Halechiniscidae (Heterotardigrada, Arthrotardigrada) of Oura Bay, Okinawajima, Ryukyu Islands, with descriptions of three new species

Abstract Marine tardigrades of the family Halechiniscidae (Heterotardigrada: Arthrotardigrada) are reported from Oura Bay, Okinawajima, one of the Ryukyu Islands, Japan, including Dipodarctus sp., Florarctus wunai sp. n., Halechiniscus churakaagii sp. n., Halechiniscus yanakaagii sp. n. and Styraconyx sp. The attributes distinguishing Florarctus wunai sp. n. from its congeners is a combination of two characters, the smooth dorsal cuticle and two small projections of the caudal alae caestus. Halechiniscus churakaagii sp. n. is differentiated from its congeners by the combination of two characters, the robust cephalic cirrophores and the scapular processes with flat oval tips, while Halechiniscus yanakaagii sp. n. can be identified by the laterally protruded arched double processes with acute tips situated dorsally at the level of leg I. A list of marine tardigrades reported from the Ryukyu Islands is provided.


Introduction
Halechiniscidae (Heterotardigrada: Arthrotardigrada) is a group of unarmoured marine tardigrades possessing cephalic appendages, including the median cirrus, and legs with four digits terminating in distal claws. More than half of the described marine species are assigned to this family, which comprises 29 genera in seven subfamilies.
In January 2014, the first Umisawa-kai (Field Workshop for Young Marine Biologists) was held to survey the invertebrate fauna of Oura Bay, Okinawajima, one of the Ryukyu Islands, Japan. During this survey, the following five species of Halechiniscidae were encountered: Dipodarctus sp., Florarctus wunai sp. n., Halechiniscus churakaagii sp. n., Halechiniscus yanakaagii sp. n. and Styraconyx sp.

Materials and methods
Specimens were found in five sediment samples (each sample was approximately 1L in volume) collected from Oura Bay, Okinawajima, one of the Ryukyu Islands, Japan by SCUBA diving. The geographical coordinates, water depth, sediment type and date of collection are listed for each sediment sample in Table 1.
The samples were freshwater-shocked (Kristensen 1983), sieved through a 32-μmmesh net and fixed in 3% formaldehyde. To extract specimens from the remaining sediment the fixed samples were treated using a modified density separation method from Burgess (2001). The sample was rinsed with distilled water to remove formaldehyde. Subsequently, the sample was thoroughly mixed with distilled water-diluted LUDOX® HS-40 colloidal silica (density slightly above 1.15 g cm -3 ) before allowing the sediment to settle (for at least 15 minutes). The supernatant was sieved through a 32-μm-mesh net to collect the specimens, and the procedure repeated three times per sample. The specimens were sorted under a stereomicroscope before being mounted in glycerol and observed under a phase-contrast microscope (Olympus BX53). The terminology for the genus Florarctus follows Hansen (2011).  Fig. 1 Material examined. Two four-clawed juveniles found in sediment sample 4 (Table 1).
Remarks. The species resembles Dipodarctus borrori Pollock, 1995 andD. susannae Jørgensen, Boesgaard, Møbjerg &Kristensen, 2014 by having digits of unequal length on legs I-III and the lack of lateral processes between legs III and IV. It is distinguished from the two species by the lateral cirrus lack of scapus, which is present in the both D. borrori and D. susannae. It is also distinguished from D. borrori by the shorter digit 1 of legs I-III and from D. susannae by its shorter papillate leg IV sense organ. These observations are based on comparing juveniles with descriptions of adults so while this species is probably an undescribed species observation of an adult specimen is required for confirmation.
Type depository. The type series is deposited in the Zoological Collection of Kyoto University (KUZ).
Description of holotype. Adult female, body length: 257 μm, excluding alae ( Fig. 2A, 3A). Cephalic region clearly separated from trunk. Dorsal surface smooth with folds. Ventral surface smooth. Lateral margin of body surrounded by aliform expansions with continuous caestus, which consists of frontal ala, pair of anterolateral alae, pair of postero-lateral alae and caudal ala. Frontal ala spreads across entire anterior margin of cephalic region. Scapi of internal cirri continuous with ala. Base of lateral cirri and primary clavae enveloped together in ala. Antero-lateral ala spreads from approximately level with median cirrus to level of leg III with four slight indentations. Antero-lateral alae caestus with small projections at level of leg I and leg II and developed projection at posterior end. Slightly overlapping anterolateral ala, postero-lateral ala spreads from level of leg III to level of cirrus E with two indentations: anterior slight indentation and posterior relatively strong indentation. Postero-lateral alae caestus with developed projection parallelogram-shaped at posterior end. Caudal ala spread between pair of cirri E with pair of lateral indentations (26 μm deep) and medial indentation (40 μm deep). Caudal caestus with pair of small projections. Unpaired median cirrus (36 μm) with scapus (10 μm), tubular portion (22 μm) and flagellum (4 μm) inserted dorsally 27 μm from frontal margin. Pair of internal cirri (46 μm) each with scapus (13 μm) tubular portion (30 μm) and flagellum (3 μm) inserted at anterior margin. Internal structure of internal cirrus arise  25 μm from frontal margin. Pair of external cirrus (44 μm) with scapus (18 μm), tubular portion (20 μm) and flagellum (6 μm) inserted ventrally 30 μm from frontal margin. Internal structure of external cirrus arise 39 μm from frontal margin. Lateral cirrus (43 μm) with scapus (15 μm), tubular portion and flagellum and primary clava arise from same cirrophore. Boundary between tubular portion and flagellum of lateral cirrus indistinct in holotype. Lateral cirrus inserted dorso-posteriorly to primary clava. Primary clava (101 μm) thicker near base with basal van der Land's body. Secondary clavae in shape of longitudinally elongated, flat sac with internally directed weak swelling on each side of ventrally protruded mouth cone (Figs 2B, 3B). Bucco-pharyngeal apparatus not visible except for pharyngeal bulb (32 μm × 24 μm). No bacterial vesicles visible. Leg I sense organ (29 μm) consists of tapering spine and distal flagellum. Leg II and leg III sense organ (28 μm and 24 μm respectively) each consists of unsegmented tapering spine. Leg IV sense organ (29 μm) consists of tapering spine with basal van der Land's body, distal constricted portion and distal pore. Pair of cirri E (46 μm) each with proximal portion and flagellum arise from between postero-lateral and caudal alarum caesti. Rosette-like female gonopore opens 27 μm anterior to anus. Pair of seminal receptacles sited laterally at a level between gonopore and anus. Seminal receptacle consists of sinuous duct, which opens 29 μm laterally from gonopore and terminates in spherical vesicle 9 μm in diameter. Each leg terminates in four digits with proximal wrinkles and distal claws. External digits with hook-shaped peduncle. Internal digits longer than external digits. Internal claws with dorsal spur. External claws with calcar and avicularia. Internal claws smaller than external claws. Etymology. The specific epithet, wunai, is a Ryukyuan word for "sister" (Nakamoto 1981) referring to the new species as a sister of Florarctus antillensis Van der Land, 1968, a species with similar morphology.
Remarks on paratypes. The adult male, KUZ Z707, was smaller than adult females, KUZ Z705 and Z706, but had longer primary clavae relative to its body length ( Table 2). The male gonopore of KUZ Z707 opens 10 μm anterior to the anus. The precise shape of male gonopore was not visible but spermatozoa were present inside the body. Excluding the lack of the genital structure, the paratypic four-clawed juvenile, KUZ Z709, was identical to the adults. A pair of bacterial vesicles is present in the paratypic four-clawed juvenile (Fig. 3C). For antero-lateral and postero-lateral alae, the number of slight indentations varied among specimens. There are two projections at the level of leg II in the paratypic specimen, KUZ Z708. The pair of small projections of the caudal alae caestus was better observed in the paratypes (Fig. 3D).
Differential diagnosis. The presence of the continuous caestus and the absence of dorsal mammilla-like ornamentation, are shared by Florarctus antillensis, F. glareolus Noda, 1987, F. pulcher De Zio Grimaldi, Lamarca, D'addabbo Gallo & Pietanza, 1999 and F. wunai sp. n. The new species is distinguished from these three species by the two small projections of the caudal alae caestus, which are long projections in F. glareolus, long projections with swollen tips in F. pulcher and absent in F. antillensis (using Renaud-Mornant [1970] for information on the caestus morphology of F. antillensis).
Type depository. The type series is deposited in the Zoological Collection of Kyoto University (KUZ).
Etymology. The specific epithet, churakaagii, is a Ryukyuan word for "beautiful woman" (Tojo 1930) Table 2 Diagnosis. Halechiniscus with no distinct cephalic lobes; only lateral cirrus and primary clava inserted on cirrophore; laterally protruded arched, double processes with acute tips at level of leg I; unsegmented cirrus E; bipartite leg I sense organ; unsegmented legs II and III sense organs; papillate leg IV sense organ; claws of internal digits with dorsal spur.
Etymology. The specific epithet, yanakaagii, is a Ryukyuan word for "ugly woman" (Tojo 1930) referring to dirty appearance of the holotype. Differential diagnosis. Halechiniscus yanakaagii sp. n. and H. tuleari are the only two Halechiniscus species with double processes at the level of leg I. The new species is distinguished from H. tuleari by the absence of distinct cephalic lobes and robust cephalic cirrophores (which are present in the latter species), the similar length, arched, double processes (23 μm, 26 μm) in contrast with a short, straight, anterior process (holotype female: 8 μm; paratype male: 4 μm) and a long, straight, posterior process (holotype female: 19 μm; paratype male: 10 μm) (see: Renaud-Mornant 1979), and the absence of processes at level of leg II and III, which are present in H. tuleari.
Remarks. The individuals found resemble Styraconyx nanoqsunguak Kristensen & Higgins, 1984 by the dorsal ridges (Fig. 8A, B). However, these specimens are distinguished by the lateral cirrus with no scapus (which is present S. nanoqsunguak), longer peduncles of the external digits and leg IV sense organs consisting of a spheri- cal papilla and a distal spine (which is an elongate papilla and a shorter distal spine in S. nanoqsunguak). While I believe this is a new undescribed species, lack of visible taxonomic characters has hindered providing a complete species description.

Discussion
With the addition of the three new species and two unidentified species reported in this study, two orders, five families, 15 named and two unidentified genera, 13 named and 14 unidentified species of marine heterotardigrades are known from the Ryukyu Islands (Table 3). Sudzuki (1979) reported the first species as Actinarctus sp., which I deem a misidentification of Florarctus sp. according to the micrograph in his paper. Subsequently, Noda (1993Noda ( , 1994aNoda ( -c, 1998 reported 21 species but, with the exception of four species, with neither exact sampling localities nor remarks on species morphology. He noted that Renaudarctus psammocryptus Kristensen & Higgins, 1984 accorded well with the original description (Noda 1994b) and considered three species to be undescribed: Stygarctidae gen. (?) sp. (Noda 1993), Renaudarctidae gen. (?) sp. (Noda 1994b) and Anisonyches sp. (Noda 1994c). Recently, Fujimoto and Miyazaki (2013) described a new species from a submarine cave off Shimoji Island, Miyako Islands. Table 3. Marine tardigrades reported from the Ryukyu Islands.
As noted above, at best the identifications are ambiguous, and verifying the identity of the species across published papers is difficult. Nonetheless, the data shows that Ryukyu Islands harbour a rich marine tardigrade fauna. With more research we can expect further species discoveries as many of the islands are unexplored and there are currently only seven species reported for the usually speciesrich sub-littoral zone.