Two new species of the genus Ophichthus from the western central Pacific Ocean, with a redescription of Ophichthus megalops Asano, 1987 (Anguilliformes: Ophichthidae)

Two new species similar to Ophichthus megalops Asano, 1987 with dark-tipped anal fins, are described on the basis of one specimen of each species. Ophichthus semilunatus sp. nov. from northeastern Taiwan is characterized by having 176 total vertebrae, three rows of teeth on the maxilla, one + three supraorbital pores, two preopercular pores, a brownish anterior-nostril tube, and a blotch on the anterior margin of anus. Ophichthus brevidorsalis sp. nov. from New Caledonia is characterized by having two preopercular pores, one + three supraorbital pores, smaller eyes 2.7 in head, a short head 9.5% of total length, a long tail 59.8% of total length, a slightly short snout 19.4% of head, and 43 predorsal vertebrae. A redescription of O. megalops is provided based on the holotype and 18 specimens newly collected from Taiwan. Selected characters of all nine Ophichthus with a dark-tipped anal fin are provided. In addition, partial COI sequences of five species is provided.


Introduction
The genus Ophichthus Ahl, 1789, the most diverse group within the family Ophichthidae, comprises more than 80 species that are known from marine waters, a part of that are deeper than 200 m (Hibino et al. 2019). Most of the deepwater species have been poorly collected and are very rare, therefore, their morphological variation and/or ontogenetic change is insufficiently known.
Ophichthus megalops Asano, 1987, the third described species having a dark tip on the anal fin, was originally known from a single specimen collected deepwater off Owase, the Pacific coast of Japan. Since Asano's description (1987), no additional specimens have been collected from Japan. Ho et al. (2010) revealed O. megalops from Taiwan but subsequently Ho et al. (2015) noted that the specimen is not O. megalops. They listed two registered specimens as the true record of the species from Taiwan with catalog numbers. During YH and YCC's visit in NMMBA, all specimens identified as O. megalops with dark-tipped anal fin were examined in detail. We recognized two species in the collection, including O. megalops and an undescribed species. Additionally, another undescribed Ophichthus was found in the ASIZ collection; it was collected from New Caledonia. Here, we provide description of the new species, and redescribe O. megalops with information about its intraspecific variation.

Materials and methods
All counts and measurements follow McCosker (2010). Measurements for total and tail lengths are taken with a 600 mm or 1000 mm ruler and others by digital caliper to the nearest 0.1 mm. Vertebral counts were made from soft X-ray photos. Mean vertebral formula (MVF) is expressed as the average of predorsal, preanal and total vertebrae (Böhlke 1982). All terminology including head structures, i.e. sensory pores and some protrusion belong upper lip, follow Hibino et al. (2019). Total and head lengths are abbreviated as TL and HL, respectively. Institutional codes for materials follow Fricke & Eschmeyer (2018).
Cytochrome c oxidase subunit I (COI) barcoding method follow Chang et al. (2016). PCR amplifications of the 5' region of the COI gene (approximately 650 bp) were performed, and all the successfully amplified sequences were aligned (Clustal W), trimmed, constructed and saved as FASTA format by using BioEdit ver. 7.2.5 (Hall 1999), then constructed a Neighbor-joining tree with 10,000 bootstrap-replicated K2P distance by using MEGA ver. 10.0.5 (Kumar et al. 2018). Twelve sequences from five species were used as the ingroup, and one of Myrophis punctatus (BOLD: AAB7198) was used as the outgroup. The tissue samples of O. brachynotopterus, O. tomioi, and O. mystacinus were provided and extracted by National Taiwan University, seven tissues of O. megalops were provided by the National Taiwan Ocean University, and the sequence of O. exourus was provided by Kyushu University. All of the accession numbers are listed in Table 3.  1A).

Ophichthus megalops
Head relatively large, branchial basket moderately expanded; head 8.7-9.9 in TL; head and trunk 2.2-2.3 in TL; snout short, weakly acute, its length generally slightly longer than eye diameter; no ventral groove on snout; lower jaw included in upper jaw, its tip slightly beyond anterior base of anterior-nostril tube; mouth large, rictus well behind a vertical from posterior margin of eye, with a short, additional fold below rictus; eye large, 2.1-2.2 in upper jaw and 4.4-7.1 in head; anterior nostril a simple tube with a shallow notch on rim anteriorly; posterior nostril a hole opening outside mouth, above upper lip, slit-like with a short slim flap anteriorly; upper lip smooth, without protrusions, numerous small papillae on inner upper and lower lips; interorbital region smooth and mostly flat; gill opening constricted, located ventrolaterally ( Fig. 2A).
Dorsal and anal fins relatively low, ending with a ridge almost equal to half of snout length; dorsal-fin origin behind pectoral-fin tip, 2.5-4.3 times pectoral-fin length (2.5 in holotype), behind level of gill opening and 1.2-1.5 times in HL (1.2 in holotype); caudal fin absent; pectoral fin short, pedal-shaped, its tip pointed but not filamentous.  Head pores small but obvious; arrangement of sensory pores on head as follows ( Fig. 2A): one + four on supraorbital, one pore above the posterior rim of posterior nostril; three + three on infraorbital, one between anterior and posterior nostrils; six on mandible and three on preopercle; midtemporal and interorbital pores present. Lateral-line pores almost complete, 8-10 (10 in holotype) in branchial basket, 28-35 (32) anterior to dorsal-fin origin, 59-64 (61) anterior to anus, and 137-146 (141) in total, canal and pores absent in 0.6-0.9 times (0.7) of HL.
Coloration when fresh ( Fig. 1): head and body bluish to deep brownish dorsally and whitish on belly; peritoneum silver gray, with numerous minute, black dots along myomeres (Fig. 1B); anus generally without surrounding, darker marking or rarely less faded melanophores (only KAUM-I. 125145); tube of anterior nostril whitish; sensory pores not marginated. Dorsal fin white without darker margin; anal fin pale except distinct black end about 1/2 HL from tail tip (Fig. 1C); pectoral fin white. After preservation in 10% formalin and transferring in 75% ethanol, body coloration becomes monotonous.
Description. Counts and measurements (in mm) of the holotype: predorsal vertebrae 29; preanal vertebrae 64; total vertebrae 176; preanal lateral-line pores 65. Total length 500; head length 46.8; preanal length 212.0; tail length 287.5; predorsal length 103.5; body depth at gill opening 16.4; body depth at mid-anus 13.0; body width at gill opening 13.3; body width at mid-anus 13.5; snout length 8.9; eye diameter 8.3; upper-jaw length 19.7; gill opening length 4.3; interorbital width 6.1; isthmus width 10.0; pectoral-fin length 14.4; pectoral-fin base 4.5. Body long, subcylindrical, its depth at gill openings 30.5 in TL; tail slightly compressed posteriorly, its depth reduced gradually, its tip elongate (Fig. 3).  Head relatively large, branchial basket moderately expanded; head 10.4 in TL; head and trunk 2.4 in TL; snout short, not acute, its length almost equal to eye diameter; no ventral groove on snout; lower jaw included in upper jaw, its tip slightly beyond anterior base of anterior-nostril tube; mouth large, rictus well behind a vertical from posterior margin of eye, with a short, additional fold below rictus; eye large, 2.4 in upper jaw and 5.6 in head; anterior nostril tubular, a simple tube with a shallow notch on rim anteriorly; posterior nostril a hole opening outside of mouth, above upper lip, the hole with an extremely tiny flap anteriorly; upper lip smooth, without protrusions, numerous small papillae on inner upper and lower lips; interorbital region smooth and mostly flat; gill opening constricted, located ventrolaterally (Fig. 4A).
Dorsal and anal fins relatively low, ending with a ridge equal to half of snout length; dorsal-fin origin behind pectoral-fin tip by 2.9 times pectoral-fin length, distance between level of gill opening to origin of dorsal fin 1.2 times in HL; caudal fin absent; pectoral fin short, its tip pointed, weakly filamentous.
Head pores small but obvious; arrangement of sensory pores on head as follows (Fig. 4A): one + three on supraorbital, no pore above the posterior rim of posterior nostril; three + three on infraorbital, one between anterior and posterior nostrils; six (seven right side) on mandible and two on preopercle; midtemporal and interorbital pores present. Lateral-line pores almost complete, 8 in branchial basket, 30 anterior to dorsal-fin origin, 65 anterior to anus, and 158 in total, canal and pores end about 0.9 HL before tail tip.
Coloration in preservative (50% isopropanol) of head and body bicolored, brown dorsolaterally and white ventrally, the border from rictus through insertion of pectoral fin, falling gradually toward end of anal fin; a dark halfmoon-shaped blotch along anterior margin of anus (Fig. 5A); tube of anterior nostril brownish but slightly paler than body; tip of lower jaw dusky; sensory pores not marginated. Dorsal fin extremely pale brown with melanophores but not marginated; anal fin pale white, except for a distinct black area around anal fin and base of the fin, its length about half of HL; pectoral fin white with a pale brown margin.
Distribution. Known only from the holotype collected from the northwestern Pacific Ocean, off northeastern Taiwan. Collecting depth unknown but estimated from deepwater.
Etymology. From the Latin, semi-(half) and lunatus (moon), relative to the half-moon blotch on the anterior margin of anus.  Tashiro et al. 2017). The new species closely resembles O. megalops in the position of the dorsal-fin origin, counts of predorsal and preanal vertebrae, the shape of the posterior nostril, the shape of the pectoral fin and coloration of the anal fin with a black tip, resulting in the holotype of O. semilunatus sp. nov. having been misidentified as O. megalops. However, the new species can be distinguished from O. megalops by having more total vertebrae (176 vs. 157-168), three rows on the maxilla (vs. two rows, rarely one more row restricted to the posterior end), one + three supraorbital pores (vs. one + four), two preopercular pores (vs. three), the brownish tube of the anterior nostril (vs. mostly pale white), and the presence of a half-moon blotch on the anterior margin of the anus [vs. generally absent, or rarely, faded melanophores are present (Fig. 5B)].
Description. Counts and measurements (in mm) of the holotype: predorsal vertebrae 43; preanal vertebrae 61; total vertebrae 164; lateral-line pores before gill opening 7; predorsal lateral-line pores 48; preanal lateral-line pores 65; total lateral-line pores c.a. 148. Total length 398; head length 38; preanal length 160; tail length 238; predorsal length 121; body depth at gill opening 17.3; body depth at mid-anus 9.8; body width at gill opening 13.5; body width at mid-anus 10.7; snout length 7.4; eye diameter 6.4; upper-jaw length 17.1; gill opening length 8.1; interorbital width 5.3; isthmus width 10.5; pectoral-fin length 13.4; pectoral-fin base 4.2. Body moderately elongate, subcylindrical, its depth at gill openings 23.1 in TL; tail slightly compressed posteriorly (Fig. 6). Head relatively small, branchial basket expanded; head 10.5 in TL; head and trunk 2.5 in TL; snout short, not acute and its tip pointed, the length somewhat longer than eye diameter; lower jaw shorter than upper jaw, its tip slightly beyond anterior base of anterior nostril; mouth not large, rictus right behind a vertical from posterior margin of eye; eye located from the range within the last 1/3 of upper jaw; eye large, 2.7 in upper jaw and 5.9 in head; tubular anterior nostril with a shallow notch on rim anteriorly; posterior nostril a hole with a tiny flap anteriorly above upper lip; both of lips smooth, but many protrusions in mouth; gill opening located laterally (Fig. 7A).
Median fins low; tail become bare from the tip about half of snout length; dorsal-fin origin behind pectoral-fin tip by 5.3 times pectoral-fin length, distance between level of gill opening to origin of dorsal fin 2.2 times in HL; pectoral fin short with a filamentous end. Head pores minute but visible; arrangement of cephalic pores as follows (Fig. 7A): one + three on supraorbital, no pore above the posterior rim of posterior nostril; three + three on infraorbital; six on mandible and two on preopercle; midtemporal and interorbital pores present. Lateral-line pores almost complete; 7 in branchial basket, 48 anterior to dorsal-fin origin, 65 anterior to anus, and c.a. 148 in total.
Coloration in preservative (75% ethanol): of body bicolored, brown dorsally and pale ventrally, the border from rictus and across through the base of pectoral fin, than falling gradually to the tail tip; anterior nostril pale; upper lip and tip of lower jaw dusky. Dorsal fin pale; anal fin whitish, its end presents a dark region, its length about a half of head; pectoral fin white without margin. The iris coloration has a dark background with numerous silver spots on it, which resembles stars in the sky.
Distribution. Known only from the holotype, from New Caledonia.   (1) to (12) are 12 specimens of five Ophichthus congeners with dark-tipped anal fin, and (13) was taken as an outgroup, respectively. (1) (3) (8) Etymology. From the Latin, brevidorsalis, meaning short dorsal fin, which refers to the diagnostic character of its dorsal-fin origin.
Remarks. The new species has the character of a posteriorly blackened of its anal fin. The position of the dorsal-fin origin of the new species is also characteristic, and can easily distinguish it from those species with their dorsal fin arising above or behind the pectoral fins, such as Ophichthus aniptocheilos, O. kunaloa, and O. tomioi. The new species is similar to O. megalops in the position of the dorsal-fin origin, and counts of preanal and total vertebrae, but the former can be separated from the latter by its number of preopercular pores (two vs. three in O. megalops) and supraorbital pores (one + three vs. one + four), smaller eyes (2.7 vs. 2.1-2.2 in head), shorter head (9.5% of total length vs. 10.1-11.5%), longer tail (59.8% of total length vs. 54.5-57.4%), slightly shorter snout (19.4% of head vs. 19.6-22.9%), and having more predorsal vertebrae (43 vs. 28-35). The new species shares the same composition of supraorbital pores with O. semilunatus sp. nov. (one + three), as compared to other black-analfin species (one + four). The new species is also similar to O. brachynotopterus by the shape of its snout, condition of its posterior nostrils, and the position of its dorsal-fin origin, but differs in the shape of its pectoral fin (filamentous in O. brevidorsalis sp. nov. vs. not elongated in O. brachynotopterus). The new species has the highest number of predorsal vertebrae among those dark anal-fin species (43 in O. brevidorsalis sp. nov.) ( Table 2).
In Table 1 . aniptocheilos), however, snout shape, pectoral-fin shape, and the condition of the posterior nostril can separate all similar species. A NJ tree constructed by partial COI gene sequences (552 bp after processed by BioEdit software) of five species with dark-tipped anal fins ( Table 3) that supported the separation of these species (Fig. 8). Besides, in Table 4, K2P distance matrix reveals that the distance lower than 0.01 among the same species, distance ranging from 0.105 to 0.160 among the 12 dark-tipped anal fin Ophichthus congeners, and the outgroup shows the distance ranging from 0.173 to 0.194. The COI sequences are currently not available for the two new species described herein.  Table 2 are collected from deepwater. In addition, most of blackened species belonging to other genera inhabit deepwater more than about 200 m.
Other material examined. Benthos program and its joint research project entitled 'Taiwan France marine diversity exploration and evolution of deep-sea fauna (TFDeepEvo)' supported by the French ANR and Taiwanese MOST (Principal investigators: S. Samadi and W.-J. Chen). This study is also supported partly by the National Museum of Marine Biology & Aquarium, the Aquatic Biology Research Fund of the California Academy of Sciences, Grant-in-Aid from the Japan Society for the Promotion of Science for JSPS Fellow to Y.H. (DC2/PD: 15J02820), grants from the program titled "Water quality, plankton ecology, database construction, and ecosystem modeling in the Yun-Zhang water: the abundance and assemblage of fish eggs and larva" from the Ministry of Science and Technology, Taiwan (MOST106-2119-M-005-004) to C.H.-M. We appreciate the assistance of colleagues from Laboratory of Aquatic Ecology (Department of Aquaculture, National Taiwan Ocean University, Taiwan). Finally, we are grateful to John E. McCosker for his critical reading.