﻿Description of two species of the genus Astrodia Verrill, 1899 (Ophiuroidea, Euryalida, Asteronychidae), including a new species from seamounts in the West Pacific

﻿Abstract Five specimens of Ophiuroidea from deep-sea seamounts in the West Pacific were collected and identified as two species, Astrodiaduospinasp. nov. and Astrodiaabyssicola. The new species, Astrodiaduospinasp. nov., can be distinguished from its congeners by having indistinct or underdeveloped oral papillae, relatively short genital slits, crescent-shaped lateral arm plates, and plate-shaped external ossicles on the aboral surface of the disc. One specimen was identified as Astrodiaabyssicola, which has been reported in the north-western Pacific and the north-eastern coast of Japan. The most recent tabular key of Astrodia was revised with two more key characteristics added, the shape and presence of oral papillae and the number of arm spines. The phylogenetic relationship of Astrodia and Asteronyx was analyzed based on 16S and COI sequences. The discovery of the two species further expanded the geographical distribution of the genus Astrodia.


Introduction
Class Ophiuroidea, as the largest group among echinoderms, with 2126 valid species , are widely distributed from the tropics to polar seas, and from the intertidal to the deep ocean. The Indo-Pacific, North Pacific, and South Pacific regions are reported to have relatively high ophiuroid species richness (Stöhr et al. 2012). Due to the technical limitations of deep sea exploration, the deep-sea ophiuroid fauna remains poorly known (Rodrigues et al. 2011). Seamounts are often of volcanic origin, with elevated topography from the deep-sea floor, which alters the flow of ocean currents and provides highly heterogeneous habitats serving as "hotspots" for deep-sea animals, especially for suspension-feeding epibenthic organisms (e.g. corals, sponges, and ophiuroids) (Yesson et al. 2011). Understanding the biodiversity of ophiuroids from seamounts will provide key information for the protection of this vulnerable ecosystem.
The order Euryalida Lamarck, 1816 comprises about 200 species from three families, Euryalidae Gray, 1840, Asteronychidae Ljungman, 1867, and Gorgonocephalidae Ljungman, 1867 ). Among these, Asteronychidae is the smallest family with only 12 extant species from four genera (Asteronyx Müller & Troschel, 1842, Astrodia Verrill, 1899, Astronebris Downey, 1967and Ophioschiza H.L. Clark, 1911. The genus Astrodia was erected by Verrill, in 1899 and currently comprises four species, Astrodia abyssicola (Lyman, 1879), Astrodia excavata (Lütken & Mortensen, 1899), Astrodia plana (Lütken & Mortensen, 1899) and Astrodia tenuispina (Verrill, 1884). Astrodia tenuispina was first described by Verrill (1884) under the name Asteronyx tenuispina, and was transferred to Astrodia by Verrill (1899). Koehler (1922) described a new species, Astrodia bispinosa, which was later regarded as a junior synonym of Astrodia tenuispina (Baker 1980). The most recent description of Astrodia plana was published by Döderlein (1927). Recently, Okanishi and Fujita (2014) reviewed this genus and transferred Ophiocreas abyssicola Lyman, 1879 to Astrodia. In their review, Okanishi and Fujita (2014) provided interspecific distinguishing characteristics including the shape and arrangements of external ossicles on the aboral surface of the disc, length of genital slits in relation to the height of the disc, the shape of the lateral arm plates, presence or absence of a projection of the lateral arm plates on the middle to the distal portion of the arms. Additionally, the geographical distribution of the four species was summarized (Okanishi and Fujita 2014).
In this study, we describe a new species, Astrodia duospina sp. nov., and redescribe Astrodia abyssicola, from seamounts of the West Pacific. New interspecific diagnostic characteristics were identified, and the tabular key of Okanishi and Fujita (2014) for the genus Astrodia was updated. DNA sequences were used to infer the phylogenetic relationship of the two species with their congeners.

Sample collection
Five specimens of Astrodia were collected by ROV HAILONG III, ROV HAILONG IV, and HOV JIAOLONG, from seamounts in the Philippine Sea and the Northwest Pacific, during several COMRA's cruises in 2013, 2020, and 2021 ( Fig. 1). All specimens were preserved in 95% ethanol on board the vessels and photographed using a digital camera (Canon EOS 5D), then deposited in the repository of the Second Institute of Oceanography, Hangzhou, China (RSIO).

Morphological analysis
Morphological characters were examined and photographed using a stereoscopic microscope (Zeiss Axio Zoom V16). Arm skeletons were examined with a Hitachi TM1000 scanning electron microscope. Skeletal elements were prepared by submerging in commercial bleach (2.5% NaOCl). Washed in distilled water and ethanol, air-dried, and mounted on a stub using dissolved carbon tapes. The following literature was used as references for the morphological analysis: Okanishi and Fujita (2014), Okanishi et al. (2018), Manso (2010), Baker (1980), andMartynov (2019).

Molecular analysis
Several arm segments were dissected from each individual for genomic DNA extraction using DNeasy Blood & Tissue Kit (QIAGEN) following the manufacturer's protocols. The COI sequences and 16S rRNA sequences were amplified with primers listed in Table 1. The PCR procedures were as follows: an initial denaturation step at 95 °C for 4 min followed by 35 cycles of 94 °C for 15 s, 50 °C for 30 s, and 72 °C for 1 min, and a final extension step at 72 °C for 10 min, for COI; an initial denaturation at 95 °C for 4 min, followed by 35 cycles of 94 °C for 15 s, 50 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 7 min, for 16S. PCR reactions were performed using 25 µL volumes containing: 1 µL of DNA template, 1 µL of each primer, 9.5 µL of dd H 2 O, and 12.5 µL of 2 × Phanta Max Master Mix (Vazyme, China). PCR products were purified with a QIAquick PCR purification kit (QIAGEN) following the protocol supplied by the manufacturer. Sequencing was performed by Sangon Biotech (Shanghai, China) on an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). Forward and reverse sequences were de novo assembled and edited using Geneious Prime 2021 (https://www.geneious.com), deposited in GenBank (COI: OP328780-OP328783; 16S: OP325290-OP325293).
Seventy-two 16S sequences and 28 COI sequences of Asteronychidae were downloaded from the NCBI. In total, 78 16S sequences and 34 COI sequences (Table 2), including four new 16S sequences and four new COI sequences were used for phylogenetic analysis, with two species of Asteroschema as the outgroup. COI and 16S were aligned using Clustal Omega (Sievers and Higgins 2014) as a plug-in in Geneious with default settings, respectively. Maximum likelihood trees were inferred based on a concatenated alignment of 16S and COI, as well as an alignment of 16S and COI respectively. IQ-TREE was used to perform the maximum likelihood bootstrap method (http://iqtree.cibiv.univie.ac.at/) (Nguyen et al. 2015), with the substitution model GTR+I+G, bootstrap support values determined by the ultrafast bootstrap algorithm for 100,000 replicates (Hoang et al. 2018). The best substitution model was selected by ModelFinder as a plug-in in IQ-TREE websites. (Kalyaanamoorthy et al. 2017).   Diagnosis. Disc raised high above the arm. Aboral disc with plate-shaped external ossicles in the center and on the periphery. Radial shield narrow, longer than wide. Teeth triangular, oral papillae indistinct or underdeveloped. Genital slits short, approximately one-fourth of the height of the disc. Lateral arm plates crescent and not projecting on arms. Arm spines no more than two.
Description of holotype. Disc pentagonal, notched interradial edges, 14 mm in diameter, 4.7 mm in height. Aboral surface almost flat, slightly depressed in the center, entirely covered by thickened skin with plate-shaped external ossicles in the center, about 220 µm long (Fig. 3A). Peripheral disc covered with a few plate-shaped external ossicles, similar to those in the center but larger, approximately twice in length. Radial  shields narrow, tumid, bar-like, without granules or spines, and almost reach center of disc (Fig. 3A, B). Approximately 7.2 mm long and 550 µm wide in the center and 1.1 mm wide at periphery.
Oral surface flat, covered by thickened skin. Oral shield small to invisible, one madreporite. Adoral shield obscured by skin (Fig. 3C). Oral interradial surface covered with several plate-shaped external ossicles (Fig. 3C). Six teeth, triangular, forming vertical row on dental plate, each jaw covered by a pair of conical oral tentacles (Fig. 3C). Oral papillae invisible or underdeveloped. Two genital slits, small, about 1/4 as long of disc height (1.3 mm long and 260 µm wide), present on oral side of each interradius (Fig. 3D). Gonads visible on each interradius (Fig. 3C, D).
Five arms, long and slender, about eight to nine times as long as disc diameter, no abrupt change in width basally (Fig. 3E). Proximal segments 2.5 mm wide and 1.7 mm high, with arched aboral surface and flattened oral surface (Fig. 3E), gradually tapering toward tip. Arm spines only present on ventral side. First to fourth tentacle pores with one arm spine and following tentacle pores with two arm spines. Outer arm spines slightly shorter than inner ones at proximal segments, but only three-fifths as long as inner spines on middle and distal segments (Fig. 3F). Color. Pink in situ, white in alcohol (Fig. 2). Ossicle morphology of holotype. Vertebrae articulation streptospondylous, wider than long in proximal segments (Fig. 4A, B), longer than wide in distal segments (Fig. 5A, B). Oral side of each vertebra with longitudinal groove along midline, deeply depressed, and no oral bridge (Figs 4C, 5C). Pair of podial basins on oral side moderate in size (Figs 4C, 5C). Aboral side of each arm vertebra with longitudinal aboral groove, moderately depressed (Figs 4D, 5D). Lateral furrow of vertebrae declining obliquely from aboral to oral side (Figs 4E-F, 5E-F). Lateral arm plates crescent-shaped, each associated with one or two arm spines and spine articulations with nerve and muscle opening separated. Spine articulation bulges outward (Fig. 6A,  C). A ridge on inner side of lateral arm plate, parallel to proximal edge (Fig. 6B, D). Arm spines cylindrical, never hooked, bearing fine thorns at tip throughout arms (Figs 3F, 6E-F).
Description of paratypes. Two paratypes (RSIO31004, RSIO61069) share the same morphological characteristics as the holotype, disc diameter 10.17 and 13.94 mm, about 1/10 and 1/9 as wide as the length of the arms, respectively. However, the radial shields of RSIO31004 are shorter than the radial shields of the holotype and of RSIO61069 (Fig. 7F). Three arm spines exceptionally occurred only once in both paratypes (RSIO31004 and RSIO61069), the innermost arm spine of RSIO61069 is the longest and the stoutest, while the middle arm spine of RSIO31004 is the stoutest. (Fig. 7A, B). The other paratype (RSIO59012) is smaller, only 6 mm in disc diameter, about 1/3 as wide as the length of the arms and may be a juvenile of this species. The radial shields and the genital silts are much shorter than in the other three specimens (Fig. 7C, D). Likewise, the arm spines are shorter than one segment (Fig. 7E) Etymology. The species name duo is derived from the Latin numeral word, meaning two, and Latin feminine noun, spina, meaning spine, referring to the presence of no more than two arm spines throughout the arm.
Remarks. This new species falls within the genus Astrodia by only possessing cylindrical unhooked arm spines. The new species resembles Astrodia abyssicola mostly by having plate-shaped external ossicles on the aboral disc and crescent-shaped lateral arm plates. However, the oral papillae are indistinct or underdeveloped in Astrodia duospina, which can be used to distinguish the two species from each other (Fig. 3D). Moreover, the genital slits are very short in Astrodia abyssicola, which are only onefifth of the height of the disc, while Astrodia duospina has larger genital slits, being longer than one-fourth the height of the disc (Fig. 3C). Astrodia duospina can easily be distinguished from A. plana and A. excavata by external ossicles and lateral arm plates. External ossicles are plate-shaped on the aboral surface of the disc in Astrodia duospina (Fig. 3A, B), but are absent in A. plana. Lateral arm plates are not projecting in the new species (Fig. 3E), but are distinctly projecting from the oral surface of the arm in A. excavata. Additionally, the new species differs from A. tenuispina by having distinctly smaller genital slits (Figs 3C, 7C).
Astrodia tenuispina is a widely distributed species and was characterized by having slender unhooked arm spines, small and short oral papillae, separated genital slits (Verrill 1884). Baker (1980) compared specimens from south of Australia and the northwest Atlantic, described this species with 2 or 3 arm spines, and imbricating punctate scales on the disc surface. Okanishi and Fujita (2014) redescribed this species as with plate-shaped external ossicles on the periphery of the aboral disc, granuleshaped on the central disc, genital slits half of the height of the disc, lateral arm plates not projecting. According to these descriptions, A. duospina sp. nov. can be differentiated from A. tenuispina by having smaller genital slits and indistinct oral papillae. Furthermore, in two of the three large specimens of the new species, three arm spines were observed exceptionally at one arm segment (Fig. 7A, B), while the other three species possess three arm spines at several successive segments in the middle part of the arms. Since only a small number of specimens were examined, this characteristic was not used to distinguish the new species from its congeners, and more specimens should be examined before a robust result can be achieved. Description. Disc pentagonal and almost flat, 10 mm in diameter, 3.2 mm in height, skin wrinkled under dry conditions (Fig. 9A, B). Aboral surface of disc lacks external ossicles (Fig. 9A, B). Radial shields narrow, slightly tumid, bar-like, with- out granules or spines, and almost reaching center of disc. (Fig. 9A). Approximately 3.8 mm long and 0.6 mm wide in center and 0.8 mm wide on periphery (Fig. 9A).
Five arms, long and slender, about nine to ten times as long as disc diameter, no abrupt change in width basally (Fig. 9E). Proximal portion of arm 1.8 mm wide and 420 µm high, with arched aboral surface and flattened oral surface. Arms tapering gradually toward tip. Arm spines only present in ventral part of arm. First to third tentacle pores without arm spines, fourth tentacle pores with one arm spine and following tentacle pores with two arm spines. Inner arm spines longer than outer arm spines. On middle and distal part of arm, outer arm spines three-fourths as long as inner spines (Fig. 9F). Three arm spines occurred once in two of the five arms. Lateral arm plates not projecting on arms. Color. Bright pink in situ, entirely white in alcohol (Fig. 8B, C). Ossicle morphology. Vertebrae articulation streptospondylous, wider than long in proximal segments (Fig. 10A, B), longer than wide in distal segments (Fig. 11A, B). Oral side of each vertebra with longitudinal groove along midline, deeply depressed, and no oral bridge (Figs 10C, 11C). Pair of podial basins on oral side moderate in size (Figs 10C, 11C). Aboral side of each arm vertebra with longitudinal aboral groove,moderately depressed (Figs 10D, 11D). Lateral furrow of vertebrae declining obliquely from aboral to oral side (Figs 10E,F,11E,F). Lateral arm plates crescentshaped, each associated with one or two arm spines. Spine articulations with separated nerve and muscle openings, bulging outwards (Fig. 12A, C). A ridge on inner side of lateral arm plate (Fig. 12D). Arm spines cylindrical, never hooked, bearing fine thorns at apex throughout arms (Fig. 12E, F).
Remarks. Ophiocreas abyssicola was first described by Lyman (1879). Okanishi and Fujita (2014) transferred O. abyssicola to the genus Astrodia and redescribed it. This specimen (RSIO68002) was identical to Astrodia abyssicola by having 0~2 arm spines, rather short genital slits and crescent-shaped lateral arm plates. However, this specimen lacks external ossicles on the disc and arms, which is different from previous descriptions of Astrodia abyssicola by Okanishi and Fujita (2014) as having plate-shaped external ossicles on the periphery. Nevertheless, the genetic distance of COI and 16S (2.9% and 1.9%) between the new collected specimen and A. abyssicola are too small to justify two different species. Therefore, this specimen was identified as A. abyssicola, thus the external ossicles on the aboral surface of the disc could be plate-shaped or absent in this species.

Key morphological characters to the species of Astrodia
The key morphological characters among the five species from the genus Astrodia based on Okanishi and Fujita (2014) were revised in this study (Table 3). Three diagnostic characteristics were proposed by Okanishi and Fujita (2014) in their key for Astrodia: the length of the genital slits related to the height of the disc, external ossicles on the aboral disc surface, and shape and existence of projections of lateral arm plates. All three characteristics were useful to distinguish the new species from its congeners. The external ossicle, being absent in the A. abyssicola specimen examined in the present study but present and plate-shaped in the previous descriptions (Okanishi and Fujita 2014), might be an intraspecific variation. Additionally, we added two morphological characters, the number of arm spines and the shape of oral papillae, as key characters for interspecific discrimination of Astrodia. Astrodia abyssicola is the only species that possesses no more than two arm spines along their arms, whereas the other four species possess up to three arm spines or occasionally four. Furthermore, oral papillae are indistinct or underdeveloped in A. duospina sp. nov., but are domed granule-shaped in the four known species. Thus, we consider the number of arm spines and the shape and existence of oral papillae important characteristics for interspecific discrimination within Astrodia (Table 3).

Molecular phylogenetic analysis
Based on the COI (583~1511 bp) and 16S (431~539 bp) sequences, the phylogenetic relationship of the two genera, Astrodia and Asteronyx, was inferred. The ML tree based on the concatenated 16S and COI sequences suggested that both Astrodia and Asteronyx were monophyletic with high bootstrap values (Fig. 13, Suppl. material 1: Fig. S1). The ML tree based on COI sequences was consistent with the tree generated Verrill (1884), Koehler (1906), Koehler (1922), Baker (1980), Gage et al. (1983) from two genes (Suppl. material 1: Fig. S2), while in the ML tree based on 16S sequences, Astrodia abyssicola clustered with Asteronyx, with a low bootstrap value (Suppl. material 3: Fig. S3). Okanishi et al. (2018) suggested that the relationship of the two genera was unclear based on COI and 16S sequences. With newly sequenced DNA data added, our results indicated that the two genera are probably monophyletic. Additionally, the genetic distances of CO1 and 16S between A. duospina sp. nov. and A. abyssicola were 9.0% and 9.1%, respectively, supporting the morphological identification results. Molecular analysis also supported that the three specimens identified as Astrodia duospina sp. nov. are the same species, and the specimen identified as Astrodia abyssicola is closely related to the published sequence of this species with very small genetic distances (2.9% for COI and 1.9% for 16S) that fall into the intraspecies genetic distance of Euryalida (Okanishi et al. 2018;Nethupul et al. 2022).

Conclusion
In this study, we described a new species of the genus Astrodia collected from seamounts in the West Pacific, and another species (Astrodia abyssicola) was redescribed. Through comparing the five species of Astrodia, the tabular key of Okanishi and Fujita (2014) was revised and two additional key characteristics, the number of arm spines and the shape of the oral papillae, were identified for interspecific discrimination of Astrodia. Maximum likelihood trees supported our morphological results and suggested that both Astrodia and Asteronyx were monophyletic. This study provided both morphological and molecular information of the two Astrodia species, and the specimens reported further expanded the known geological distribution of the genus.