevicaudata; Bc, Bathytoshia centroura; Bl, Bathytoshia lata. Figure 6. The complete mitogenome of Bathytoshia brevicaudata. The middle and innermost circles represent depth distribution and GC content, respectively. The outermost circle shows gene arrangements, with green for PCGs fragments, orange for rRNAs and red for tRNAs.