A new species of snailfish of the genus Paraliparis (Liparidae) from the western North Pacific, with a redescription of the poorly known species Paraliparis mandibularis

Abstract A new snailfish, Paraliparis flammeus, is described on the basis of 18 specimens collected off the Pacific coast of Tohoku District, northern Japan at depths of 422–890 m. The new species is distinguished from 28 species of Paraliparis described from the North Pacific by the following combination of characters: mouth oblique; uppermost pectoral-fin base below horizontal through posterior margin of maxillary; 60–63 vertebrae, 54–58 dorsal-fin rays, 50 or 51 anal-fin rays, six principal caudal-fin rays, and 17–20 pectoral-fin rays. A maximum likelihood tree based on 106 COI gene sequences (492 bp) of Paraliparis recovered a monophyletic group comprising P. flammeus, Paraliparis cephalus, and Paraliparis dipterus. Paraliparis cephalus is similar to P. flammeus in having an oblique mouth, but it has four caudal-fin rays (vs six rays) and the uppermost pectoral-fin base above a horizontal through the maxillary posterior margin. Paraliparis dipterus differs from P. flammeus in having a horizontal mouth, 12–14 pectoral-fin rays, and lacking pyloric caeca (present in P. flammeus). Paraliparis flammeus is most similar to the eastern North Pacific Paraliparis mento in having an oblique mouth and the uppermost pectoral-fin base below a horizontal through the posterior margin of the maxillary. However, P. flammeus differs from P. mento in having six caudal-fin rays (vs five rays) and greater preanal length (29.9–35.3% SL vs 26.7–28.5% SL). A poorly known species, Paraliparis mandibularis, previously known from only two specimens collected from Tosa Bay, southern Japan, is redescribed based on the holotype and seven newly collected specimens. It is also similar to the new species but has 27–30 pectoral-fin rays and a shorter pectoral-fin lower lobe (13.8–15.9% SL in P. mandibularis vs 16.7–23.4% SL in P. flammeus).

A poorly known species, Paraliparis mandibularis, previously known from only two specimens collected from Tosa Bay, southern Japan, is redescribed based on the holotype and seven newly collected specimens. It is also similar to the new species but has 27-30 pectoral-fin rays and a shorter pectoral-fin lower lobe (13.8-15.9% SL in P. mandibularis vs 16.7-23.4% SL in P. flammeus).

Keywords
Japan, Paraliparis cephalus, Paraliparis flammeus sp. nov., Paraliparis mento, taxonomy, Tohoku introduction Members of the family Liparidae (snailfishes), comprising over 430 species in ca 30 genera, exhibit great diversity in morphology, as well as in geographic and habitat range (Chernova et al. 2004;Nelson et al. 2016;Orr et al. 2019), and they occur worldwide in warm-temperate to cold water habitats ranging from the intertidal to depths exceeding 8,000 m (Nelson et al. 2016;Gerringer et al. 2017). Paraliparis Collett, 1879 and related genera have been variously synonymized in previous studies. Although Kido (1988) combined 11 genera under Paraliparis following a phylogenetic analysis based on osteological characters, later authors, e.g., Mecklenburg et al. (2002), Chernova et al. (2004), and Nakabo and Kai (2013), considered six of the included genera, e.g., Elassodiscus Gilbert & Burke, 1912, Rhinoliparis Gilbert, 1896, and Lipariscus Gilbert, 1915, as valid. In a recent molecular phylogenetic study, Orr et al. (2019) confirmed that Paraliparis was paraphyletic, requiring further taxonomic revision. Nevertheless, present members of the genus are generally distinguishable by the following characters: single nostril; one suprabranchial pore, six branchiostegal rays, more than two rays in the lower lobe of the pectoral fin, and the absence of a pelvic disk, a pseudobranch, a coronal pore and a barbel or a skin flap on the head (Stein et al. 2001;Stein 2012;Murasaki et al. 2018). In its present concept, Paraliparis is one of the most speciose genera of the family with ca 140 species, mostly known from depths greater than 200 m (Chernova et al. 2004;Murasaki et al. 2019a, b).
Along the Pacific coast of Tohoku District, northern Honshu Island, Japan, continuous surveys for resource assessments of ground fishes by the Tohoku National Fisheries Research Institute, Japan Fisheries Research and Education Agency, have resulted in the discovery of several new species (Shinohara et al. 2009), including the recent collection of 18 specimens of a previously unknown snailfish of the genus Paraliparis. Most closely resembling Paraliparis mento Gilbert, 1892 and Paraliparis mandibularis Kido, 1985, the specimens have an oblique mouth and a pectoral fin below a horizontal through the posterior maxillary margin but are clearly distinguishable from the latter two species in other morphological characters, as well as DNA barcoding sequence data. They are accordingly described herein as members of a new species. Paraliparis mandibularis, a rare species previously known only from the holotype and one non-type specimen (Kido 1988), is redescribed here in detail on the basis of the holotype and seven newly collected specimens from southern Japan.

Materials and methods
Methods for counts and measurements follow Baldwin and Orr (2010), with the descriptive terminology of Stein et al. (2001). Counts of median-fin rays and vertebrae were taken from radiographs. Cephalic pores were observed by staining with Aniline Blue (Wako Chemicals). Selected specimens were cleared and double stained (C&S) for bone and cartilage examination following the protocol of Kawamura and Hosoya (1991), and using an incident-light fluorescence unit (OptoCode; LED 470 MS-EPI) and stereomicroscope. Osteological characters of the holotype were examined by an industrial x-ray and a computed tomography scanning system (Nikon Corporation), with data visualized by VGStudio Max 3.1 (Volume Garaphics GmbH). The specimens examined in this study are deposited in the fish collections of the Faculty of Science and Technology, Kochi University, Japan (BSKU); Kyoto University, Kyoto and Maizuru, Japan (FAKU); the Marine Science Museum, Tokai University, Shizuoka, Japan (MSM); the Smithsonian Institution, National Museum of Natural History, Suitland, USA (USNM); and the Burke Museum, University of Washington, Seattle, USA (UW).
For DNA barcoding, we attempted to include all the available sequences of congeneric or related species (see Orr et al. 2019) for the robust phylogenetic inference. Total DNA of the present new species and the following species, Paraliparis atramentatus Gilbert & Burke, 1912, Paraliparis cephalus Gilbert, 1892, Paraliparis dipterus Kido, 1988, Paraliparis hokuto Murasaki, Takami & Fukui, 2019a, Paraliparis mento, Paraliparis ruficometes Murasaki, Takami & Fukui, 2018, Paraliparis variabilidens Murasaki, Takami & Fukui, 2019b, Rhinoliparis barbulifer Gilbert, 1896, was extracted from fin clips preserved in 99.5% ethanol, using the Wizard Genomic DNA Purification Kit (Promega Inc.). The partial Cytochrome Oxidase subunit I (COI) gene was amplified using the primers designed by Folmer et al. (1994) (LCO1490: 5′-GGT CAA CAA ATC ATA AAG ATA TTG G -3′; HCO2198: 5′-TAA ACT TCA GGG TGA CCA AAA AAT CA -3′). The PCR proceeded for 30 cycles, with denaturation at 94 °C for 15 sec, annealing at 45 °C for 15 sec, and extension at 72 °C for 30 sec, using the KAPA2G Robust PCR Kit (KAPA Biosystems). After the PCR products were purified using ExoSAP-IT Express (ThermoFisher Scientific), they were sequenced on an automated DNA sequencer (ABI Prism 310 Genetic Analyzer; ThermoFisher Scientific) using amplification primers and the BigDye Terminator v. 1.1 Cycle Sequencing Kit (ThermoFisher Scientific). All sequences determined here are available from INSDC (International Nucleotide Sequence Database Collaboration) under accession numbers LC556300-LC556314. Together with previously determined sequences of species of Paraliparis and the "Paracareprocta" clade of Orr et al. (2019), as well as an outgroup taxon (Nectoliparis pelagicus Gilbert & Burke, 1912: see Orr et al. 2019 available from INSDC and BOLD (Barcode of Life Data System), the present sequences were aligned using MAFFT v. 7 (Katoh and Standley 2013). From the aligned sequences, the uncorrected p-distance among specimens was calculated with MEGA X (Kumar et al. 2018). In addition, in order to reconstruct a maximum likelihood (ML) tree, the best evolutionary model was found by MEGA X, the Tamura and Nei (1993) model with gamma shape parameter and invariant sites being selected. Branch support was measured using nonparametric bootstrapping with 1,000 replications, based on the same algorithm (Felsenstein 1985 Diagnosis. Paraliparis flammeus is distinguished from other species of Paraliparis by the following combination of characters: mouth oblique; uppermost pectoralfin base below a horizontal through posterior margin of maxillary; 60-63 vertebrae, 54-58 dorsal-fin rays, 50 or 51 anal-fin rays, 6 principal caudal-fin rays, and 17-20 pectoral-fin rays; pectoral radials 4, moderately large and located medially.
Description. Measurements are shown in Table 1. Paratype data are given in parentheses if different from the holotype.
Pectoral fin moderately notched, with 19 (17-20) rays; upper lobe with 14 (12-15) rays, extending beyond (or just reaching) anal-fin origin; lower lobe elongate, with 5 (3-7) rays, uppermost ray of lower lobe longest, extending beyond anus, not reaching (reaching) anal-fin origin. Uppermost pectoral-fin base below a horizontal through posterior margin of maxillary. Lowermost pectoral-fin base below anterior rim of orbit (or below midway between tip of snout and anterior rim of orbit). Rays between upper and lower lobes widely spaced.
Selected osteological characters. Roof of cranium comprising frontal and supraoccipital incompletely closed; frontal and supraoccipital poorly ossified; parietal absent ( Fig. 2). Opercle well ossified, sharpened posteriorly, supporting upper margin of opercular flap. Subopercle thin, comprising two spines forming a V-shape; lower spine supporting lower margin of opercular flap. Subopercle and interopercle separated. Dorsal portion of cleithrum elongated. Proximal pectoral radials 4, rounded, moderately large and located medially (Fig. 3C). No interradial fenestrae between proximal radials. Scapula with strong helve, posterior margin with a small slit. Coracoid triangular with broad lamina. Distal radials present at base of all pectoral-fin rays, except for uppermost and lowermost rays. Coloration. In fresh specimens, head and body pale pink with fine melanophores; margin of preopercle silvery; anteroventral portion silvery, with dark peritoneum visible through thin skin; dorsal and anal fins crimson, distal margins somewhat darker; pectoral fin crimson (Fig. 1A). In preserved specimens, head, body, and fins pale with fine melanophores, somewhat larger posteriorly on body; peritoneum black, stomach dark brown (or black); orobranchial cavity pale with scattered melanophores.
Etymology. The specific epithet flammeus is from Latin, meaning "flame", and refers to the crimson fin coloration of the species.
Geographical distribution. Western Pacific Ocean, off the Pacific coast of Tohoku District, northern Honshu, Japan, in depths of 422-890 m (Fig. 4A).
Remarks. Alignment of the COI gene sequences (492 bp) determined herein with previously determined sequences of Paraliparis resulted in a maximum likelihood tree based on 101 aligned sequences and the recovery of a monophyletic group comprising P. flammeus, P. cephalus, and P. dipterus (Fig. 5). Monophyly of the above species was supported by high bootstrap probability (95%). The uncorrected p-distance within P. flammeus was less than 0.006, strongly contrasting with values ranging from 0.030 to 0.067 for the above two species. Paraliparis cephalus is similar to P. flammeus in having an oblique mouth, but has 4 caudal-fin rays (vs 6 rays) and the uppermost pectoral-fin base above a horizontal through the maxillary posterior margin (Stein 1978; this study). Paraliparis dipterus, known only from the holotype collected from Suruga Bay, Japan,   until the recent description of a developmental series by Takami and Fukui (2012), differs from P. flammeus in having a horizontal mouth, 12-14 pectoral-fin rays, and lacking pyloric caeca (present in P. flammeus) (Kido 1988;Takami and Fukui 2012;this study). The position of P. mandibularis is unknown due to the unavailability of sequence data.

Diagnosis.
Paraliparis mandibularis is distinguished from other species of Paraliparis by the following combination of characters: mouth oblique; uppermost pectoral-fin base below a horizontal through posterior margin of maxillary; 63-66 vertebrae, 58-61 dorsal-fin rays, 52-54 anal-fin rays, 6 principal caudal-fin rays, and 27-30 pectoral-fin rays. Proximal pectoral radials 4, enlarged and moved to anterior edge of basal lamina. Parietals present. Among North Pacific species, it is similar to P. flammeus sp. nov., which differs from the former in having 17-20 pectoral-fin rays, and to P. mento, which has 5 principal caudal-fin rays.
Description. Measurements are shown in Table 1. Body compressed, elongate, deepest at nape, taping posteriorly (Fig. 5). Skin thin, fragile. Head compressed, dorsal profile strongly sloping from nape to snout. Snout deep, abruptly angled, its length almost equal to orbit diameter; not projecting anterior to upper jaw. Mouth strongly oblique, lower jaw slightly protruding beyond (or almost same length as) upper jaw; premaxillary tooth plates matching mandibular tooth plates; maxilla extending to posterior margin of orbit; oral cleft extending to middle of orbit. Premaxillary teeth simple, in 3-6 oblique rows; diastema narrow between premaxillae. Mandibular teeth simple, in 4 or 5 oblique rows (Fig. 3B); diastema absent at lower jaw symphysis. Orbit of moderate size, rounded. Nostril single, without distinct tube, slightly above level of mid-orbit. Cephalic sensory pores small: nasal pores 2, maxillary pores 6, preoperculomandibular pores 7, suprabranchial pore 1; cephalic pore pattern 2-6-7-1. Chin pores paired, openings well separated on skin surface. Coronal pore absent. Gill slit moderately large, upper margin level with mid-orbit, extending ventrally to just above pectoral fin or to level of 1-3 uppermost pectoral-fin rays. Gill rakers 10-12, blunt and small. Tip of opercular flap sharp, directed posteriorly, level with mid-orbit or posterior margin of maxillary.
Pectoral fin moderately notched, with 27-30 rays; upper lobe with 17-19 rays, extending beyond (or just reaching) anal-fin origin; lower lobe elongate, with 8-13 rays, uppermost ray of lower lobe longest, extending beyond anus, not reaching anal-fin origin. Uppermost pectoral-fin base below a horizontal through posterior margin of maxillary. Lowermost pectoral-fin base below anterior rim of orbit or below midway between tip of snout and anterior rim of orbit. Rays between upper and lower lobes widely spaced.
Selected osteological characters. Roof of cranium without distinct crest comprising well ossified frontals, supraoccipital, and parietals. Opercle well ossified, sharpened posteriorly, supporting upper margin of opercular flap. Subopercle thin, comprising two spines forming a V-shape; lower spine supporting lower margin of opercular flap. Subopercle and interopercle attached. Cleithrum broad and robust, dorsal portion elongated. Proximal pectoral radials 4, enlarged occupying almost entire width of cartilaginous basal laminae and moved to anterior edge of basal lamina (Fig. 3D). No interradial fenestrae between proximal radials. Scapula with strong helve. Coracoid narrowly triangular with narrow lamina. Distal radials absent.
Coloration. In fresh specimens, head and body pale pink, posterior half of body reddish; dark peritoneum visible through thin skin; dorsal and anal fins pale pink, distally reddish; caudal and pectoral fins red (Fig. 6). In preserved specimens, head, body, and fins pale; peritoneum black, stomach pale or white; orobranchial cavity pale with scattered melanophores.
Geographical distribution. Western Pacific Ocean; Tosa Bay and Bungo channel, off Shikoku Island, Japan, in depths of 600-1,092 m (Kido 1988; this study).
Remarks. Paraliparis mandibularis was originally described by Kido (1985) on the basis of a single specimen collected from Tosa Bay, Japan. Subsequently, Kido (1988) redescribed the species on the basis of the holotype and an additional non-type specimen. However, a number of details, including osteology and fresh coloration have remained unknown to date. Whereas the pectoral girdle in Paraliparis species generally has a reduced number and size of radials (Andriashev 1998;Orr et al. 2019), that of P. mandibularis has four broad robust radials (Fig. 3D). The species is also characterized by a pair of parietals in the cranium, such being absent in some other species of Paraliparis (Kido 1988). Although Kido (1988) described P. mandibularis as having a diastema at the symphysis of both the upper and lower jaws, we could find no obvious diastema at the lower jaw symphysis in the specimens examined here, including the holotype. In addition to the differences described above between P. mandibularis and P. flammeus, the former is further distinguished from the latter by the enlarged pectoral radials occupying almost entire width of the cartilaginous basal laminae and moved to the anterior edge of basal lamina in the pectoral girdle (vs moderately large and medial) and the parietals in the cranium (vs. absent). Paraliparis mandibularis differs from other species with an oblique mouth and a low positioned pectoral fin, viz. P. angustifrons, P. membranaceus, and P. molinai (see Remarks under P. flammeus), in having 27-30 pectoral-fin rays (vs 37 in P. angustifrons, ca 25 in P. membranaceus, 24 in P. molinai). Paraliparis membranaceus and P. molinai have similar pectoral-fin ray numbers to P. mandibularis but have a reduced caudal fin (4 rays in P. molinai, 2 or 3 in P. membranaceus vs 6 in P. mandibularis) (Stein 2005).

Discussion
Jordan and Evermann (1896) designated the subgenus Amitrichthys under Paraliparis, the former including P. mento, P. cephalus, Paraliparis rosaceus Gilbert, 1890, Paraliparis copei Goode & Bean, 1896, and Paraliparis dactylosus Gilbert, 1896. Recently, however, Orr et al. (2019 included on the basis of their molecular phylogenetic tree only P. mento and P. cephalus in Amitrichthys. In the molecular phylogenetic tree presented herein, P. flammeus has a monophyletic relationship with P. cephalus and P. dipterus, whereas P. mento is included in a clade comprising P. rosaceus, P. copei, and some other, undescribed species (Fig. 5). Because the positions of P. mandibularis, P. angustifrons, P. membranaceus, and P. molinai are still unknown due to a lack of available COI sequences, the bootstrap probabilities supporting each node of both the current phylogenetic tree and that of Orr et al. (2019) are generally low, and the significant difference in osteological characters between P. flammeus and P. mandibularis may be indicative of a distant relationship, we remain conservative in avoiding a redefinition of the subgenus Amitrichthys at present. Further studies are required, based on longer sequences and comprehensive taxon sampling, for clarification of the phylogenetic relationships within Paraliparis.
Paraliparis flammeus is characterized by a poorly ossified cranium, a characteristic recognized elsewhere in some snailfishes, including Nectoliparis pelagicus, Pseudoliparis swirei Gerringer & Linley, 2017and Rodichthys regina Collett, 1879(Kido 1988Wang et al. 2019). Although Wang et al. (2019) reported a genetic change associated with adaptation to the deep sea in P. swirei (known only from hadal depths in the Mariana Trench, 6,198-8,098 m;Gerringer et al. 2017) as resulting in reduced cranial structure, P. flammeus is known from 422-890 m, N. pelagicus from ~549 m (Love et al. 2005), and R. regina from 400-2,365 m (Chernova et al. 2004;Mecklenburg et al. 2018), suggesting that incomplete cranial structure is not necessarily a result of adaptation for hadal depths. It should be noted that, unlike demersal P. swirei, characterized by a large pelvic disk (Gerringer et al. 2017), P. flammeus, N. pelagicus, and R. regina lack a pelvic disk and are apparently mesopelagic (Mecklenburg et al. 2002(Mecklenburg et al. , 2018. Similarly, a mesopelagic genus of bobtail snipe eel, Neocyema Castle, 1978, is known to have an incompletely ossified cranium, possibly suggesting neoteny or a trade-off between reduction of some bony structures (Poulsen 2015).