A new species of the genus Henricia (Asteroidea, Spinulosida, Echinasteridae) from South Korea

Abstract A new species of the genus Henricia Gray, 1840 that belongs to the family Echinasteridae is described from South Korea. Henricia epiphysialissp. nov. has epiphyseal ossicles at the ends of the abactinal and lateral plates, and the abactinal and lateral spines form a hooked crown. The partial sequence of the mitochondrial COXI gene (537 bp) of H. epiphysialissp. nov. was obtained, and the new species was morphologically and genetically compared with other related Henricia species.

In DNA barcoding, sequence variation in a 658 bp region of the mitochondrial cytochrome c oxidase subunit I (COXI) gene is used for specimen identification and species discovery (Hebert et al. 2003). An integrative approach to taxonomy (i.e., using morphological characteristics from preserved specimens as well as one to several genes) has become necessary for assessing species diversity and species boundaries (Puillandre et al. 2012).
In this study, we identified a new species that belongs to the genus Henricia collected from waters adjacent to the East Sea, South Korea, and performed detailed morphological and molecular mitochondrial sequence analyses. This paper aims to extend the taxonomical insights to Henricia species in South Korea by providing a complete description of this new species.

Materials and methods
In May and December 2014, sea stars were collected from the East Sea in South Korea by using fishing nets (Fig. 1). The collected specimens were preserved in 95% ethanol and deposited at the National Institute of Biological Resources (NIBR) and Marine Echinoderm Resource Bank of Korea (MERBK), South Korea. The external features of the specimens were observed using a stereomicroscope, and the specimens were identified on the basis of morphological characteristics such as the size of the disk, R/r ratio (R: length of arm; r: radius of the disk), size of the upper and proximal portions of arms, number of abactinal spines, shape of abactinal and actinal skeletons, and number of adambulacral spines. For observing the detailed structures of the specimens such as the shape of the spines and skeletal plates, sodium hypochlorite (5.25% solution) was applied carefully to dissolve the skin (Shin 2010). Then, the specimens were washed with water and observed using the stereomicroscope. The important morphological characteristics of the specimens were photographed using a scanning electron microscope (JEOL JSM-6510), stereomicroscope (Nikon SMZ1000), and digital camera (Nikon D7000). Abbreviations for the measurements were those used by Shin and Ubagan (2015a, b). Total genomic DNA was isolated from ethanol-preserved tube feet tissue by using a DNeasy blood and tissue DNA isolation kit (Qiagen), according to the manufacturer instructions. The genomic DNA quality and concentration were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). 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, LCOech1aF1 (Layton et al. 2016) and HCO2198 (Folmer et al. 1994). PCR was performed using a 25 µL reaction mixture containing 2.5 µL of 10× Ex Taq Buffer containing 20 mM MgCl 2 (Clontech), 1 µL of 2.5 mM dNTPs (Clontech), 1 µL of each primer at 10pmol, 1.5 µL of the template DNA, 0.3 µL of 5 U/µL Taq polymerase (Clontech), and 17.7 µL of distilled water. The PCR conditions were as follows: initial denaturation at 95 °C/3 min, followed by 35 cycles of denaturation at 95 °C/30 s, annealing at 52 °C/90 s, extension at 72 °C/90 s, and a final extension at 72 °C/7 min. The PCR product quality was determined using electrophoresis with a 1.5% agarose gel stained with ethidium bromide. The PCR products were directly sequenced in both directions by using ABI Big Dye Terminator kits (Applied Biosystems) and ABI 3730XL DNA Analyzer. To construct a neighbor-joining (NJ) tree, COX1 sequences (189 and 537 bp) from the Henricia species dataset, including one sequence of the new species (GenBank accession No. MT086587), were used ( Table   Table 1. List of Henricia species and GenBank accession numbers of COX1 gene used in this study. 1). Four species of genus Echinaster were used as outgroups (Table 1). Pairwise distances were calculated using MEGA 7.0 and the Kimura-2-parameter model (Kumar et al. 2016). The gaps and missing data were removed, and the bootstrap analysis was performed with 1000 replicates. Diagnosis. Regular size, R/r = 4.9-5.4, abactinal plates crowded with 11-40 spines, abactinal and lateral spines forming distinct hooked crown, epiphyseal ossicles formed at ends of abactinal and lateral plates, one to three papulae, marginal and ventrolateral series distinguishable, adambulacral plates bearing 10-14 slender spines.

Phylum
Description. Holotype.  Size. R = 51 mm, r = 10 mm, R/r = 5.1. Arms five, semi-cylindrical, gradually tapering to tips ( Fig. 2A-B). Abactinal paxillae formed in group with evenly spaced spinulation, bearing 11-40 spines with serrated tips (Fig. 2C). Denuded abactinal spines forming hooked crown composed of nine to 11 large hook-shaped spinules enclosing nine to 12 small connected apical tips (Fig. 4A-C). Paxillae on lateral side of arms similar to abactinal paxillae (Fig. 2D). Denuded abactinal plates reniform, usually connected to end of other plate in mid convex part of plate, larger than papular areas, partially enclosing papular area on concave side of plate. Papular areas narrow, containing one to three papulae in an area. Some papular areas divided by small ossicles (Fig. 3A). Almost every skeletal plate aside from adambulacral plates was observed bearing epiphyseal ossicles at ends of plate (Fig. 3A, C, D). Madreporite circular in form, slightly elevated, bearing spines same as adjacent spines (Fig. 3B). Shape of spines on lateral side nearly similar to that of abactinal spines (Fig. 4D-F). Superomarginal, inferomarginal, and ventrolateral plates well defined forming elongated cross shape and arranged in rows show- ing consistent series (Fig. 3D). Superomarginal plates bearing 12-28 spines, bend upward toward base of arm in crescentic form, and reach tip of arm (Figs 3C-D, 4D). Intermarginal plates forming small elongated shape, extending near half of arm (Fig. 3D). Inferomarginal plates longer than superomarginal and ventrolateral plates, bearing 34-45 spines, reaching tip of arm (Figs 3C-D, 4E). Ventrolateral plates forming a rounded cross shape, bearing 21-25 spines, reaching near tip of arm, epiphyseal ossicles forming a knob-like connection to adambulacral plates, extending to middle part of arm (Figs 3D, 4F). Adambulacral plates forming semi-rounded shape, bearing 10-14 slender, thorny spines, arranged in two transverse series (Figs 3D-E, 4G), articulated with ambulacral plates (Fig. 3E). Furrow spine single, somewhat curved (Fig. 4H). Oral part bearing two slender, bluntly pointed oral spines, with six or seven marginal spines, and five or six sub-oral spines similar to adambulacral spines (Fig. 2F). Paired oral plates forming a slightly elongated triangular shape, articulated with first pair of adambulacral plates. Plates of inter-radial area slightly compact (Fig. 3F).
Paratypes. Size. R = 39 mm, r = 8 mm, R/r = 4.9; R = 60 mm, r = 11 mm, R/r = 5.4. Etymology. The specific name is derived from the Latin "epiphysialis," which means the end part of a long bone.
Ecology. This species is found on hard substrates (rocky bottom) from a shallow water of a depth of 40 m to 100 m.

Molecular results
We determined the phylogenetic relationships based on two COX1 datasets with 27 and 31 species of the genus Henricia respectively, including H. epiphysialis sp. nov., on the basis of 189 and 537 bp of the mitochondrial COX1 gene by using the NJ method. All Henricia species formed a monophyletic group with congeneric species and were clearly distinguished from the sister taxa (Fig. 5). Henricia epiphysialis sp. nov. was clearly monophyletic in the COX1 dataset of 189 bp (Fig. 5A), but the 189 bp COX1 dataset was too short for species-level identification using DNA barcoding analysis. Therefore, we reanalyzed phylogenetic relationships using a longer COX1 dataset (537 bp) including 27 species of Henricia (Table 1, Fig. 5B). The phylogenetic tree of the 537 bp dataset also revealed that H. epiphysialis sp. nov. presented a monophyletic node according to short and long COX1 datasets analysis (Fig. 5). In addition, each Henricia species examined was grouped at the species level. The intra-and inter-specific variations of Henricia species from the 537 bp COX1 dataset were calculated by the Kimura 2-parameter model and presented in Table 3. According to the p-distance result, the average value between Henricia spp. and Echinaster spp. was 0.208 (Table 3). The range of inter-specific distance in the genus Henricia was 0.002-0.188, and the average value of inter-specific distance was 0.110 (Table 4). The inter-specific values of H. kinkasana and Henricia sp. 3-7 was lower than the relationships of other Henricia species (Table 3, 4). The range of Henricia species, except for H. kinkasana and Henricia sp. 3-7, was 0.040-0.188 and the average was 0.114 (Table 4). The range and average of H. epiphysialis sp. nov. and other Henricia species were 0.056-0.154 and 0.098, respectively (Table 4). Therefore, the molecular analysis results show that H. epiphysialis sp. nov. is a new species in the genus Henricia.

Discussion
The diagnostic combination of the morphological characteristics of the genus Henricia such as spination of the abactinal (primarily on the disk and proximal portion of the arm), adambulacral, and oral plates was found to be consistent and reliable for determining the species (e.g., Hayashi 1940;Djakonov 1950;Clark and Jewett 2010). The shape of the abactinal spines and plate formation exhibited the distinct morphological characteristics of Henricia epiphysialis sp. nov. (Figs 3A, C, D, 4A-C). Henricia epiphysialis sp. nov. was compared with nine related Henricia species (Table  2). This new species has very peculiar abactinal spines that form a hooked crown and  . Henricia epiphysialis sp. nov. is morphologically distinguishable from its congeners primarily by the presence of conspicuous epiphyseal ossicles in almost every plate, and also by the distinctive arrangement of the epiphyseal ossicles of the ventrolateral plates, forming a knob-like connection to the adambulacral plates (Fig. 3D). The knob-like form of epiphyseal ossicles in the ventrolateral plates is rarely seen in related Henricia species having slender arms with imbricated plates. Our new species, H. epiphysialis sp. nov.    Other morphological characteristics of H. epiphysialis sp. nov., such as the ratio of arm to disk and number of adambulacral spines, are similar to those of H. kinkasana which is a slender-rayed species; however, this new species differs mainly in the number of abactinal spines and shape of both abactinal and lateral spines. Henricia epiphysialis sp. nov. has 11-40 robust abactinal spines on the abactinal plate, whereas H. kinkasana has five to 18 fine, delicate abactinal spines. Moreover, the conspicuous epiphyseal ossicles at the ends of the abactinal and lateral plates are exclusively present in H. epiphysialis sp. nov. Therefore, the extension of ossicles in the plate and hooked crown shape of the spines are diagnostic characteristics for this new species.
In this study, we identify a new Henricia species based on its morphological characteristics and DNA barcoding. Henricia epiphysialis sp. nov. has distinct morphological features and was classified as a new species after comparison with related species. Moreover, the molecular analysis showed that H. epiphysialis sp. nov. clearly formed a monophyletic node in a large clade of the genus Henricia species (Fig. 5), and the minimum value for the inter-specific distance was significantly higher than the interspecific distance reported in a previous asteroid DNA barcoding study (Table 3) (Ward et al. 2008). Therefore, the molecular analysis clearly supported the diagnostic morphological identification of H. epiphysialis sp. nov. as a new species under the genus Henricia. The mitochondrial COX1 gene is especially useful and effective for the DNA barcoding analyses of Henricia species.