A new species of hagfish, Eptatretus wandoensis sp. nov. (Agnatha, Myxinidae), from the southwestern Sea of Korea.

Abstract Four specimens of the five-gilled white mid-dorsal line hagfish, Eptatretus wandoensissp. nov. were recently collected from the southwestern Sea of Korea (Wando). This new species has five pairs of gill apertures, 14–18 prebranchial slime pores, 4 branchial slime pores, a dark brown back with a white mid-dorsal line and a white belly. These hagfish are similar to Eptatretus burgeri and Eptatretus minor in having a white mid-dorsal line, but can be readily distinguished by the numbers of gill apertures (5 vs. 6–7), gill pouches (5 vs. 6), and prebranchial slime pores (14–18 vs. > 18), as well as the body color (dark brown back vs. gray or brown pale). In terms of genetic differences, Eptatretus wandoensis could be clearly distinguished from E. burgeri (0.9% in 16S rRNA and 8.5% in cytochrome c oxidase subunit I sequences) and E. minor (4.5% and 13.9%).


Introduction
Myxinidae (hagfishes) are currently classified into six genera and 81 species worldwide (Fernholm et al. 2013; Froese and Pauly 2019). They are characterized by an eel-like body shape and 1-16 pairs of gill apertures and gill pouches; however, they have no jaws, eyes, or fins (Fernholm 1998). Recent research using morphological and molecular characteristics revealed that hagfishes comprise three subfamilies: Eptatretinae, Myxininae, and Rubicundinae (Fernholm et al. 2013). There have been several unresolved issues regarding the number of recognized genera in the subfamily Eptatretinae; however, its genera were recently reorganized taxonomically based on morphological and molecular data (Fernholm et al. 2013;Song 2019). Therefore, Eptatretinae currently includes a single genus, Eptatretus, which is characterized by the presence of more than two pairs of gill apertures; notably, Eptatretus is the most species-rich myxinid genus, currently comprising 51 valid species in the northwestern Pacific Ocean (e.g., Korea, Taiwan, and Japan) and coastal waters around Asia (e.g., China, Philippines, and Vietnam) (Froese and Pauly 2019). Surveys of the deep sea and other hard-to-reach areas using special-purpose submarines are increasingly revealing new or cryptic species worldwide (Fernholm and Quattrini 2008;Mincarone and Fernholm 2010;Zintzen et al. 2015). Based on examinations of both morphological and genetic characteristics of hagfish specimens from the southwestern Sea of Korea, we herein describe a new species, Eptatretus wandoensis sp. nov., and compare it with other members of the Eptatretus genus in around northeastern Asia.

Materials and methods
We obtained four specimens (202.0-292.0 mm total length) from Yeoseo-ri, Wandogun in Korean waters in 2018, caught by fishing trapping and bought to the fish markets (Fig. 1). The specimens have been deposited in the Marine Fish Resource Bank of Korea (MFRBK) at Pukyong National University (PKU), Busan-si, Korea. We performed morphological and molecular analyses to clarify their taxonomic status, the former based on a total of 11 counts and 13 measurements. Morphological methods and terminology followed Fernholm and Hubbs (1981) and Wisner and McMillan (1988). Each body part was measured to the nearest 0.1 mm using digital Vernier calipers, and the data were converted to percentages of the total length (TL). We counted the numbers of anterior (outer) unicusps (AUC), posterior (inner) unicusps (PUC), multicusps (= fused cusps), and total cusps according to Fernholm (1998), using a stereomicroscope (SZX-16; Olympus, Tokyo, Japan). Images were analyzed using an image analyzer (Shinhan Active Measure; Shinhan Scientific Optics, Seoul, Korea), and features were sketched using a camera lucida (SZX-DA; Olympus). We examined the anatomical characters such as the arrangement between gill pouch (GP) and efferent branchial duct (EBD). The terminology of anatomical structures followed Mok and McMillan (2004): afferent branchial arteries (ABA), efferent branchial artery (EBA), ventral aorta (VA), medial section of ventral artery (MVA), and side branchial artery (SBA).We examined (and added to) the morphological description of nasal-sinus papillae following Mok (2001) and Zintzen et al. (2015).
Coloration when fresh: Body uniformly dark brown or purplish dorsally and white ventrally; white mid-dorsal line conspicuous, beginning from the upper region of the first prebranchial slime pore to around the tail. Eyespots conspicuous; whole barbels (rarely the tip) pale, and pale around mouth. Each gill aperture and pharyngocutaneous duct aperture with white margin; most slime pores blackish (except for tail region), tail slime pores same as surrounding color. White around cloaca; ventral fin-fold with a white line along the ventral midline; posterior margin of caudal fin pale (Fig. 2). Coloration when preserved: Body brown to dark brown dorsally and murky white ventrally (more conspicuous than fresh specimen). Eyespots conspicuous; all slime pores surrounded by conspicuous white ring. Each gill aperture and pharyngocutaneous duct aperture conspicuous; ventral fin-fold pale; white mid-dorsal line inconspicuous.
Distribution. Southwestern Sea of Korea. Biology. Attains a maximum TL of 292.0 mm (fresh specimen); this specimen is female, without mature eggs in the body cavity. A female specimen of 290.0 mm TL carries approximately 20 developing eggs, which have no terminal anchor filaments or hooks; each egg approximately 4-7 mm in diameter and 10-12 mm in length.
Etymology. The specific name, wandoensis, refers to the type locality, in Korea.

Genetic comparisons
Differences among mtDNA sequences obtained from the holotype and paratypes of Eptatretus wandoensis sp. nov. were consistent with species-level divergences in other hagfish species (Fernholm et al. 2013). The phylogenetic relationships of myxinid species, inferred from neighbor-joining trees, showed large genetic distances between similar hagfish species using mtDNA 16S rRNA (477 bp) and cytochrome c oxidase subunit I (COI) (466 bp) sequences. Eptatretus wandoensis sp. nov. is separated from other congeneric species by high genetic divergences of 0.9%-7.5% in 16S rRNA sequences and 4.9%-13.9% in COI sequences (Fig. 5). The respective genetic distances between this species and E. burgeri and E. minor were 0.9% and 4.5% in 16S rRNA sequences and 8.5% and 13.9% in COI sequences. In addition, phylogenetic analysis of 16S rRNA sequences showed that E. wandoensis sp. nov. is well separated from other five-gilled hagfishes (E. cheni, E. nelsoni, and E. yangi), with genetic differences of 7.5%, 1.4%, and 1.6%, respectively. Eptatretus cheni is located at a basal position of hagfishes and well nested in the Eptatretus clade.

Discussion
Eptatretus wandoensis sp. nov. is one of many new hagfish species recently discovered in the northwest Pacific Ocean. Thus far, six hagfish species with five gill apertures have been reported worldwide (McMillan and Wisner 2004;Kuo et al. 2010;Zintzen et al. 2015); most are included in the genus Eptatretus (Fernholm et al. 2013). However, three species have tubular nostrils and pink coloration; thus, they are regarded as Rubicundus species (Fernholm et al. 2013;Zintzen et al. 2015). This new species is the third member of the genus with a white mid-dorsal line, after Eptatretus burgeri and E. minor (Girard 1855;Fernholm and Hubbs 1981). This new species was initially confused with E. burgeri because it may have been considered a morphological variation of E. burgeri, due to the presence of five gill apertures. However, they are well distinguished by the body color, prebranchial slime pores, total slime pores, and ventral fin-fold. In addition, we found a female specimen with ripe eggs on June 26, 2018. Recent study revealed that the minimum mature size Eptatretus burgeri with ripe eggs is more than 500.0 mm TL (Song 2019); however, this female specimen was 290.0 mm TL. Recently, specific anatomical structures such as cusps, nasal-sinus papillae, and heart have been regarded as useful characters for clarifying interrelationship among hagfish (Mok 2001;Icardo et al. 2016a;Icardo et al. 2016b). Indeed, Mok (2001) suggested that the absence of nasal-sinus papillae may be an apomorphic character of most eptatretines. Interestingly, all three Eptatretus species have no nasal-sinus papillae (Song and Kim 2020), and so therefore well supports the hypothesis of Mok (2001). Phylogenetic trees indicated that the new species is sister- group to E. walkeri (supported by CO1 gene), but E. walkeri becomes the sister-group of E. burgeri (supported by 16S rRNA gene). Naylor and Brown (1998) mentioned that genes yielding correct results might vary among data sets and thus this discordance might be influenced by stochastic error associated with a different number of species and data sets. Kawaguchi et al (2001) suggested that taxonomic sampling and comprehensive sequencing may clarify intra-and interrelationships of fish using mitochondrial data. In terms of geographic distribution, Eptatretus minor occurs in the Gulf of Mexico and Atlantic Ocean, while E. burgeri coexists with this new species in the same region of coastal Korea. In the comparison of depths, Eptatretus wandoensis sp. nov. is collected from depths between 60 to 80 m, and E. burgeri is known as between 5 and 270 m, and E. minor is known as between 300 and 400 m (Fernholm and Hubbs 1981;Moller and Jones 2007;Knapp et al. 2011;Angulo and Moral-Flores 2016). Among them, Eptatretus minor is deeper than the other two species. Interestingly, most Korean hagfishes tend to be distributed in quite shallow waters (within 100 m water depth) (Song 2019;Song and Kim 2020). In a recent morphological and molecular taxonomic review of Eptatretus atami from the coast of Japan, the specimens with 3/2 multicusps from the western coast of Honshu were identified as E. walkeri, whereas eastern specimens with 3/3 multicusps matched E. atami (Kase et al. 2017;Kitano et al. 2019). Later, Song and Kim (2020) revealed for the first time the existence of E. walkeri previously misidentified as E. atami in Korea, and confirmed that three species are currently distributed in Korea.