Echinoderes xiphophorus sp. nov. – the first deep-water representative of Echinoderidae in the Sea of Japan (Kinorhyncha: Cyclorhagida)

A new species of echinoderid kinorhynchs, Echinoderes xiphophorus sp. nov. collected from oxidized brown silt at the deepest depression in the Sea of Japan, North-West Pacific, is described and illustrated using light and electron microscopy. This new representative of the most speciose kinorhynch genus is characterized by the unique set of spines and tubes and can easily be distinguished from most of its congeners. The second trunk segment bears three pairs of tubes in subdorsal, midlateral and ventrolateral position in both sexes; one pair of tubes on trunk segment 5 in lateroventral position and on trunk segment 8 in sublateral position; aciculate lateroventral spines on trunk segments 6–9; aciculate middorsal spines on trunk segments 4, 6, 8. This species is well recognized by very long tergal extensions of the posteriormost segment, some of the longest within the family Echinoderidae. Males of Echinoderes xiphophorus sp. nov. are well distinguished from all the congeners by extremely long sword-like appendages dorsally to three pairs of penile spines. The species constitutes the first deep-sea representative of the Echinoderidae in the Sea of Japan and the deepest representative of the Kinorhyncha in this sea.

As reported in some recent papers, kinorhynchs are very common and numerous in the abyss and even in the hadal zone (see Neuhaus 2013;Adrianov & Maiorova 2015Sørensen et al. 2018;Yamasaki et al. 2018aYamasaki et al. , 2018bYamasaki et al. , 2018c. Nevertheless, information on the species composition in the deep sea is still very limited. Only six species of Echinoderes Claparède, 1863 have been described from the Sea of Japan: E. filispinosus Adrianov, 1989 andE. multisetosus Adrianov, 1989 from the Peter the Great Bay, and E. ulsanensis Adrianov, 1999, E. koreanus Adrianov, 1999, E. obtuspinosus Sørensen et al., 2012and E. songae Sørensen & Chang, 2020 off the east coast of the Korean Peninsula (see Adrianov & Malakhov 1999;Sørensen et al. 2012Sørensen et al. , 2020. Three other species, E. tchefouensis Lou, 1934;E. cernunnos Sørensen et al., 2012 andE. microaperturus Sørensen et al., 2012, were found in the Korean Strait (between Tsushima Island and the Korean mainland) between the Sea of Japan and the East-China Sea, and E. sensibilis Adrianov, Murakami & Shirayama, 2002 was reported from Tsugaru Strait between the Sea of Japan and the Pacific (see Sørensen et al. 2012Sørensen et al. , 2020Yamasaki et al. 2014). All these echinoderids are shallowwater species, found in intertidal and subtidal environments only (from 0 to 140 m depth).
On the slope of the deepest depression in the Sea of Japan, near the Russian coast, at a depth of 1530 m, we found a new species of echinoderid kinorhynchs with unique morphological characters. The purpose of this paper is to describe the new species as the first deep-sea representative of the genus Echinoderes in the Sea of Japan and the first bathyal species of the genus in the North-West Pacific.

Material and methods
The specimens here described as a new species, Echinoderes xiphophorus sp. nov., were collected from an oxidized brown clay taken by multiple corer (MUC) on a slope at the deepest depression in the western part of the Sea of Japan at a depth 1530 m (44.7942° N and 137.2550° E). Sediments were collected during the SoJaBio voyage (Russian-German deep-sea expedition) of the RV "Akademik M.A. Lavrentyev" in August-September 2010 alongside the deep-water transect from 500 to 3660 m near the Russian coast of the Sea of Japan.
Water above the sediment in the core was filtered through a 32 µm mesh sieve. Filtered material was checked under a stereo microscope to collect live specimens.
All specimens were fixed in 10% buffered formalin in seawater.
Six adult specimens were transferred to 70% ethanol-5% glycerol-25% deionized water solution. After evaporation of the ethanol and water, the material was preserved in anhydrous glycerol. All specimens were mounted individually in VECTASHIELD mounting medium for fluorescence microscopy between two circular slips (coverglasses with 18 and 12 mm in diameter, respectively) which enable viewing from both sides. The slips were positioned on Higgins-Shirayama plastic slide frames for further examination using differential interference contrast microscopy (DIC or Nomarski microscopy). The specimens were studied with a Zeiss (Imager.Z2) microscope equipped with an AxioCamHR Rev3 camera.
Four adult specimens were selected for scanning electron microscopy (SEM). These specimens were transferred by an Irwin Loop from 10% formalin to a vessel of distilled water and washed using a detergent to clean the body surface. The cleaned specimens were dehydrated through a graded series of ethanol, transferred to acetone, and critical-point dried. The dried specimens were mounted on aluminum stubs. Uncoated stubs were initially imaged at low vacuum with a Zeiss Sigma scanning electron microscope using a SE2 and a VSPG4 detectors. Stubs were subsequently coated with gold and observed using a regular scanning electron microscope protocol by SEM (Zeiss Sigma).
All microscope facilities were provided by the Far Eastern Center for Electron Microscopy at the NSCMB FER RAS.
In the examination procedures, we followed the standard protocol described by Higgins (1983) and modified more recently by several authors (see Neuhaus 2013). Measurements are summarized in Table 1.
Positions of cuticular structures on the trunk segments follow the terminology used in several recent papers (see Neuhaus 2013;Adrianov & Maiorova 2018a. Presence and position of cuticular structures are summarized in Table 2. Type material is deposited at the Museum of the A.V. Zhirmunsky National Scientific Center of Marine Biology of the Far Eastern Branch of the Russian Academy of Sciences (NSCMB FER RAS) (MIMB). Trunk length (TL) 290-305 µm; lateral terminal spines (LTS) 142-185 µm; trunk segments 1-2 consisting of closed cuticular rings, and trunk segments 3-11 of one tergal and two sternal plates; trunk segment 2 with three pairs of well-developed tubes in subdorsal, midlateral, and ventrolateral positions; trunk segment 5 with tubes in lateroventral position; trunk segment 8 with tubes in sublateral position; middorsal spines on trunk segments 4, 6, 8; lateroventral acicular spines on trunk segments 6-9; trunk segment 11 with remarkably long tergal extensions (TE) about 15-16% of TL; males with three pairs of penile spines and remarkably long sword-like extensions (about 80% of TE) dorsally to penile spines; females with pair of laterodorsal tubes on trunk segment 10.

Etymology
The species name is derived from the Greek ʻxiphosʼ (sword) and ʻphorusʼ (carry) because of very long sword-like appendages in males dorsally to penile spines.

Description
Body. Adult specimen consists of head, neck and 11 trunk segments. Measurements (µm) and ratios (%) are provided in Table 1. Head consists of retractable mouth cone and eversible introvert. Inner oral styles are arranged in two circlets (5 large tooth-like outer styles + 10 minute innermost styles). External mouth cone bears nine articulated outer oral styles. Head bears seven rings of spinoscalids and one ring of six trichoscalids. Neck consists of 16 placids. All placids trapezoid in shape, distinctly articulating with the first trunk segment. Midventral placid widest; remaining ones narrower. Two ventral and four dorsal trichoscalid plates present, ventral ones being broader than four dorsal ones.
Trunk segment 1. Closed cuticular ring, with three pairs of sensory spots located close to the anterior segment margin in subdorsal and laterodorsal and more posteriorly in ventromedial position ( Trunk segment 2. Closed cuticular ring, without any partial intra- or extracuticular fissures; three pairs of tubes in subdorsal, midlateral and ventrolateral positions; each tube with short and smooth basal part and longer distal part with two wing-like lateral projections (Fig. 6B); one sensory spot in middorsal position and three pairs in laterodorsal, lateroventral and ventromedial position, sensory spots on this and following segments with 10-15 very short petals and 1-2 very long posteriormost petals similar to cilia (Figs 6B, 7C-E); one pair of glandular cell outlets of type 1 in ventromedial position anterior to ventromedial sensory spots (Fig. 3B); ventral hairs on the trunk segment 2 seem to be more regularly Trunk segment 3. One tergal and two sternal plates, with distinct tergosternal and midventral articulations, as in all remaining trunk segments; with two pairs of sensory spots in subdorsal and midlateral position (Figs 1A-B, 2A, 3B, 6B); single glandular cell outlet of type 1 in middorsal position located close to the anterior segment margin and hidden under preceding segment; with a pair of glandular cell outlets in ventromedial position; long cuticular hairs absent in ventromedial position, being replaced by much shorter cuticular hairs (Fig. 7B); other cuticular hairs and pectinate fringe as in the preceding segment.   Trunk segment 8. With acicular spine in middorsal position (Figs 2A, 4A, 6E); with a pair of tubes in sublateral position (Figs 2E, 4C, 7D); with one pair of sensory spots in paradorsal position (Figs 6E, 7C); with two pairs of glandular cell outlets of type 1 in paradorsal and ventromedial position (Fig. 2B, D); other spines, pectinate fringe and cuticular hairs as in the preceding segment.  appendages (similar in shape to Japanese swords katana or wakizashi) in laterodorsal position, dorsally to penile spines, only slightly shorter than tergal extensions (about 80% of TE) (Fig. 8A-B); females with a pair of short tubes / spines in laterodorsal position corresponding to sword-like appendages of male ( Fig. 8C-D); other characters as in the preceding segment.
Trunk segment 11. One pair of long lateral terminal spines (Fig. 1C); tergal extensions very long, about 15-16% of TL (Figs 4-5, 8); tergal plate with nearly rounded middorsal elevation (Fig. 8B, D); with two pairs of sensory spots in subdorsal and ventromedial position, and additional pair of sensory spots present at the base of tergal extensions (Fig. 8A, C-D); with two glandular cell outlets of type 1 in middorsal position and one pair ventrolateral position (Figs 2C, E, 5); males with three pairs of penile spines, first and third pairs thin and flexible, spine of second pair shorter and cone-shaped with terminal tuft of cuticular hairs (Figs 5, 8A-B); female with a pair of long and thin lateral terminal accessory spines; sexes can be also recognized by observation of gonads.

Taxonomic remarks
Echinoderes xiphophorus sp. nov. is well distinguished from all congeners by a unique combination of characters: three pairs of tubes on trunk segment 2 and one pair of tubes on trunk segments 5 and 8 middorsal spines on trunk segments 4, 6, 8; acicular lateroventral spines on trunk segments 6-9; extremely long tergal extensions of 11 th trunk segment; very long sword-like appendages dorsal to penile spines in male and short spines at the same position in female (Fig. 1A-B).
Among the more than 130 species of the genus Echinoderes described so far, only five possess three pairs of tubes on trunk segment 2. Three shallow-water species, E. newcaledoniensis Higgins, 1967 from South-West Pacific, E. peterseni Higgins & Kristensen, 1988 from the Greenland Sea, North-West Atlantic, and E. ohtsukai Yamasaki & Kajihara, 2012 from North Pacific, possess three pairs of tubes in subdorsal, laterodorsal and ventrolateral position (see Higgins 1967;Higgins & Kristensen 1988;Adrianov & Malakhov 1999;Yamasaki & Kajihara 2012). The first two species are also characterized by having middorsal spines on trunk segments 4, 6 and 8, and additional lateral tubes on trunk segment 8, but well differ from the new species by the shape of much shorter tergal extensions. In addition, E. peterseni bears additional lateral tubes on trunk segment 9. Echinoderes ohtsukai is well distinguished by the presence of only a single minute middorsal spine on trunk segment 4. Echinoderes belenae Pardos, Herranz & Sanchez, 2016 from the Pacific coast of Panama possesses three pairs of tubes in subdorsal, midlateral and ventrolateral position (see Pardos et al. 2016). This species well differs from the new one by having very short and stubby lateral terminal spines. The fifth species, E. hispanicus Pardos, Higgins & Benito, 1998 from Spain, bears tubes in subdorsal, sublateral and ventrolateral position (see Pardos et al. 1998) and well differs from the new species by the shape and length of the terminal tergal extensions.
The bathyal E. balerioni Grzelak & Sørensen, 2019 from the Arctic Ocean, close to Spitsbergen Archipelago, also possesses three pairs of tubes on trunk segment 2 in laterodorsal, sublateral and ventrolateral position (see Grzelak & Sørensen 2019). Echinoderes balerioni and E. xiphophorus sp. nov. are characterized by similar spine / tubes formula. Nevertheless, E. balerioni is well distinguished from the new species by having a tube on trunk segment 8 in the lateral accessory position, opposite to the sublateral position in E. xiphophorus sp. nov., and the shape of the sternal plates of the posteriormost segment (see Grzelak & Sørensen 2019: fig. 2). Also, this species has no sword-like appendages dorsally to three pairs of penile spines in males.
The shallow-water Mediterranean E. capitatus (Zelinka, 1928) even possesses four pairs of tubes on trunk segment 2 in subdorsal, laterodorsal, midlateral and ventrolateral position, but is easily distinguished by the single middorsal spine on trunk segment 4, a different spine formula and rather short triangular tergal extensions (see Yamasaki & Dal Zotto 2019 fig. 2b, d). The new species of Echinoderes is externally very similar to Meristoderes okhotensis by the spine / tubes formula and by presence of very long tergal extensions. The latter species also bears three pairs of tubes on trunk segment 2 in subdorsal, laterodorsal and ventrolateral position; lateroventral tubes on trunk segment 5, lateral tubes on trunk segment 8, and lateroventral acicular spines on trunk segments 6-9 (see Adrianov & Maiorova 2018a). However, M. okhotensis can easily be distinguished from the new species by the presence of only two middorsal spines on trunk segments 6 and 8, and by different arrangement of sensory spots and glandular cell outlets.
Similar to M. okhotensis, the new species is also characterized by very small sensory spots on trunk segments 3-10, just about 1 µm in length and only with a few petals around one pore. One of the remarkable characters of these sensory spots in both species is the presence of 1-2 remarkably long posterior petals, 3-4 times as long as the sensory spots themselves (see Adrianov & Maiorova 2018a: fig. 4c-h) (Fig. 7D-E).
Interestingly, M. okhotensis is the only other species of kinorhynchs having sword-lake appendages dorsally to three pairs of penile spines in males (see Adrianov & Maiorova 2018a: fig. 4f). These appendages in M. okhotensis are about twice as short as the tergal extensions, and similar in shape as the Roman gladius. In E. xiphophorus sp. nov., these sword-like appendages are much longer, constituting about 80% of the tergal extensions, and more similar in shape to the Japanese katana or wakizashi ( Fig. 8A-B). These appendages cannot be misinterpreted with ordinary penile spines usually situated more laterally at the boundary between two posteriormost segments. In contrast to the flexible penile spines that appear round in cross-section in both species, these appendages are remarkably flattened and not flexible. Probably, these sword-like appendages can be homologized with tubes of trunk segment 10, known in many species of Echinoderes, but not with ordinary penile spines. Nevertheless, these structures, present only in males, are suspected to be use in copulation process, but their real function is still not understood (see Adrianov & Maiorova 2018a).
Echinoderes xiphophorus sp. nov. constitutes the second bathyal representative of the genus Echinoderes in the North-West Pacific, the first deep-sea representative of the Echinoderidae in the Sea of Japan and the deepest representative of the Kinorhyncha in this sea.