A new marine tardigrade genus and species (Arthrotardigrada, Styraconyxidae) with unique pockets on the legs

A marine heterotardigrade Cyaegharctus kitamurai gen. et sp. nov. (Arthrotardigrada, Styraconyxidae) is described from Daidokutsu, a submarine cave off Iejima island, Okinawa Islands, Ryukyu Archipelago, Japan. It is easily distinguished from all other styraconyxids by its pocket organs (putative sensory structures) on all legs in addition to the usual leg sensory organs. Its combination of other character states, such as the dorso-ventrally flattened body, ovoid primary clavae, conical secondary clavae, large terminal anus, internal digits with proximal pads and peduncles, external digits with developed peduncles and all digits with three-pointed claws in adult female, supports the erection of a new genus and species.


Introduction
Marine tardigrades, specifically arthrotardigrades, exhibit remarkable morphological diversity (see comprehensive drawings of arthrotardigrade genera in Fontoura et al. (2017) and drawings of additional genera in Fujimoto and Yamasaki (2017) and Fujimoto and Ohtsuka (2019)). Styraconyxidae Kristensen and Renaud-Mornant, 1983 is one of the arthrotardigrade families and it is comprised of 38 species and subspecies of ten genera: Angursa Pollock, 1979 (eight species), Bathyechiniscus Steiner, 1926 (monotypic), Lepoarctus Kristensen and Renaud-Mornant, 1983 (monotypic), Paratanarctus D'Addabbo Gallo et al., 1992 (monotypic), Pleocola Cantacuzène, 1951 (monotypic), Raiarctus Renaud-Mornant, 1981a (five species), Rhomboarctus Renaud-Mornant, 1984 (three species), Styraconyx Thulin, 1942 (15 species and subspecies), Tetrakentron Cuénot, 1892 (monotypic) and Tholoarctus Kristensen and Renaud-Mornant, 1983 (three species and subspecies) (Guidetti and Bertolani 2005;Degma and Guidetti 2007;Degma et al. 2019;Pérez-Pech et al. 2020). In addition to these ten genera,  reported an undescribed genus related to Styraconyx and Tetrakentron from a submarine cave in Japan (for detail of this cave see Yamamoto et al. 2009). However,  did not make any remarks on the undescribed genus' morphology and only provided two voucher micrographs of a specimen used for their molecular phylogenetic study (provided by MorphoBank (O'Leary and Kaufman 2012) at http://doi.org/10.7934/ P2234). Herein, we describe this taxon as a new genus and species, based on detailed morphological observation of additional material collected from the same cave. sediment samples were stirred with tap water and the supernatants were concentrated using a 30 μm opening mesh net to separate coarse sediment and to wash away seawater. Subsequently, the meiofauna and fine sediment were separated using LUDOX HS-40 colloidal silica (Sigma-Aldrich Co., St. Louis) and a 32 μm opening mesh net (e.g. Giere 2009). The type material was sorted under a stereomicroscope and fixed in 2-4% buffered formaldehyde. Specimens for light microscopy were mounted in distilled water for brief observation (only for the holotype) and mounted in glycerol. Differential interference contrast microscopy (DIC) was conducted using an Olympus BX53 and phase contrast microscopy (PhC) was conducted using an Olympus BX41. One specimen for scanning electron microscopy (SEM) was post-fixed in 2% OsO 4 for 2 h, dehydrated through a series of ethanol and acetone, critical point dried (BAL-TEC CPD-030), osmium coated (Filgen OPC40) and observed using JEOL JSM-7001F. Type material was deposited in the Zoological Collection of Kyoto University (KUZ). Adobe Illustrator CS6 and Photoshop CS6 were used to prepare figures and to obtain morphometric data.

Diagnosis.
Styraconyxidae with dorso-ventrally flattened body; cuticle smooth; epicuticular pillars present; cephalic region with complete set of cephalic cirri, ovoid primary clavae and compact conical secondary clavae; median cirrus and internal cirri at anterior margin of cephalic region; external cirri latero-ventral to internal cirri; lateral cirri and primary clavae sharing common base at antero-lateral position of cephalic region; secondary clavae between internal and external cirri; buccal apparatus with stylet supports; cirri E spine-like; seminal receptacle ducts opening anterior to gonopore; terminal anus with pair of large longitudinally elongate lobes; dorsal side of each leg with usual sensory organ on proximal part of femur and pocket organs at distal margin of femur; internal digits each with proximal pad and thin peduncle; external digits with proximal developed peduncles; claw sheaths present; adult female with three-pointed claws on all digits; four-claw juvenile with three-pointed claws on internal digits and single-pointed claws on external digits; three-pointed claws each with accessory and secondary hooks less developed compared to primary hook.
Etymology. The genus name is masculine and derives from two words, Cyäegha, a deity of darkness and caves in the Cthulhu Mythos from Eddy C. Bertin's short story Darkness, My Name Is and arctus, a Latinised Greek word meaning bear.
Remarks. The presence of pocket organs distinguishes the new genus and species from all other members of the phylum (see Description of holotype and Discussion for further information on the new structure). For this uniqueness, it is difficult to infer the new taxon's taxonomic position, based solely on this morphology. Here we use other comparable morphological characters to infer its taxonomic position. The new taxon's three-pointed claws and peduncles indicate its affinity to the following four genera of Styraconyxidae: Raiarctus, Styraconyx, Tetrakentron and Tholoarctus (van der Land 1975;Kristensen and Higgins 1984;Jørgensen et al. 2014).
The new taxon's epicuticular pillars are not developed to the degree of Raiarctus's characteristic pillars (Jørgensen et al. 2014). In addition, the new taxon has its seminal receptacles opening near the gonopore, in contrast to those of the latter genus opening laterally apart from the gonopore (Jørgensen et al. 2014).
Styraconyx harbours species covering a wide range of character states and has been regarded as a non-monophyletic group (Kristensen and Renaud-Mornant 1983;Kristensen and Higgins 1984). Due to this problematic state, many of the new taxon's character states fall within the range of Styraconyx, i.e. morphology of cephalic cirri, primary clavae, usual leg sensory organs, claws and digits (including peduncles) and seminal receptacles (Kristensen and Higgins 1984). However, the new taxon's compact conical secondary clavae differ from Styraconyx's flat sac to dome-shaped ones (Kristensen and Higgins 1984). Further, although it is not included in the generic diagnosis (Kristensen and Higgins 1984), it is apparent from all the original descriptions of Styraconyx species that they have a cylindrical body shape differing from the new taxon's dorso-ventrally flattened body and also the anus is not known to be developed as the large bi-lobed anus of the new taxon (Thulin 1942;Robotti 1971;Kristensen 1977;Renaud-Mornant 1981b;Pollock 1983;Kristensen and Higgins 1984;D'Addabbo Gallo et al. 1984, 1989Chang and Rho 1998;Bartels et al. 2015;Pérez-Pech et al. 2020).
The new taxon and Tetrakentron both have a dorso-ventrally flattened body, but the latter genus has short legs with strongly developed claws (van der Land 1975). The latest diagnosis of Tetrakentron by van der Land (1975) is brief and we here present some other differences, based on information that van der Land (1975) did not implement in the diagnosis and additional information provided by subsequent literature (Kristensen 1980;Kristensen and Hansen 2005). One difference is that, in contrast to the new taxon, Tetrakentron has unique peduncles, which van der Land (1975) called 'drop-shaped' (already illustrated in its original description (Cuénot 1892); for detailed drawing see van der Land (1975)).
Another difference is that the new taxon and Tetrakentron have differently arranged cephalic appendages: i) the new taxon's primary clavae are positioned laterally apart from the secondary clavae, whereas the two clavae are situated close together in Tetrakentron and ii) the new taxon's secondary clavae are positioned posterior from the anterior margin of the cephalic region, whereas those of Tetrakentron are at the antero-lateral margin (see detailed drawings and SEM in Kristensen (1980)). The presence of epicuticular pillars in the new taxon further differentiates it from Tetrakentron without pillars (Kristensen 1980). On the other hand, the new taxon and Tetrakentron have similar secondary clavae morphology and they both have their seminal receptacles opening anterior to their gonopores (Kristensen and Hansen 2005). It is also noteworthy that these two taxa, as well as Angursa, have a terminal anus (Kristensen and Hansen 2005;Fujimoto and Hansen 2019). Whether these morphological similarities between the new taxon and Tetrakentron are phylogenetic signals or not need to be verified with molecular data.
The new taxon has a dorso-ventrally flattened body differing from Tholoarctus's cylindrical body with a large detached outer epicuticle (Jørgensen et al. 2014), Further, the new taxon has ovoid primary clavae and conical secondary clavae, in contrast to Tholoarctus with cone-shaped primary clavae and large secondary clavae surrounding the internal and external cirri (Jørgensen et al. 2014).
For the morphological differences indicated above, the new taxon's designation to any of the four genera is not plausible and the erection of a new genus and species is justified. Diagnosis. Cyaegharctus with median, internal and external cirri each with basal swelling (cirrophore?) and short subdivision; lateral cirri each with short subdivision; cirri E on cirrophores arising from round lateral processes between legs III and IV; seminal receptacle ducts run postero-laterally with U-shaped bend (vesicles unknown); leg I sensory organs spine-like, each with distal subdivision and terminal pore; legs II and III sensory organs spinelike without subdivisions; leg IV sensory organs papillate with short distal tubes. (Figs 1, 2A-J). Dorso-ventrally flattened body 202 μm in length and 117 μm wide at level of leg III (Figs 1A, 2A). Outer epicuticle loose from pillar layer as artefact of formaldehyde fixation (for further information see Remarks on paratypes). Fine epicuticular pillars recognised.
Paired spine-like cirri E (38 μm) (Figs 1A, 2E) on cirrophores arise from round lateral processes at level between legs III and IV.
Rosette-like gonopore 9 μm anterior to anus (Figs 1A, 2E). Gonopore morphology disturbed when cleaning specimen and only contour recognisable after permanent preparation (8 μm in diameter). Anus 16 μm wide at posterior most part of body between legs IV and consists of paired longitudinally elongate lobes. Paired seminal receptacles open immediately anterior to gonopore. Seminal receptacle ducts first run laterally, bent in U-shape and run postero-laterally. Terminal vesicles of seminal receptacles not recognised.
Four pairs of legs (Figs 1, 2F-J) each with usual leg sensory organ on dorsal side of femur's proximal portion, pocket organ on dorsal side of femur's distal margin and four digits terminating in claws. Legs I each with spine-like sensory organ (10 μm) with distal subdivision. Legs II each with spine-like sensory organs  Habitus (ventral view). B. Leg IV pocket organ. an anus, bt buccal tube, ca cavity, cE cirrus E, db dense body, ec external cirrus, go female gonopore (detail not available), ic internal cirrus, lc lateral cirrus, mc median cirrus, pc primary clava, pl placoid, op opening, sc secondary clava, so I, IV legs I and IV sensory organs, sr seminal receptacles, ss stylet support, st stylet.
which lack subdivisions (length not measured due to bad orientation). No sensory organs recognised on legs III but presence likely (recognised in paratypes; see Remarks on paratypes). Legs IV each with sensory organ (7 μm) consisting of papilla (4 μm) with distal tubular portion (2 μm). Pocket organ consists of small opening, cavity and apparent dense body. No difference recognised amongst legs I-IV pocket organs besides protruded opening of leg II pocket organ. Leg IV pocket organ in good orientation for observation of whole structure . an anus, bt buccal tube, ca cavity, cE cirrus E, db dense body, ec external cirrus, go gonopore, ic internal cirrus, lc lateral cirrus, mc median cirrus, pc primary clava, pe i,e peduncles of internal and external digits, pl placoid, po I-IV legs I-IV pocket organs, pp proximal pad, sc secondary clava, so I-IV legs I-IV sensory organs, sr seminal receptacles, ss stylet support, st stylet. (Fig. 1B). Internal digits each with proximal pad and thin peduncle. External digits each with broad peduncle that do not reach claws, subdivided into proximal and distal portion. Internal digits longer than external digits. Each claw of both internal and external digits with three points: primary hook and less-developed, secondary and accessory hooks. Digits and peduncles slightly longer than those of preceding leg pairs (legs I: internal digits 13 μm (peduncles 3 μm), external digits 10 μm (peduncles 5 μm); legs II: 14 μm (3 μm), 10 μm (5 μm); legs III: 15 μm (4 μm), 10 μm (5 μm); legs IV: 16 μm (4 μm), 10 μm (6 μm)).

Remarks on paratypes.
Fixation of specimens using formaldehyde seems to have introduced an artefact in the cuticle, i.e. the detached (or loose) outer epicuticle. When the specimens were sorted in distilled water before fixation, the outer epicuticle did not look loose at 63× magnification (all four specimens) and also at 400× magnification (only observed for KUZ Z2627) as it would appear in Tholoarctus. Although these are only brief observations and we did not conduct any experiment to test this artefact, we consider the outer epicuticle's detached state as an artefact and excluded this character state from the diagnoses of this genus and species. Leg III digits and claws. ah accessory hook, an anus, cE cirrus E, ec external cirrus, fe femur, ic internal cirrus, lc lateral cirrus, mc median cirrus, pc primary clava, ph primary hook, po III, IV legs III and IV pocket organs, pp proximal pad, sc secondary clava, sh secondary hook, so I-IV legs I-IV sensory organs, ti tibia.
The SEM of a four claw juvenile (Fig. 3) confirmed the results of light microscopy and also provided further detail. However, this SEM specimen also seems to have its outer epicuticle detached. If the outer epicuticle is attached to the underlying layer, a pattern is recognised on the surface of the body due to the pillar layer, but no such indentations were found, suggestive of the detached state of the outer cuticle (for ultrastructure of cuticle, see Kristensen and Neuhaus 1999). The proximal part of each leg has an inflated appearance (Fig. 3A) not recognised in light microscopy. The view of the cephalic region (Fig.  3B, C) revealed the three-dimensional morphology and the arrangement of the cephalic appendages and also confirmed the presence of terminal pores on the cephalic cirri and the primary clavae. The conical shape of the secondary clavae seems not as evident as in light microscopy probably due to the overlying outer epicuticle. The large anus is not on the ventral surface and rather direct posteriorly (Fig. 3D). The leg sensory organs were recognised (Fig. 3E-H) and those of legs I and IV revealed to have terminal pores. The pocket organs (Fig. 3I, J) were recognised on all legs, however, with slightly different degrees of protruded appearances. The protruded state might be an artefact caused during specimen preparation since specimens on microscope slides do not always have these appearances (Figs 1B, 2F, I, J) or the pocket organs are capable of moving. In the latter case, since no muscles seem to be attached to the structures, a passive movement is likely. Regarding claw/digit morphology, the peduncles (internal structures) were not recognised but the three hooks of the internal claws and the single-pointed external claws were recognised (Fig. 3J).
Etymology. The species is dedicated to Prof. Dr. Akihisa Kitamura (Shizuoka University), who has been studying Daidokutsu and its bivalve assemblage to understand the paleo-environment (e.g. Kitamura et al. 2007).

Discussion
We consider the dense body inside the pocket organs of Cyaegharctus kitamurai gen. et sp. nov. to be related to the van der Land's body, often situated at the base of the primary clavae and leg IV sensory organs and suggest that the pocket organs are chemoreceptors. However, Kristensen (1981) is the only one who investigated the function and ultrastructure of arthrotardigrade sensory organs by transmission electron microscopy and, with our poor knowledge on arthrotardigrade sensory organs, this remains a matter of speculation. Another possibility for the new structure is a secretory organ. However, no gland was recognised in its vicinity and there is no evidence supporting this hypothesis. To understand the true functions and evolutionary significance of the pocket organs, comparative ultrastructure studies of arthrotardigrades (including C. kitamurai gen. et sp. nov.) are necessary.