Morphological Description and Molecular Analysis of Newly Recorded Asteroid, Henricia djakonovi Chichvarkhin, 2017 (Asteroidea: Spinulosida: Echinasteridae), from Dokdo Island, Korea

: We recently collected the samples of Henricia from adjacent waters of Dokdo Island, Korea, using trimix SCUBA diving. Based on a combined result of morphological and molecular analysis, we identiﬁed our specimen as Henricia djakonovi Chichvarkhin, 2017, which is newly recorded in Korea. Morphologically, H. djakonovi has crescent abactinal plates bearing numerous pillar-shaped abactinal spines with a droplet-like apical tip. Moreover, molecular analysis based on the mitochondrial COI gene occurred that clearly distinguished H. djakonovi from other species of Henricia in the pairwise genetic distance and maximum likelihood analysis. Accordingly, 15 species of Henricia are recorded in Korean fauna, including H . djakonovi .


Introduction
The family Echinasteridae Verrill, 1867 is the only family in the order Spinulosida Perrier, 1884 of the class Asteroidea de Blainville, 1830. This is a small but significant family with just eight genera and a variety of taxa assigned to it [1]. Echinasterids are epifaunal animals that live on the seabed, rock, sand, gravel, or mud at a depth of 6 to 904 m [2]. The genus Echinaster Müller and Troschel, 1840 and Henricia Gray, 1840 are the genera in which the majority of the species are found. Henricia is found mostly in cold waters, including polar habitats and abyssal locations [3]. In the north Pacific Ocean, most of the echinasterids, including Henricia, are directly associated with the huge encrusting sponge fauna [4]. Henricia in the Atlantic are usually found in rocky subtidal zones [5]. The global diversity of sea stars is believed to be considerably underestimated due to taxonomic issues and sampling biases [6]. In three coastal regions (East Sea, Korea Strait, and Yellow Sea) of Korea, Henricia diversity patterns show a higher species richness in the East Sea and lower richness levels recorded in the Yellow Sea [7,8]. A total of 14 [7,[9][10][11][12]. According to studies of previous taxonomists, identifying species of Echinasteridae is particularly difficult for a variety of reasons, including: (1) the characteristics of these sea stars are highly variable; (2) they are capable of interbreeding with each other where they overlap to produce intermediates; and (3) they are capable of interbreeding [13,14].
DNA barcoding sequence variation in a 658-base pair (bp) region of the mitochondrial cytochrome c oxidase subunit I (COI) gene is a potent tool for the identification and Taxonomy 2023, 3 47 discovery of species [15,16]. The region of the COI sequence has been validated as an efficient tool for species discrimination in echinoderms [16][17][18]. This research aimed to add knowledge to the taxonomy of the Henricia species in South Korea and the surrounding regions by providing a complete morphological description and molecular analysis of H. djakonovi.

Materials and Methods
A specimen of Henricia was collected by trimix SCUBA diving from a depth of 33.7 m on Dokdo Island of Korea on 8 July 2022. The collected specimen was preserved in 95% ethyl alcohol solution immediately. Morphological characteristics such as the size of the disk, the upper and proximal portions of arms, the number of abactinal spines, the shapes of abactinal and actinal skeletons, and the number of adambulacral spines were examined. The morphological features of the specimen were photographed using a scanning electron microscope (JSM-6510; JEOL Ltd., Tokyo, Japan), a stereomicroscope (Nikon SMZ1000; Nikon Co., Tokyo, Japan), and a digital camera (Nikon D7000). The abbreviations used for measurements were the same as those used by Ubagan and Shin [19].
Total genomic DNA was isolated from ethanol-preserved tube feet tissues using a DNeasy blood and tissue DNA isolation kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Genomic DNA quality and concentration were determined using a Nanodrop One-C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All genomic DNA samples were stored at −20 • C until further use. The partial sequence of the mitochondrial COI gene (658 bp) was amplified using a pair of primers conserved in echinoderms, Hen_LCO1490m (unpublished) and Hen_HCO2198m (unpublished). PCR was performed using an AccuPower PCR PreMix kit (Bioneer, Daejeon, Republic of Korea) in a total volume of 20 µL in accordance with the manufacturer's instructions. After 1.5 µL of template DNA, 1 µL of each primer at 10 pmol, and 16.5 µL of distilled water were added to AccuPower PCR PreMix, polymerase chain reaction (PCR) was performed with the following amplification parameters: an initial denaturation at 94 • C for 3 min, 35 cycles of denaturation at 94 • C for 30 s, annealing at 48 • C for 45 s, and extension at 72 • C for 1 min, followed by a final extension step at 72 • C for 7 min. The PCR product quality was determined by 1.5% agarose gel electrophoresis staining followed by staining with an EcoDye TM Nucleic Acid Staining Solution (Biofact, Daejeon, Republic of Korea). PCR products were directly sequenced in both directions using an ABI Big Dye Terminator kit and an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
Mitochondrial COI sequences were assembled with Geneious R11 (Biomatters Limited, Auckland, New Zealand). Pairwise genetic distances were calculated using MEGA 7 [20] with the Kimura-2 parameter genetic distances model [21]. Gaps and missing data were completely deleted. The best-fit model of nucleotide substitution for the COI dataset was selected by jModelTest v. 2.1.1 [22] using the Akaike Information Criterion for Maximum Likelihood (ML). The ML tree was constructed using PhyML 3.0 [23] under the GTR+I+G model for the COI dataset. Bootstrap analysis was performed with 1000 replicates, the proportion of invariable sites was 0.487, and the gamma distribution parameter was 0.742. For aligned COI sequences (420 bp), 10 species of Henricia were used including H. djakonovi. Two species of genus Luidia Forbes, 1839 were used as an outgroup.     Description. Arms five, long, large disk, gradually tapering to tips ( Figure 1A,B). Abactinal paxillae forming elongated, crescent-shaped with smaller paxillae bearing seven to 18 spinelets; larger paxillae bearing 126-144 robust spinelets (Figures 1C and 2I). Denuded abactinal spines pillar formed with droplet-like apical tip ( Figure 1J). Paxillae on the lateral side of arms similar to abactinal paxillae. Abactinal skeleton closely meshed, crescent-shaped, closely united into fine meshed network, abactinal plates larger than papular areas ( Figure 1G). Papular areas narrow with one to three papulae in an area ( Figure 1H). Some papular areas divided by small ossicles. Madreporite circular in form, slightly sunken, bearing spines similar to adjacent spines ( Figure 1F). Three regular series of plates adjacent to adambulacral (superomarginal, inferomarginal, and ventrolateral) plates well defined. Superomarginal forming elongated cross, reaching tip of arm. Inferomarginal plates longer than superomarginal and ventrolateral plates, bearing 130-175 spines, reaching tip of arm. Ventrolateral plates rounded cross-shaped, bearing 55-78 spines, reaching tip of arm. Actinal papular areas bearing one or two papulae. Adambulacral plates forming semi-rounded shape, bearing 13-16 spines, flat tip spines near furrow edge, stout spines near ventrolateral spines, arranged in two transverse series ( Figure 1D,I,K). Furrow spine single. Oral part bearing two slender oral spines, three marginal spines, and four or five sub-oral spines ( Figure 1E).
Remarks. Henricia djakonovi is easily distinguishable from other Henricia species due to its characteristic of possessing spotted live coloration [24]. Morphological analysis showed that Korean H. djakonovi differed morphologically from three related Henricia species (Table 1) [24]. Our specimen possessed a higher number of abactinal spines (up to 144). However, differences in the number of abactinal spines alone cannot be regarded as a stable character for Henricia species identification [25]. Therefore, we consider that Korean H. djakonovi is the same species as the holotype specimen. H. djakonovi is reported for the first time in the Korean fauna. Moreover, we obtained a partial sequence of the mitochondrial COI gene and deposited it into GenBank of NCBI (GenBank accession no. OP522340). This is the first registration of the COI data for H. djakonovi. Pairwise genetic distances (p-distance) were calculated by the Kimura-2 parameter ( Table 2). The mean interspecific p-distance in Henricia was 12.0%, ranging from 5.0% (H. leviuscula-H. sanguinolenta) to 16.7% (H. oculata-H. regularis). The mean interspecific p-distance between H. djakonovi and other Henricia species was 13.8%, ranging from 11.0% to 16.4% (Table 2). DNA barcoding analysis of Henricia in a previous study revealed that the interspecific p-distance was relatively high (mean: 13.7%, range: 2.6-18.3%) but the intraspecific p-distance was relatively low (mean: 0.3%, range: 0.0-1.6%) [26]. The p-distance showed that the Korean H. djakonovi was clearly separated from other Henricia species (Table 2). The molecular analysis clearly distinguished H. djakonovi from other Henricia species. Thus, the mitochondrial COI gene is especially useful and effective for DNA barcoding analyses of Henricia species. The maximum likelihood tree also showed that H. djakonovi was clearly separated from other species of Henricia ( Figure 3). The values of bootstrapping were used to indicate support for stable groupings and assess differences between the resulting topologies. A clade of Henricia in the ML tree was clearly monophyletic. However, polyphyly occurred between H. reniossa and H. nipponica, which has a low bootstrap value, 26% (Figure 3). The results of the molecular analyses strongly supported that our Henricia specimen is clearly different from other species of Henricia. Thus, we newly reported a sea star, H. djakonovi, in Korean fauna based on morphology and molecular analyses.