A taxonomic review of the genus Astrocladus (Echinodermata, Ophiuroidea, Euryalida, Gorgonocephalidae) from Japanese coastal waters

Japanese species of the genus Astrocladus (Echinodermata, Ophiuroidea, Euryalida, Gorgonocephalidae) are reviewed. Astrocladus coniferus recently has two junior synonyms, A. dofleini Döderlein and A. pardalis Döderlein, however, status of these species has long been questioned. These species concepts have not been reviewed in recent years and no molecular phylogenetic analyses have been performed. Observations of the lectotype of A. coniferus, as well as the lectotype and four paralectotypes of A. dofleini and the holotype of A. pardalis have revealed that A. coniferus and A. pardalis are conspecific and morphologically distinguishable from A. dofleini. Astrocladus coniferus and A. dofleini are supported as distinct species by our molecular data. Additionally, we re-describe A. exiguus and A. annulatus, based on recently collected specimens and the holotype. We conclude that four species, A. annulatus, A. coniferus, A. dofleini, and A. exiguus are present in Japanese waters.


Morphological observation and terminology
Ossicles from Astrocladus coniferus (NSMT E-13118), A. dofleini (NSMT E-13124) and A. exiguus (NSMT E-13126) were isolated by immersion in domestic bleach (approximately 5% sodium hypochlorite solution), washed in deionized water, air-dried, and mounted on SEM stubs using double-sided conductive tape. The ossicles were observed and photographed with a Jeol JSM 5200LV SEM at Misaki Marine Biological Station, The University of Tokyo. A part of photographs were focus-stacked using the software CombineZM v. 1.0.0 (https://www.softpedia.com/get/Multimedia/Graphic/ Graphic-Editors/CombineZM.shtml). The size of external ossicles, represented by the length of the longest axis, it is referred to as "length" in this study.

DNA extraction, amplification and sequencing
Genomic DNA was extracted using DNeasy Blood and Tissue extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. We sequenced mitochondrial COI gene for phylogenetic analysis. The method of DNA extraction and PCR parameters followed Okanishi & Fujita (2013). A primer set of COI005 (5′-TTAGGTTAAHW AAACCAVYTKCCTTCAAAG-3′) and COI008 (5-CCDTANGMDATCATDGCRT ACATCATTCC-3′) (Okanishi & Fujita, 2013) was used for PCR of COI. The optimum cycling parameters for those COI primers consisted of an initial denaturation step of 94 C/2 min followed by 41 cycles of 94 C/30 s,48 C/90 s and 72 C/60 s with final extension step at 72 C/10 min was followed by storage at 4 C. The PCR products were separated from excess primers and oligonucleotides using Exo-SAP-IT (GEHealthcare, Chicago, IL, USA) by following manufacturer's protocol. All samples were sequenced bidirectionally and sequence products were run on the 3730xI DNA Analyzer of Misaki Marine Biological Station. The accession numbers of the DNA Data Bank of Japan (DDBJ) are shown in Table 1.

Phylogenetic analysis
We sequenced 2 specimens of Astrocladus coniferus, 5 specimens of Astrocladus dofleini and 2 specimens of A. exiguus, but sequence data from a single specimens of A. dofleini (referred as A. coniferus in Okanishi & Fujita (2013, 2018) and A. exiguus obtained by Okanishi & Fujita (2013, 2018 were also used. A. exiguus was added to the analysis to compare genetic distances and to determine the taxonomic rank of each phylogenetic group within Astrocladus. For outgroups, we selected 6 species of the subfamily Gorgonocephalinae with the shortest genetic distance from Astrocladus to avoid long branch attraction (Bergsten, 2005;Okanishi & Fujita, 2018). These species were also used in a previous molecular phylogeny (Okanishi & Fujita, 2013, 2018. All sequences were aligned using the Clustal W algorithm in MEGA X (Thompson, Higgins & Gibson, 1994;Kumar et al., 2018). All missing sequences were scored as gaps. Overall average of substitution rate was computed using MEGA X according to the Kimura 2-parameter model (Kimura, 1980) to compare the evolutionary rate of each gene. The K2P, HKY and TN93 with gamma distributions were selected as best-fit models of the first, second and third codons, respectively (Kimura, 1980;Hasegawa, Kishino & Yano, 1985;Tamura & Nei, 1993), by using the "Find best fit models" option of MEGA X. Seaview ver. 4.3.0 (Gouy, Guindon & Gascuel, 2010) were used in preparing the data matrices in PHYLIP format.
Figtree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) was used in exploring tree files, in preparing NEWICK format and exploring alternative tree topologies. The phylogenetic tree was constructed with RAxML ver. 8.1.20 (Stamatakis, 2014) for maximum likelihood analysis (ML) to obtain bootstrap support values and with MrBayes v. 3.1.2 (Ronquist & Huelsenbeck, 2003) to obtain Bayesian posterior probabilities (BPP). The Markov-Chain Monte-Carlo (MCMC) process was run with four chains for 3,000,000 generations, with trees being sampled every 100 generations. The first 7,500 trees were discarded as burn-in. Data sets were partitioned by each codon for the maximum likelihood analysis to allow for separate optimization per-site substitution rates. The best-supported likelihood tree was found by performing 1,000 replications.
K2P genetic distances were computed within each clade and between clades using MEGA X to compare the evolutionary rate of COI gene.

Diagnosis
Arms five, branching. Number of arm segments less than six before first branch. No rows of calcareous plates on edge of disc margin. One madreporite situated on oralmost portion of interradial lateral disc. Arm spines present before the first branch. Hooklets on dorsal arms with one secondary tooth. Disc covered by variously shaped external ossicles and/or large tubercles (Döderlein, 1927;Baker, 1980;McKnight, 2000).

Remarks
Our molecular and morphological results have confirmed that Astrocladus pardalis ) is a junior synonym of A. coniferus  which can be separated from A. dofleini (Döderlein, 1910). Therefore, 11 species are now known in this genus (see list of "Included species" below); A. annulatus, A. coniferus, A. dofleini and A. exiguus are distributed in Japan.

Notes on lectotype
In the original description , Astrophyton coniferum (=Astrocladus coniferus) was thought to have been described based on two specimens which are listed in a table (Döderlein, 1902, P326). They were collected in Kagoshima Bay at, ca. 30 m depth and subsequently, one of them was figured by the same author in 1911 as "Typus" of Astrocladus coniferus (Döderlein, 1911, Taf 4, 2-2a). This is in accordance with §75.4 of the International Code of Zoological Nomenclature, and it can be regarded as the lectotype. The morphological traits of ZSM 20000897 concur with this figured specimen. Therefore, ZSM 20000897 is the lectotype.

External morphology of the lectotype (ZSM 20000897)
Disc. Disc five-lobed with notched interradial edges, 60 mm in diameter (Fig. 2C). Dorsal disc wholly covered by external ossicles in contact with each other. Radial shields completely covered by granules and conical external ossicles, approximately 110-450 mm in length; other areas covered by smaller granules, approximately 100 mm in length (Fig. 2F). Radial shields tumid, bar-like, approximately 50 mm in length, width gradually decreasing from 10 mm at disc periphery to 2.5 mm almost at disc center (Fig. 2E). One large conical tubercle on peripheral edge of each radial shield, 2.5-3.4 mm in length (Fig. 2E).
Ventral surface of disc completely covered by skin and polygonal plate-like external ossicles, fully in contact, approximately 600 mm in length (Fig. 2G). Oral shields, adoral shields, oral plates and ventral arm plates completely concealed by ossicles (Fig. 2G). Teeth uniformly spiniform, situated on top of dental plates (Fig. 2G). Teeth arranged in a cluster covering ventral-most part of dental plate approximately 10 in number (Fig. 2G), and in a vertical line, on dorsal part of dental plates, 3 or 4 in number. Spiniform oral papillae situated in 1 or 2 transverse rows on ventral edge of each oral plate, 4 to 5 in number (Fig. 2G). Size of teeth varying in position on jaw, approximately 400-1000 mm in length and oral papillae approximately 400 mm in length (Fig. 2G).
Interradial surface of lateral disc covered by granules fully in contact, approximately 100 mm in length (Figs. 2G and 2H). Two genital slits (5 mm long and 1-3 mm wide) in each interradius (Fig. 2G). One large, elliptical madreporite situated on ventral interradius, approximately 5.5 mm in width and 3.75 mm in length (Fig. 2H).
Arms. Arms branching. On proximal portion before first branch, arm 8.5 mm wide with arched dorsal surface and flattened ventral surface (Fig. 3A). Between first branch and second branch, arm width abruptly decreases to 5.8 mm. Subsequently, arms tapering gradually toward arm tip (Fig. 3).
On dorsal and lateral surface, each arm segment covered by single annular row of large hooklet-bearing plates (Figs. 3A and 3B). Before second branch, each plate separated by granules. Plates fully in contact from third branch and subsequent distal segments (Fig. 3B). With exception of hooklet-bearing plates dorsal and lateral surface of arm completely covered by granules, fully in contact, approximately 200-400 mm in length (Fig. 3A). Before first branch, ventral surface covered by polygonal plate-like external ossicles, similar to those on ventral disc, approximately 100 mm in length (Fig. 3C). After first branch, ossicles become into round granules, slightly in contact, and decreasing in size gradually toward arm tip (Fig. 3D). With exception of the articulations with arm spines and/or hooklets, lateral arm plates and ventral arm plates completely concealed by external ossicles on entire arm (Figs. 3C and 3D). Tentacle pore with single arm spine before first branch; 2 or 3 spines after first branch; up to 4 spines on subsequent pores (Figs. 3C and 3D). Number of arm spines decrease gradually to 2 towards arm tip. Arm spines approximately one-seventh to one-eighth (ca. 12-13%) of length of the corresponding arm segment on proximal portion of arm, and one-thirds to one-fourth length of the corresponding arm segment on middle to distal region of arm (Figs. 3C and 3D).
Living color of NSMT E-13118: Dorsal disc (diameter = 30 mm) vivid orange with yellow patches, arms with yellow transverse bands on dorsal side (Fig. 4G). Ventral side of arms and disc uniformly creamy white, with orange arm tip (Fig. 4H).
Ossicle morphology of NSMT E-13118: All arm hooklets with single inner tooth and reticular structures (Figs. 5A-5C). Inner tooth on distal portion of arm smaller and more rudimentary than those from proximal to middle portion of arm. Hooklet-bearing plates with 9 or 10 tubercle-shaped articulations for hooklets in proximal portion of arm; articulations forming 2 parallel rows (Fig. 5D). On proximal to middle portion of arm, lateral arm plates longer than high, curved to distal side (Figs. 5E, 5F, 5I and 5J). On proximal portion of arm, simple muscle openings besides border structures on distal edge (Fig. 5F), and on middle portion of arm, nerve openings on internal side of the muscle openings (Fig. 5I). One perforation present on internal side (Fig. 5J). On distal portion of arms, plate square, at least one nerve opening of articulation for arm spine beside border structure and one articulation for hooklet on distal side (Fig. 6A). No perforations recognizable on internal side (Fig. 6B). On proximal and middle portion of arm, arm spines ovoid, with three or four terminal projections, approximately one-third to one-fourth length of the height of arm spine (Figs. 5G, 5H, 5K, and 5L). In distal portion, arm spines transformed into hooks with one inner secondary tooth (Fig. 6C). Hook-shaped arm spines distinguished from hooklets by lack of reticular structure (Figs. 5A-5C and 6C).
All vertebrae with hourglass-shaped streptospondylous articulations (Figs. 6D, 6F, 6G, 7C, and 7D). In middle portion of arm, surfaces of lateral furrows smooth, with tubercle shaped ornamentations on dorsal side (Fig. 7B). In distal portion of arm, depressions for tube feet openings in distal part of ventral-lateral side of vertebrae but these are unrecognizable on proximal to middle portion of arm (Figs. 7E). A pair of channels for passage of lateral nerves opening inside ventral furrows (Figs. 6E, 7A, and 7E). In middle portion of arm channels for passage of lateral canals also opening on distal side of canals of lateral nerves (Fig. 7A). Channels for lateral nerve obscured in distal portion of arm (Fig. 7E).

Notes on lectotype
In the original description (Döderlein, 1910), this species was based on a specimen in the Peabody Museum of Natural History (Yale University) which was reported as A. verrucosus by Verrill (1899), plus several specimens collected from Japan. Subsequently, one of them was figured by , and named the "Typus" of Astrocladus coniferus. In accordance with §75.4 of the International Code of Zoological Nomenclature, the author "unambiguously selected a particular syntype to act as the unique name-bearing type of the taxon", namely the lectotype. The morphological traits of one of the five syntypes, ZSM 20000901/1 concur with the specimen figured by   Döderlein, 1910, Okinose, Sagami Bay, 600 m, Japan. These four specimens of ZSM were probably collected with Zuso-Maru (see also Doflein, 1906

Description of external morphology of the lectotype (ZSM 20000901/1)
Disc. Disc circular with slightly notched interradial edges, 53 mm in diameter (Fig. 8C). Radial shields tumid (Fig. 8C). Dorsal disc wholly covered by granules in contact each other and domed large tubercles (Fig. 8C). Radial shields covered by granules, approximately 200-330 mm in length (Fig. 8C). and large domed tubercles, 20-25 on each radial shield in number, each approximately 2.5-3.4 mm in length (Fig. 8C). Radial shields bar-like, approximately 25 mm in length, and the width gradually decreasing from 4.6 mm at disc periphery to 1.5 mm almost reaching to disc center (Fig. 8C).
Ventral surface of disc covered by polygonal plate-shaped external ossicles, fully in contact. Oral shields, adoral shields, oral plates and ventral arm plates concealed by ossicles (Fig. 8E). Teeth uniformly small, granule-like, situated on the top of dental plates, forming a cluster, approximately 6 to 8 in number (Fig. 8E). Oral papillae on the ventral edges of oral plates, forming a transverse row on each plate, 1 or 2 in number (Fig. 8E). Interradial surface of lateral disc covered by granules fully in contact, approximately 170 mm in length, and domed tubercles, approximately 200-500 mm in length (Fig. 8E). Two pore-like genital slits (6.5 mm long, 3 mm wide) in each interradius (Fig. 8E).
Arms. Arms branching. On proximal portion before first branch, arm 16 mm wide with an arched dorsal surface and flattened ventral surface (Figs. 8D and 8E). Between first branch and second branch, arm width abruptly decreasing to 10 mm. Subsequently, arms tapering gradually toward arm tip (Figs. 8F and 8H).
On dorsal and lateral surface, each arm segment covered by single annular row of hooklet-bearing plates. Before third branch, each plate separated by granules. The plates fully in contact from fourth branch and subsequent distal segments. With exception of hooklet-bearing plates, dorsal and lateral surface of arm completely covered by granules, fully in contact, approximately 100 mm in length. Before the first branch, ventral surface covered by polygonal and plate-shaped external ossicles, similar to those on ventral disc. After the first branch, the ossicles transforming granules, slightly in contact, and decreasing in size gradually toward the distal arm tip. With exception of the articulations with arm spines and/or hooklets, lateral arm plates and ventral arm plates concealed by external ossicles on entire arm (Figs. 8E, 8G, and 8H). Tentacle pore with single arm spine after the first branch; 1 or 2 spines after second branch; and up to 4 spines for the subsequent pores (Figs. 8G and 8H). Distally, the number of arm spines decreasing gradually to 2 toward arm tip. Arm spines approximately one-fourth to one-fifth of length (ca. 20-25%) of corresponding arm segment on proximal portion of arm, and one-thirds to one-fourth length of corresponding arm segment on middle to distal arm segment (Figs. 8G and 8H).
Color. Uniformly dull brown with whitish large tubercles in ethanol preserved specimen (Fig. 8).

Description of other materials
Ossicle morphology of NSMT E-13124: Each hooklet on proximal and middle portion of arm with single inner secondary tooth, distal portion of arm with two inner secondary teeth. All hooklets with reticular structures (Figs. 9A, 9B, 9D). Hooklet-bearing plates with 6, 8 and 6 tubercle-shaped articulations for hooklets in proximal, middle and distal portion of the arm, respectively; articulations forming 2 parallel rows (Figs. 9C, 9E, and 10B).
On proximal portion of arm, lateral arm plates long, with straight both proximal and distal edges (Figs. 9G and 9H); edges ellipse-shape in middle (Figs. 9J and 9K) and distal (Figs. 10B and 10C) portion of arms. No perforation observed on ventral side (Figs. 9H, 9K, and 10C). Pairs of simple nerve and muscle openings of articulation for arm spine with border structures on external edge (Figs. 9G, 9J, and 10B). On middle to distal portion of arms, lateral arm plates carrying 5 or 6 articulations for hooklets on external edge beside the articulation for arm spine (Figs. 9J and 10B). On proximal to middle portion of arm, arm spines ovoid, with three projections, approximately one-thirds to one-fifth to one-sixth length of the height of spine (Fig. 9F). In distal portion, arm spines transformed into hooks with two inner secondary teeth (Fig. 10A). Hook-shaped arm spines distinguished from hooklets on dorsal and lateral surface of arm by lack of reticular structure (Figs. 9A, 9B, 9D, and 10A).
All vertebrae with hourglass-shaped streptospondylous articulations (Figs. 10D, 10E, 11A, 12F, and 12G), and distal side of branching vertebra slightly wider than non-branching vertebra due to their possession of 2 articulation surfaces (Figs. 10H, 11C, and 12C). Lateral furrows of vertebrae ornamented by tubercles in proximal to middle portion of arm, but smooth in distal portion of arm (Figs. 10F, 11D, and 12B). Depressions for tube feet openings in distal part of ventral-lateral side of vertebrae (Figs. 10G, 10H, 11C, and 12A). In proximal to middle arms, a pair of the channels for passages of lateral canals opening inside of ventral furrow, near depression of tube feet entire arms, and distal side of the channels, a pair of the channels for passage of lateral nerves opening (Figs. 10G, 10H, and 11C). They are unrecognizable at distal portion of the arm (Figs. 10G, 10H, 11C, 12A, and 12C).

Discussion
Döderlein (1902) described A. coniferus and A. pardalis on the basis of presence/absence of a large conical tubercle on the distal end of each radial shield. Subsequently, he determined that the presence/absence was an intra-specific character and the synonymized A. pardalis with A. coniferus .
In our study, although examinations of the lectotype of A. coniferus (ZSM 20000897) and the holotype of A. pardalis (ZSM 20000898) confirmed these morphological differences between the two specimens (Figs. 2E and 4C), monophyly of two additional specimens which morphologically agree with the lectotype of A. coniferus (NSMT E-13118) and the holotype of A. pardalis (NSMT E-13119) was confirmed by our molecular phylogeny (see also "Molecular phylogeny" below). Thus, we follow Döderlein's decision to synonymize these two species.  also distinguished Astrocladus coniferus and A. dofleini as follow: A. coniferus possesses conical external ossicles but lacks large tubercles on dorsal surface of proximal portion of arms, whereas A. dofleini possesses only granules and large tubercles on the same position of the arms. Matsumoto (1917) made the two species conspecific based on the existence of specimens showing intermediate features (Matsumoto, 1917) and Fujita & Kohtsuka (2003) followed this classification. Irimura (1982) distinguished A. coniferus (as "A. coniferus coniferus") and A. dofleini (as "A. coniferus var. dofleini") based on presence/absence of large tubercles on dorsal surface of proximal arms. However, Irimura (1982) did not recognize any morphological features between the "A. coniferus var. dofleini" and A. pardalis (as "A. coniferus var. pardalis") except color differences.
In addition to the types of A. coniferus and A. pardalis, we also studied four paralectotypes (ZSM 20000901/2, ZSM 20000901/3, ZSM20000901/4 and ZMB 5923) of A. dofleini and confirmed that the Döderlein's diagnostic characters can not distinguish these species, because: Ossicles on dorsal surface of proximal portion of arms were granular and conical in A. coniferus (Figs. 3A and 4F) and granular in A. dofleini (Fig. 8D). Both A. coniferus (Fig. 3A) and A. dofleini (Fig. 8D) possess large tubercles on dorsal surface of proximal arms.
Instead, A. coniferus can be distinguished from A. dofleini by the following three morphological characters: (1) Shape of ossicles: Ossicles on periphery of radial shields were conical in A. coniferus (Figs. 2F and 4D) but those of A. dofleini (Fig. 8C) were granules.
(3) Distribution of the large tubercles: Although the large tubercles were only on the peripheral edges of radial shields (Fig. 2F) or absent (Fig. 4C) in A. coniferus, those of A. dofleini were scattered on the dorsal surface of the disc (Fig. 8C).
These differences were also recognized in other examined materials: 2 specimens of A. coniferus (NSMT E-13118 and NSMT E-13119); and 6 specimens of A. dofleini. Therefore, we conclude that A. coniferus and A. pardalis are conspecific and distinct from A. dofleini. Our molecular phylogenetic analysis also supports this conclusion (see "Molecular Phylogeny" below).
Additionally, color may also differ in these specimens. On the dorsal side, the 2 examined specimens of A. coniferus are vivid orange with yellow patches and arm bands, and the 6 NSMT specimens of A. dofleini are uniformly black with small black patches and arm bands, or yellow with light yellowish small patches and arm bands (Fig. 7). However, we refrain from employing these color variations as diagnostic characters because other color patterns for these species have been recorded (Irimura, 1982).

Description of external morphology (NSMT E-13126)
Disc. Disc five-lobed with notched interradial edges, approximately 26 mm in disc diameter. Radial shields and surrounding plates slightly tumid (Fig. 13B). Dorsal disc covered by variously sized conical ossicles, which bear spiny projections on their apices (Figs. 13A and 13B). The larger conical external ossicles separated and scattered, approximately 140-1150 mm in length, having several thorny apical projections (Fig. 13A). Radial shields concealed by ossicles (Figs. 13B and 13C). One large tubercle situated on distal edge of each radial shield, approximately 1.7 mm in diameter (Fig. 13A).
Ventral surface of disc covered by polygonal plate-like ossicles, fully in contact, approximately 170-500 mm in length. Ossicles on ventral plates granule-shaped, approximately 130 mm in length (Fig. 13C). Oral shields, adoral shields, oral plates and ventral arm plates concealed by ossicles (Figs. 13C-13E). Teeth uniformly spiniform, situated on top of dental plates and edges of ventral plates (Fig. 13C). Teeth arranged in 1 or 2 transverse rows on ventral plates, approximately 10 in number (Fig. 13C), in a cluster covering ventral-most part of dental plate, approximately 15 in number (Fig. 13C), and in a vertical line, on other areas of dental plates, 2 in number. Size of teeth varying in position on oral and dental plate, approximately 0.3-1 mm in length, 0.3 mm in greatest width on dental plates, and 1 mm in length, approximately 0.2 mm in width on ventral plates (Fig. 13C).
Interradial surface of lateral disc covered by conical ossicles similar to those on dorsal disc (Fig. 13D). They are fully in contact, approximately 40-100 mm in length (Fig. 13D). Two genital slits (0.9 mm long and 0.2 mm wide) in each interradius (Fig. 13D). One large, elliptical madreporite situated on ventral interradius, approximately 0.65 mm in width and 0.35 mm in length (Fig. 13D).
Arms. Arms branching. On proximal portion of arm, before first branch, arm 12.0 mm wide and 5.5 mm high, with an arched dorsal surface and flattened ventral surface. Between first branch and second branch, arm width and height abruptly decreasing to 4.3 mm in width and 3.0 mm in height. Subsequently, arms tapering gradually toward arm tip (Figs. 13E-13J).
On dorsal and lateral surface of middle to distal portion of arms, each arm segment covered by single annular, ring-like row of large oblong plates, approximately 700 mm in transverse length (Figs. 13I and 13J). Before third branch, each plate separated by granules. Plates fully in contact from fourth branch and on subsequent distal segments (Fig. 13I). Before third branch, no hooklets (Fig. 13H), after fifth branch, plates appearing and forming an annual band (Fig. 13I). With exception of hooklet-bearing plates, dorsal and lateral surface of arm completely covered by conical, plate-shaped and domed granule-shaped ossicles (Figs. 13H and 13I). Proximal portion of dorsal arm covered by conical ossicles similar to those on dorsal disc, approximately 0.3-1.5 mm in length slightly separated, and plate-shaped external ossicles, fully in contact, approximately 200 mm in length (Fig. 13H). Middle portion of dorsal arm covered by domed granules, approximately 170-220 mm in length, and plate-shaped ossicles, approximately 110 mm in length (Fig. 13I). The larger conical ossicles sometimes carry spiny projections. Distal portion of dorsal arm covered by granule-shaped external ossicles, approximately 50 mm in length (Fig. 13J). In proximal to middle portion of arms, ventral surface covered by polygonal and plate-shaped ossicles, similar to those on ventral disc, fully in contact, approximately 150-250 mm in length at proximal region, and 60-260 in length distally ( Fig. 13H and 13I). Distal portion of ventral arm covered by granule-shaped external ossicles, slightly in contact, approximately 40 mm in length (Fig. 13J). With exception of the articulations with arm spines and/or hooklets, lateral arm plates and ventral arm plates concealed by skin and ossicles (Figs. 13E-13G). First to fifth tentacle pore without single spine; sixth pores with 1 spine, seventh and subsequent pore with 2 or 3 spines (Fig. 13E). Distally, the number of arm spines decrease gradually to 2 toward arm tip (Fig. 13G). Arm spines approximately one-third to one-fourth of length (ca. 25-35%) of corresponding arm segment, covered by thin integument (Figs. 13E-13G).
Color. Dorsal surface dark brown with whitish patches on disc and bands on arms. Ventral surface whitish but slightly brown on disc.
Ossicle morphology of NSMT E-13126: Each hooklet with single inner tooth and reticular structure (Figs. 14A and 14C). Hooklet-bearing plates with 4 tubercle-shaped articulations for hooklets in proximal portion of the arm (Fig. 14B), approximately 5 articulations in distal portion (Fig. 14D); articulations forming 2 parallel rows (Figs. 14B and 14D). Lateral arm plates long, both distal and proximal edges straight (Fig. 14E). On proximal portion of arm, lateral arm plates without perforation-like structures on dorsal side and pairs of simple nerve and muscle openings on ventral-external side (Figs. 14E and 14F) and on distal portion of arms, no perforation-like structure on dorsal side and a pair of nerve and muscle openings of articulation for arm spine beside boarder structure and 4 articulations for hooklets on ventral surfaces (Figs. 14G and 14H). Arm spines in proximal portion of arm ovoid, with four small projections, approximately one-third the height of the height of spine (Fig. 14I). In distal portion, arm spines transformed into hooks with 2 inner secondary teeth, respectively (Fig. 15A).
All vertebrae with hourglass-shaped streptospondylous articulations (Figs. 15B, 15C, 15G, and 15H), and distal side of branching vertebra slightly wider than in non-branching vertebra and with 2 articulation surfaces (Figs. 15E and 16B). Surfaces of lateral furrows smooth, with no special ornamentation (Figs. 15F and 16A). Depressions for tube feet openings in distal part of ventral-lateral side of vertebrae (Figs. 15D, 15E, and 16C). Two pairs of the channels for passages of lateral canals and lateral nerves opening on ventral groove of vertebrae in proximal portion of arm (Figs. 15D and 15E). In distal portion of arm, only radial water canal observed (Fig. 16B). External ossicles on dorsal periphery of radial shields conical, approximately 150 mm in length and 200 mm in height with a spiny apical projection, approximately 100 mm in length (Fig. 16D).
Distribution. Widely distributed in Indo-Western Pacific Ocean. Depth range 18-494 m.

Description of holotype (UMUTZ-Ophi-26)
Disc. Disc five-lobed with notched interradial edges, 22 mm in diameter. Dorsal disc covered by granules, approximately 140-280 mm in length (Fig. 17B) Radial shields and their surrounds tumid, concealed by ossicles (Fig. 17A), approximately 1.1 mm in length, almost reaching disc center (Fig. 17A). Large domed tubercles, approximately 450 mm in length scattered on radial shields (Fig. 17B). Ventral surface of disc covered by polygonal plate-like ossicles, fully in contact, approximately 160-200 mm in length (Fig. 17D). Oral shields, adoral shields, oral plates and ventral arm plates concealed by ossicles (Fig. 17D). Teeth uniformly spiniform, on top of dental plates and edges of ventral plates (Fig. 17D). Teeth approximately 8, arranged in 1 or 2 transverse rows on ventral plates in a cluster covering ventral-most part of dental plate, approximately 10 in number (Fig. 17D). Size of teeth variable, approximately 1 mm in greatest length on dental plates, approximately 0.5 mm on oral plates (Fig. 17D). Interradial surface of lateral disc covered by thick skin (Fig. 17E). Two genital slits (4.5 mm long and 1 mm wide) in each interradius (Fig. 17E). One small, elliptical madreporite on ventral interradius.
Arms. Arms branching. On the proximal portion, before first branch, arm 4.3 mm wide and 3.5 mm high, with an arched dorsal surface and flattened ventral surface (Figs. 17A and 17C). Between first and second branch, arm width and height abruptly decreasing to 3 mm in width and 1.8 mm in height. Subsequently, arms tapering gradually toward arm tip (Figs. 17A and 17C).
On dorsal and lateral surface, each arm segment covered by single annular row of large oblong plates (Figs. 17G and 17H). With exception of hooklet-bearing plates, dorsal and lateral surface of arm completely covered by polygonal plate-like ossicles, approximately 170-290 in length at proximal portion of arms, and subsequently decreasing in size to arm tip (Figs. 17G and 17H). Ventral side of arms covered by skin which completely conceals the external ossicles, lateral arm plates and ventral arm plates, with exception of the part where lateral arm plates and arm spines articulating (Figs. 17I and 17J). Tentacle pores without arm spines before first branch; 3 or 4 spines after second branch. Distally, number of arm spines decrease gradually to 1 towards arm tip (Fig. 17J). In proximal portion, arm spines approximately one-fourth to one-fifth of length of corresponding arm segment, and covered by thin integument; subsequently relative length increase, exceeding half length of corresponding arm segment on distal portion of arm (Fig. 17J).

Discussion
Astrocladus annulatus was originally described by Matsumoto (1912a) based on the holotype collected from off Misaki, Sagami Bay. It has never been re-collected from the type locality and never re-described so far. In our examination of the holotype, we confirmed the diagnostic character of this species, namely granules on dorsal surface of body (Fig. 17B) and continuous hooklet-bearing plates on proximal portions of arms (Matsumoto, 1912a; Table 2). between A. dofleini and A. exiguus, and 14.6% between A. coniferus and A. exiguus, respectively. Intra-clade distance (0.67 to 1.3%) was about ten folds smaller than inter-clade distance (13 to 14.7%).

DISCUSSION
Our molecular phylogenetic analyses suggest that A. exiguus, A. coniferus and A. dofleini should be assigned to separate taxa. Genetic distance analysis showed that the inter-clade distances exceed intra-clade values. In previous studies of ophiuroids, genetic distance corresponding to species differences range from approximately 2.2-23% (Okanishi & Fujita, 2018). Therefore, the distances between current clades (13-14.7%) are within this range. In our analysis, we found that A. coniferus and A. dofleini form a clade (Clade 3). Therefore, a possible classification would be to unite A. coniferus and A. dofleini as the same species (A. coniferus) and subdivide A. coniferus coniferus and A. coniferus dofleini under A. coniferus, as has been done in the past (Fedotov, 1926;Irimura, 1982). However, since the genetic distance between A. dofleini and A. coniferus is comparable to the distance of the two species from A. exiguus, which is considered to be a separate species in terms of morphology, A. dofleini and A. coniferus are herein shown to be separate species.

CONCLUSIONS
In the present study, morphological observations of type and non-type specimens revealed that Astrocladus pardalis ) is a junior synonym of A. coniferus . Morphological observations and molecular phylogenetic analysis revealed that A. coniferus and A. dofleini (Döderlein, 1910) are different species. Therefore, 4 species, A. annulatus, A. coniferus, A. dofleini and A. exiguus occurin Japan. Additional molecular analyses including A. annulatus and examination of type specimens of A. exiguus are required to finally clarify the taxonomy of Japanese basket stars of the genus Astrocladus. by the integrated research, "Geological, biological, and anthropological histories in relation to the Kuroshio Current" and "Spatiotemporal Analyses on Origins and Properties of the Biodiversity Hotspots in Japan", conducted by the National Museum of Nature and Science. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.