Bull. Kitakyushu Mus. Nat. Hist. Hum. Hist., Ser. A, 19: 41–51, March 31, 2021 41 Redescription of Amphipholis kochii (Echinodermata: Ophiuroidea: Ophintegrida) al. (2019). Amphipholis kochii Lütken, 1872: 10; Lyman, 1882: 146; DNA extraction, PCR amplification, and DNA sequencing. H.L. Clark, 1915: 241; Matsumoto, 1917: 192; 1941: 338; Koehler, collected from Sagami Bay, including ossicle morphology and COI sequence We partially sequenced mitochondrial COI genes, including “DNA 1922: 163; Murakami, 1942: 10; 1944: 265; Djakonov, 1954: 59; barcoding region” for one examined specimen (KMNH IvR Irimura, 1969: 41; 1979: 3; Saba et al., 1982: 28; Shin and Rho, Masanori Okanishi*, Mayuko Nakamura, Hana Tamura and Hisanori Kohtsuka 600015). The method of DNA extraction and PCR parameters 1996: 425; Fujita and Kohtsuka, 2003: 30; Fujita et al., 2004: 196; followed that by Okanishi and Fujita (2013). Primer sets of COI005 Liao, 2004: 183; Komatsu et al., 2007: 11. (5′-TTAGGTTAAHWAAACCAVYTKCCTTCAAAG-3′) and Amphiura kochii. Lyman, 1875: pl. 5(72) [This binomen is Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo, COI008 (5′-CCDTANGMDATCATDGCRTACATCATTCC-3′) only mentioned in the figure of this reference]. 1024 Koajiro, Misaki, Miura 238-0225, Japan (Okanishi & Fujita, 2013) was used for PCR. The PCR products * Corresponding author. Email: [email protected] were separated from excess primers and oligonucleotides using Examined specimens. KMNH IvR 600015: one specimen, Exo-SAP-IT (Applied Biosystems), following the manufacturer’s Arai Beach, Misaki, Miura, Kanagawa Prefecture, central-eastern (Received July 1, 2020; accepted September 29, 2020) protocol. All samples were sequenced bidirectionally and sequence Japan, snorkeling, approximately 0.5–1 m in depth at the lowest products were run on a 3730xI DNA Analyzer (Thermo Fisher tide, collected by M. Nakamura (second author) and sorted from ABSTRACT − Amphipholis kochii Lütken, 1872 is redescribed based on two discovered specimens from the Scientific). Its accession number of DNA Data Bank of Japan calcareous algae (probably Corallina pilulifera) by M. Nakamura Sagami Bay, central-eastern Japan. Detailed morphology of internal ossicles as well as external features and (DDBJ) is LC556229. and H. Tamura (third author), 9 April 2020. KMNH IvR 600016: sequence of mitochondrial COI gene of A. kochii is provided for the first time. one specimen, Arai Beach, Misaki, Miura, Kanagawa Prefecture, central-eastern Japan, by hand, intertidal zone, collected by H. Kohtsuka (last author), 5 July 2011. KEY WORDS: Taxonomy, Amphiuridae, brittle star, Cytochrome c oxidase I, Japan RESULTS AND DISCUSSION Diagnosis. Arms approximately 4 to 5 times longer than disc Systematics diameter; disc surface covered by imbricated, densely and smooth scales; radial shields separate proximally each other, approx- INTRODUCTION MATERIALS AND METHODS Family Amphiuridae Ljungman, 1867 imately three times longer than wide; arm spines up to three, the Genus Amphipholis Ljungman, 1866 longest one in proximal portion of arm is flattened, 1.5 times longer Recent descriptions of ophiuroids have included a Specimens examined, morphological observations and Amphipholis kochii Lütken, 1872 than the corresponding arm segment; two oral papillae sensu lato; comprehensive range of photographs and/or drawings of relevant terminology. Two specimens of Amphipholis kochii were collected [Japanese name: Suna-kumohitode] two tentacle scales; lateral arm plates with parallel lines of stereom Description of external morphology (KMNH IvR 600015, gradually decreasing to outer side, in contact with each other (Fig. diagnostic morphological characters as well as scanning electron at a depth of approximately 0.5–1.0 m at Arai Beach near the (Figs 2–7) structure on the external side; base of arm spines whitish. 600016) 3E). Oral shields diamond-shaped, slightly rounded on distal side, microscope (SEM) images of ossicles, which provide a consistent Misaki Marine Biological Station, the University of Tokyo Color. In life (KMNH IvR 600016), dorsal disc was gray approximately 350 μm in length, 300 μm in width (Fig. 3E). One suite of characters for comparison and identification (e.g., (MMBS) in Misaki, Miura, Kanagawa Prefecture, Japan (Fig. 1). with dark brown spots scattered periphery but peripheral edges of larger, approximately 1.5 times than others in both length and Martynov, 2010; Thuy and Stöhr, 2016; Okanishi and Fujita, The examined specimens were directly immersed in 99% radial shields were white (Fig. 2). Proximal dorsal arms appear width, suggesting its serves as a madreporite (Fig. 3E). Tooth 2018a). ethanol without anesthetization. Photographs were taken in situ for gray-yellow basic color with irregular alternative transverse bands triangular, forming a vertical row on dental plate, a pair of The genus Amphipholis (Ophiuroidea: Ophintegrida: Amphi- one specimen (KMNH IvR 600016; Fig. 2) and in fixed status for of dark brown color on proximal portion of arm and scattered spots infradental papilla on the ventral top of the dental plate (Fig. 3E). lepidida: Amphiuridae) was erected by Ljungman (1866), and another specimen (KMNH IvR 600015; Figs 3, 4). For observation on middle to distal portion of arms. Ventral side of the body whole Two (probable) adoral shield spines situated at oral edge of each currently comprises 26 species (Stöhr et al., 2020). However, by SEM, KMNH IvR 600015 was dissected to remove arm creamy white. adoral shield, inner one fan-shaped, as long as wide, and outer one accumulation of detailed morphological data mentioned above for ossicles which were isolated using domestic bleach (approximately In the ethanol-fixed specimen (KMNH IvR 600015) dark rectangular, approximately two times as wide as long (Fig. 3E). Amphipholis has started only recently for three species (e.g., Stöhr, 5% sodium hypochlorite solution), washed in deionized water, gray color remain and other parts turn into gray white (Figs 3A, B; Second oral tentacle pore opening within the slit of mouth, thus not corresponding arm segment (Fig. 4A, D). On the middle portion of ventral side of distal portion of arm (Figs 5D, H; 6A). On proximal 2001, 2011; Kroh and Thuy, 2013). dried in air and mounted on SEM stubs using double-sided 4A–C). visible from outside (Fig. 3E). arm, three spines all spiniform, approximately 1.5 times longer than portion of arm, a keel well developed on distal side (Fig. 5C, E) and Amphipholis kochii Lütken, 1872 is known to be widely conductive tape. The preparations were sputter-coated with Disc. Five lobed, 5 mm in diameter (Fig. 3A, B), surfaces Arms. Five in number, sinuous, up to 23 mm in length. the corresponding arm segment (Fig. 4B, E). On the distal portion a pair of channels of lateral canals opening inside of ventral groove distributed from Hokkaido to Kyushu Islands in Japan (Lyman, gold-palladium and examined and photographed with Jeol JSM completely covered by smooth and imbricating scales, Proximally 0.4 mm in width and 0.3 mm in height, rectangular in of arm, the spines spiniform, almost the same length with the (Fig. 5D); depression for tentacles opening on ventral distal side of 1882; H.L. Clark, 1915; Murakami, 1942, 1944; Irimura, 1969, 5510LV SEM of the Misaki Marine Biological Station of The approximately 90–180 μm in length, and primary plates consisting cross section. Arms tapering gradually toward the distal arm (Figs corresponding arms segment, and the number finally decreasing to the vertebra (Fig. 5D, H); protrusions on ventral middle portion of 1979; Saba et al., 1982; Fujita and Kohtsuka, 2003; Fujita et al., University of Tokyo (Figs 5–7). The examined specimens were of round centerodorsal one and five separate semicircular ones, 3F; 4). Each arm segment carrying a dorsal arm plate, two lateral two on arm tip (Fig. 4C, F). On the proximal and middle portion of vertebrae (Figs 5D, E, H; 6A). A large hole opening on dorsal 2004; Komatsu et al., 2007). Prior to this study, although external deposited in the Kitakyushu Museum of Natural History & Human each 300 μm in length (Fig. 3C). Radial shields and their surrounds arm plates and a ventral arm plate (Figs 3F; 4). On proximal portion the arm, each tentacle pore bears two tentacle scales, inner one fan middle portion of vertebrae on distal portion of arm (Fig. 6A). features with sketches showing diagnostic characters of A. kochii History (KMNH) (KMNH IvR 600015, 600016). depressed, thin and semicircular, approximately three times longer of arm, dorsal arm plates diamond-shaped with round edge, shaped and outer one larger and triangular (Fig. 4D, E). On the On proximal portion of arm, lateral arm plates as high as long, were provided in six studies (Lütken, 1872; Lyman, 1875; Morphological terminologies follow Thuy and Stöhr (2011), than wide; the length approximately a quarter of the radius of the slightly in contact with each other, 1.5 times wider than long (Fig. distal portion of arm, the scales decreasing in size and disappearing distal side convex, both dorsal and ventral edges projecting toward Matsumoto, 1917, 1941; Djakonov, 1954; Liao, 2004), no data for Stöhr et al. (2012), Okanishi and Fujita (2018b), and Hendler disc (Fig. 3A, D). Interradial ventral disc covered by imbricating 4A). On middle portion of arm, the dorsal arm plates transform to on arm tip (Fig. 4F). proximal side (Fig. 6C–E). External view smooth, stereom on microstructure of ossicles of A. kochii has been provided so far. (2018). Terminologies of oral papillae sensu lato in Hendler (2018) scales, similar to those on dorsal surface, approximately 80–120 an octagonal shape, slightly wider than long and separated (Fig. Ossicle morphology (KMNH IvR 600015) central portion of proximal side slightly denser than remaining Here, we present photographs of many parts of the body and were mainly determined based on ontogenetic and anatomical μm in length (Fig. 3E, F). Genital slits long, ranging from the edge 4B). On distal portion of arm, the plates fan-shaped, separated, as Vertebrae with zygospondylous articulation, muscle flanges areas and forming parallel horizontal striations (Fig.