Redescription of Amphipholis Kochii (Echinodermata: Ophiuroidea: Ophintegrida) Al

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. 6C). On SEM images of microstructure of ossicles for A. kochii, based on observations. In this study, we did not observe ontogenetic series of of oral shield to lateral edge of the disc, approximately 0.25 mm in wide as long, and gradually decrease in size toward the arm tip on dorsal-proximal side elongate, larger, wing-like (Fig. 5A) and internal side, two knobs, composed of more densely meshed two specimens from Misaki, Sagami Bay, Japan. Additionally, examined species and did not dissect mouth ossicles to prevent width and 1.5 mm in length (Fig. 3F). On ventral surface, adoral (Fig. 4C). Lateral arm plates, widely separated by dorsal and plates square, as wide as long, in contact with each other (Fig. 4D). proximal portion of arm, three spiniform arm spines on each lateral those on ventral-proximal side smaller, approximately half width stereom than remaining area, situated at center (Fig. 6D). In the two sequence of mitochondrial Cytochrome c oxidase subunit I (COI) destruction of the precious specimens. Thus, we marked “?” for shields trapezoid, approximately 1.5 times wider than the longest ventral arm plates proximally (Figs 3F; 4A), in contact with each On middle portion of arm, the plates pentagonal, slightly longer arm plate; the dorsal most one slightly flattened and longest, of the dorsal-distal ones (Fig. 5B). In proximal portion of arm, central knobs, a single perforation next to a more distal one and gene including DNA barcoding region from A. kochii is provided oral papillae sensu lato which we are not sure of their origin (Fig. length, in contact with each other (Fig. 3E). Oral plates triangular, other after middle portion of the arms on both ventral and dorsal than wide and in contact each other (Fig. 4E). On distal portion of approximately 1.5 times longer than the corresponding arm “T”-shaped groove on dorsal side (Fig. 5C). Longitudinal groove tentacle notch that extends to distal ventral side (Fig. 6D). Three for future phylogenetic analysis. 3E). Systematics follows O’Hara et al. (2018) and Christodoulou et approximately the same width as length inner side, the length sides (Fig. 4B, C, E, F). On proximal portion of arm, ventral arm arm, the plates triangular, as wide as long, separated (Fig. 4F). On segment; the other two spines almost the same length of the on ventral side of proximal portion of arm and on both dorsal and spine articulations on distal side, with nearly horizontal and parallel

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, 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, 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 COI008 (5′-CCDTANGMDATCATDGCRTACATCATTCC-3′) only mentioned in the figure of this reference]. (Okanishi & Fujita, 2013) was used for PCR. The PCR products 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 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 Scientific). Its accession number of DNA Data Bank of Japan calcareous algae (probably Corallina pilulifera) by M. Nakamura (DDBJ) is LC556229. and H. Tamura (third author), 9 April 2020. KMNH IvR 600016: one specimen, Arai Beach, Misaki, Miura, Kanagawa Prefecture, central-eastern Japan, by hand, intertidal zone, collected by H. RESULTS AND DISCUSSION Kohtsuka (last author), 5 July 2011. Diagnosis. Arms approximately 4 to 5 times longer than disc diameter; disc surface covered by imbricated, densely and smooth approximately Systematics 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 Fig. 2. Amphipholis kochii (KMNH IvR 600016). Dorsal view in situ status, a probable piece of shell on the disc. 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. 6C). On SEM images of microstructure of ossicles for A. kochii, based on observations. In this study, we did not observe ontogenetic series of of oral shield to lateral edge of the disc, approximately 0.25 mm in wide as long, and gradually decrease in size toward the arm tip on dorsal-proximal side elongate, larger, wing-like (Fig. 5A) and internal side, two knobs, composed of more densely meshed two specimens from Misaki, Sagami Bay, Japan. Additionally, examined species and did not dissect mouth ossicles to prevent width and 1.5 mm in length (Fig. 3F). On ventral surface, adoral (Fig. 4C). Lateral arm plates, widely separated by dorsal and plates square, as wide as long, in contact with each other (Fig. 4D). proximal portion of arm, three spiniform arm spines on each lateral those on ventral-proximal side smaller, approximately half width stereom than remaining area, situated at center (Fig. 6D). In the two sequence of mitochondrial Cytochrome c oxidase subunit I (COI) destruction of the precious specimens. Thus, we marked “?” for shields trapezoid, approximately 1.5 times wider than the longest ventral arm plates proximally (Figs 3F; 4A), in contact with each On middle portion of arm, the plates pentagonal, slightly longer arm plate; the dorsal most one slightly flattened and longest, of the dorsal-distal ones (Fig. 5B). In proximal portion of arm, central knobs, a single perforation next to a more distal one and gene including DNA barcoding region from A. kochii is provided oral papillae sensu lato which we are not sure of their origin (Fig. length, in contact with each other (Fig. 3E). Oral plates triangular, other after middle portion of the arms on both ventral and dorsal than wide and in contact each other (Fig. 4E). On distal portion of approximately 1.5 times longer than the corresponding arm “T”-shaped groove on dorsal side (Fig. 5C). Longitudinal groove tentacle notch that extends to distal ventral side (Fig. 6D). Three for future phylogenetic analysis. 3E). Systematics follows O’Hara et al. (2018) and Christodoulou et Fig. 1. Sampling localities of examined specimens. approximately the same width as length inner side, the length sides (Fig. 4B, C, E, F). On proximal portion of arm, ventral arm arm, the plates triangular, as wide as long, separated (Fig. 4F). On segment; the other two spines almost the same length of the on ventral side of proximal portion of arm and on both dorsal and spine articulations on distal side, with nearly horizontal and parallel

ventral and dorsal lobes which are almost in similar size (Fig. 6C, revealed that their parallel striations on their external surfaces (Fig. for their providing critical comments. This work was partly Kroh, A. and Thuy, B. 2013. A new Philippine ophiuroid symbiotic O’Hara, T. D., Stöhr, S., Hugall, A. F., Thuy, B. and Martynov, A. Stöhr, S. 2011. New records and new species of Ophiuroidea E). Surrounds of the articulations slightly sunken but no areoles of 6C) are thicker than those of other species of Amphiuridae (see also supported by KAKENHI Grant Number JP17K07549. on a cassiduloid echinoid species. Zoologischer Anzeiger, 2018. Morphological diagnoses of higher taxa in Ophiuroidea (Echinodermata) from Lifou, Loyalty Islands, New Cale- denser stereom (Fig. 6C, E). Muscle and nerve openings clearly Thuy and Stöhr, 2011; lateral arm plates of Amphiura chiajei 252 (3): 279–288. (Echinodermata) in support of a new classification. European donia. Zootaxa, 3089: 1–50. separated by stereom protuberances (Fig. 6E). Forbes, 1843, fig. 12 (2a, 3a) and Amphiura sundevalli (Müller and Lee, T. and Shin, S. 2019. A new record of the brittle star, Journal of Taxonomy, 416: 1–35. Stöhr S. and Muths D. 2010. Morphological diagnosis of the two On distal portion of arm, lateral arm plates slightly longer Troshcel, 1842), fig. 12 (8a, 9a)), indicating that it possibly be a REFERENCES Amphistigma minuta (Ophiuroidea: Amphilepidida: Okanishi, M. and Fujita, T. 2013. Molecular phylogeny based on genetic lineages of Acrocnida brachiata (Echinodermata: than high, proximal edge slightly convex, external and internal new diagnostic character of genus level for the Amphiuridae. The Amphiuridae), from Jeju Island, Korea. Animal Systematics, increased number of species and genes revealed more robust Ophiuroidea) with description of a new species. Journal of the sides basically smooth (Fig. 6F, G). 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Available at http://www.marinespecies.org/ with sharpen proximal edge, as wide as long on distal portion of densely scales; radial shields separate each other, approximately DOI: 10.1371/journal.pone.0210331 lingar, Stockholm, 23 (6): 163–166. Ophiozonella (Echinodermata: Ophiuroidea: Amphilepidida). ophiuroidea on 2020-06-10. DOI:10.14284/358 (accessed on arm (Fig. 7A, B). Intersections of mesh structure of stereom of the three times longer than wide; arm spines up to three, the longest Christodoulou, M., O’Hara, T. D., Hugall, A. F. and Arbizu, P. M. Ljungman, A. V. 1867. Ophiuroidea viventia huc usque cognita Zootaxa, 4377 (1): 1–20. 10 June 2020) dorsal arm plate protruding on external side and smooth, not one in proximal portion of arm flatten, 1.5 times longer than the 2019. Dark ophiuroid biodiversity in a prospective abyssal enumerat. Öfversigt af Kongliga Vetenskaps-Akademiens Okanishi, M. and Fujita, Y. 2018b. A new species of Ophioconis Sundberg, P., Vodoti, E. T. and Strand, M. 2010. DNA barcoding protruding on internal side (Figs 6I, J; 7A, B). corresponding arm segment; two oral papillae sensu lato; two mine field. Current Biology, 29 (22): 3909–3912.e3. DOI: Förhandlingar, Stockholm, 23 (9): 303–336. (Echinodermata Ophiuroidea) from Ryukyu Islands, south- should accompany taxonomy – the case of Cerebratulus spp Ventral arm plates pentagonal with sharp proximal edge and tentacle scales; arm spines with whitish basal portion. 10.1016/j.cub.2019.09.012 Lütken, C. F. 1872. Ophiuridarum novarum vel minus cognitarum western Japan. Proceedings of the Biological Society of (Nemertea). Molecular Ecology Resources, 10: 274–281. slightly pointing distal-lateral edges, as wide as long on proximal In our molecular study, we obtained 825 bp of COI gene Clark, H. L. 1915. Catalogue of recent ophiurans: based on the descriptiones nonnullae. ogle nye eller mindre bekjerdte Washington, 131: 163–174. Thuy, B. and Stöhr, S. 2011. Lateral arm plate morphology in portion of arm, and slightly longer than wide on distal portion of region for Amphopholis kochii (KMNH IvR 600015; LC556229) collection of the Museum of Comparative Zoology. Memoirs Slangestjernerne beskrevne – Med nogle Bemaerkninger om Okanishi, M., Senatoku, A., Martynov, A. and Fujita, T. 2018. A brittle stars (Echinodermata: Ophiuroidea): new perspectives ·· arm (Fig. 7C–F). Intersections of mesh structure of stereom of the and so far, any DNA sequence of A. kochii has not been registered of the Museum of Comparative Zoology at Harvard College, Selvdelingen hos Straaldyrene –. Oversigt over det Kongelige new cryptic species of Asteronyx Müller and Troschel, 1842 for ophiuroid micropalaeontology and classification. Zootaxa, ventral arm plate protruding on external side and smooth, not in GenBank. A BLAST search of the sequence (LC556229) at the 25 (4): 165–376. Danske Videnskabernes Selska bs forhandlinger, 77: 75–158. (Echinodermata: Ophiuroidea), based on molecular phylo- 3013: 1–47. protruding on internal side (Fig. 7C–F). DDBJ website (http://blast.ddbj.nig.ac.jp/blastn?lang=ja) yielded Djakonov, A. M. 1954. Ophiuroids of the USSR Seas. In: Lyman, T. 1875. Zoological Results of the Hassler Expedition. 2. geny and morphology, from off Pacific Coast of Japan. Thuy, B. and Stöhr, S. 2016. A new morphological phylogeny of the On proximal portion of arm, dorsal most arm spine Amphioplus daleus (Lyman, 1882) (accession no. KU895008) as Pavlovskii, E. N. (ed.) Keys to fauna USSR. Wiener Bindery Ophiuridae and Astrophytidae. Illustrated catalogue of the Zoologischer Anzeiger, 274: 14–33. Ophiuroidea (Echinodermata) accords with molecular evidence cylindrical (Fig. 7G), others spiniform (Fig. 7H), on distal portion the closest sequence with 20.8% K2P genetic distance. It falls Ltd., Jerusalem, pp. 1–123. Museum of Comparative Zoology, at Harvard College, 8 (2): Saba, M., Tomida, Y. and Kimoto, T. 1982. Echinoderms fauna of and renders microfossils accessible for cladistics. PLoS ONE, of arm, all arm spines spiniform (Fig. 7I, J). Spines with simple and within the range of interspecific differences (2.2% to 23%) for COI Fell, H. B. 1960. Synoptic key to the genera of Ophiuroidae. 1–34. Ise Bay, and the northern and middle parts of Kumano-nada. 11 (5): e0156140. DOI: 10.1371/journal.pone.0156140 fine stereom with minute thorns which are pointing to external side in ophiuroids (e.g., Okanishi et al., 2018). Thus, it is reasonable to Zoology Publications from Victoria University of Wellington, Lyman, T. 1882. Report on the Opiuroidea. Report on the Scientific Bulletin of the Mie Prefectural Museum, 4: 1–82. on proximal portion of arms (Fig. 7G, H), and are to the lateral side assume that we provide the first DNA sequence of A. kochii 26: 1–44. Results of the Voyage of H.M.S. Challenger during the years Shin, S. and Rho, B.-J. 1996. Illustrated Encyclopedia of Fauna & on distal portion of arm (Fig. 7G–J). including DNA barcoding region. Although DNA barcoding data Fujita, T. and Kohtsuka, H. 2003. Ophiuroids (Echinodermata) 1873-76, Zoology, 5 (1): 1–386. Flora of Korea. Vol. 36. Echinodermata. The Ministry of Distribution. Amphipholis kochii has been recorded from the has been used in various taxa as a powerful tool for species from Notojima Islands and its adjacent waters in the Sea of Martynov, A. 2010. Reassessment of the classification of the Education, Seoul. coastal areas in Japan, from north of Kumamoto Prefecture to identification, it has been noted that without proper taxonomic Japan. Report of the Noto Marine Center, 9: 25–38. (In Ophiuroidea (Echinodermata), based on morphological Stöhr, S. 2001. Amphipholis linopneusti n. sp., a sexually dimor- Rishiri Island, north of Hokkaido (Matsumoto, 1917, 1941; background and knowledge and corroboration with other kinds of Japanese with English Abstract) characters. I. General character evaluation and delineation of phic amphiurid brittle star (Echinodermata: Ophiuroidea), Murakami, 1942, 1944; Irimura, 1969, 1979; Saba et al., 1982; data, this approach may fail to identify species accurately Fujita, T., Ishida, Y., Kato, T. and Irimura, S. 2004. Ophiuroids the families Ophiomyxidae and Ophiacanthidae. Zootaxa, epizoic on a spatangoid sea urchin. In: Barker, M. (ed.) Fujita and Kohtsuka, 2003; Komatsu, et al., 2007; This study), (Sundberg et al., 2010). To avoid this, DNA barcoding sequence (Echinodermata) collected from the Oki Islands in the Sea of 2697: 1–154. Echinoderms 2000. Proceedings of the 10th International Peter the Great Bay and Tatar Strait in Russia (Djakonov, 1954), should accompany morphological data for identification, but these Japan. Bulletin of the National Science Museum. Series A Matsumoto, H. 1917. A monograph of Japanese Ophiuroidea, Echinoderm Conference, Dunedin, New Zealand. AA Balke- the East Sea in Korea (Shin and Rho, 1996), and Dalian, “integrative data” are provided for few species of ophiuroids (e.g., (Zoology), 30 (4): 191–218. arranged according to a new classification. Journal of the ma Publishers, Amsterdam, pp. 317–322. northern-eastern China (Liao, 2004), intertidal to 70 m in depth Stöhr and Muthus, 2010; Okanishi et al., 2018; Alitto et al., 2019; Hendler, G. 2018. Armed to the teeth: A new paradigm for the College of Science, Imperial University of Tokyo, 38: 1–408. (see also Fujita et al., 2004). Lee and Shin, 2019). In this study, we provide the integrative data buccal skeleton of brittle stars (Echinodermata: Ophiuroidea). Matsumoto, H. 1941. Report of the Biological Survey of Mutsu Remarks. Morphologically, the two examined specimens for A. kochii for the first time, and the accumulation of such study Contributions in Science, 526: 189–311. Bay. 36. Ophiuroidea of the Mutsu Bay and Vicinities. fall within the genus Amphipholis by virtue of having smooth and should lead to a more accurate classification of ophiuroid in the Irimura, S. 1969. Supplementary report of Dr. Murakami’s paper Science Reports of the Tôhoku Imperial University, 16 (3): naked scales on disc, a pair of infradental papillae on ventral most future. on the ophiurans of Amakusa, Kyushu. Publications from the 331–344. top of jaw, adoral shield spine twice as wide as long, arm spine Amakusa Marine Biological Laboratory, 2 (1): 37–48. Murakami, S. 1942. Ophiurans of Izu, Japan. Journal of the articulations with two smooth separated, parallel, straight dorsal Irimura, S. 1979. Ophiuroidea of Sado Island, the Sea of Japan. Department of Agriculture, Kyushu Imperial University, 7: and ventral lobes, lateral arm plates which are commonly ACKNOWLEDGEMENTS Annual report of the Sado Marine Biological Station, Niigata 1–36. sickle-shaped with pointed dorsal-proximal tip, inner side of lateral University, 9: 1–6. Murakami, S. 1944. Note on the ophiurans of Amakusa, Kyusyu. arm plates with two central knobs (Fell, 1960; O’Hara et al., 2018). We are most grateful to Mamoru Sekifuji and Michiyo Koehler, R. 1922. Contributions to the biology of the Philippine Journal of the Department of Agriculture, Kyushu Imperial However, according to O’Hara et al. (2017), most of the genera of Kawabata of Misaki Marine Biological Station, for their kind Archipelago and adjacent regions. Ophiurans of the University, 7(8): 259–81. Amphiuridae, including Amphipholis appeared to be polyphyletic. assistance during sampling. We are also grateful to Dr. Toru Miura Philippine seas and adjacent waters. Smithsonian Institution O’Hara, T. D., Hugall, A. F., Thuy, B., Stöhr, S. and Martynov, A. Traditionally, external morphology, such as teeth have been used and Dr. Manabu Yoshida of Misaki Marine Biological Station, The United States National Museum Bulletin, 100 (5): 1–486. V. 2017. Restructuring higher taxonomy using broad-scale as taxonomic characters of the genus in Amphiuridae. However, University of Tokyo for their assistance in photography of the Komatsu, M., Shibata, D., Wakabayashi, K., Kogure, Y., Kano, Y. phylogenomics: the living Ophiuroidea. Molecular Phylo- this molecular evidence indicates that they should be reviewed. specimens with JEOL JSM-5510LV and OLYMPUS SZX7, and and Takahashi, N. 2007. Echinoderms in the littoral region of genetics and Evolution, 107: 415–430. DOI: 10.1016/ Our observation of lateral arm plates on Amphipholis kochii Dr Tim O’Hara of Museum Victoria and an anonymous reviewer Rishiri Island. Rishiri Studies, 26: 1–14. j.ympev.2016.12.006 44 Masanori Okanishi, Mayuko Nakamura, Hana Tamura and Hisanori Kohtsuka Redescription of Amphipholis kochii from Sagami Bay 45 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, 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, 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 COI008 (5′-CCDTANGMDATCATDGCRTACATCATTCC-3′) only mentioned in the figure of this reference]. (Okanishi & Fujita, 2013) was used for PCR. The PCR products 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 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 Scientific). Its accession number of DNA Data Bank of Japan calcareous algae (probably Corallina pilulifera) by M. Nakamura (DDBJ) is LC556229. and H. Tamura (third author), 9 April 2020. KMNH IvR 600016: one specimen, Arai Beach, Misaki, Miura, Kanagawa Prefecture, central-eastern Japan, by hand, intertidal zone, collected by H. RESULTS AND DISCUSSION Kohtsuka (last author), 5 July 2011. 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 Fig. 4. Amphipholis kochii (KMNH IvR 600015). A–C, dorsal views of proximal (A), middle (B) and distal (C) portion of arms; 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). D–F, ventral view of proximal (D), middle (E) and distal (F) portion of arms. Abbreviations: AS, arm spine; DAP, dorsal arm 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 plate; LAP, lateral arm plate; TS, tentacle scale; VAP, ventral arm plate. Scale bars, 1 mm. 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 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). 2004; Komatsu et al., 2007). Prior to this study, although external deposited in the Kitakyushu Museum of Natural History & Human Fig. 3. Amphipholis kochii (KMNH IvR 600015). A, dorsal disc and proximal portion of arms; B, ventral disc and proximal portion 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, features with sketches showing diagnostic characters of A. kochii History (KMNH) (KMNH IvR 600015, 600016). of arms; C, dorsal central disc; D, dorsal peripheral disc; E, ventral disc; F, interradial ventral disc. An oral shield serving as 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 were provided in six studies (Lütken, 1872; Lyman, 1875; Morphological terminologies follow Thuy and Stöhr (2011), madreporite is indicated by an arrow. Abbreviations: AdSh, adoral shield; AdShSp?, probably adoral shield spine; AS, arm 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 Matsumoto, 1917, 1941; Djakonov, 1954; Liao, 2004), no data for Stöhr et al. (2012), Okanishi and Fujita (2018b), and Hendler spine; GS, genital slit; IPa, infradental papilla; OP, oral plate; OSh, oral shield; PP, primary plate; RS, radial shield; TS, 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 microstructure of ossicles of A. kochii has been provided so far. (2018). Terminologies of oral papillae sensu lato in Hendler (2018) tentacle scale; VAP, ventral arm plate. Scale bars, 1 mm. 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. 6C). On SEM images of microstructure of ossicles for A. kochii, based on observations. In this study, we did not observe ontogenetic series of of oral shield to lateral edge of the disc, approximately 0.25 mm in wide as long, and gradually decrease in size toward the arm tip on dorsal-proximal side elongate, larger, wing-like (Fig. 5A) and internal side, two knobs, composed of more densely meshed two specimens from Misaki, Sagami Bay, Japan. Additionally, examined species and did not dissect mouth ossicles to prevent width and 1.5 mm in length (Fig. 3F). On ventral surface, adoral (Fig. 4C). Lateral arm plates, widely separated by dorsal and plates square, as wide as long, in contact with each other (Fig. 4D). proximal portion of arm, three spiniform arm spines on each lateral those on ventral-proximal side smaller, approximately half width stereom than remaining area, situated at center (Fig. 6D). In the two sequence of mitochondrial Cytochrome c oxidase subunit I (COI) destruction of the precious specimens. Thus, we marked “?” for shields trapezoid, approximately 1.5 times wider than the longest ventral arm plates proximally (Figs 3F; 4A), in contact with each On middle portion of arm, the plates pentagonal, slightly longer arm plate; the dorsal most one slightly flattened and longest, of the dorsal-distal ones (Fig. 5B). In proximal portion of arm, central knobs, a single perforation next to a more distal one and gene including DNA barcoding region from A. kochii is provided oral papillae sensu lato which we are not sure of their origin (Fig. length, in contact with each other (Fig. 3E). Oral plates triangular, other after middle portion of the arms on both ventral and dorsal than wide and in contact each other (Fig. 4E). On distal portion of approximately 1.5 times longer than the corresponding arm “T”-shaped groove on dorsal side (Fig. 5C). Longitudinal groove tentacle notch that extends to distal ventral side (Fig. 6D). Three for future phylogenetic analysis. 3E). Systematics follows O’Hara et al. (2018) and Christodoulou et approximately the same width as length inner side, the length sides (Fig. 4B, C, E, F). On proximal portion of arm, ventral arm arm, the plates triangular, as wide as long, separated (Fig. 4F). On segment; the other two spines almost the same length of the on ventral side of proximal portion of arm and on both dorsal and spine articulations on distal side, with nearly horizontal and parallel

Fig. 5. Amphipholis kochii (KMNH IvR 600015). SEM photographs of vertebrae at proximal (A–E) and distal portion (F–H) of the Fig. 6. Amphipholis kochii (KMNH IvR 600015), SEM photographs of ossicles. A, B, vertebrae from distal portion of arm, dorsal arms, proximal (A, F), distal (B, G), dorsal (C), ventral (D, H) and lateral (E) views, blue indicate “T” shaped or longitudinal (A) and lateral (B) views; C–H, lateral arm plates from proximal (C–E) and distal (F–H) portion of arm, external (C, F), grooves. Arrows indicate orientation: d, dorsal side; dis, distal side; pro, proximal side; v, ventral side. Abbreviations: DF, internal (D, G) and distal (E, H) views; I, J, dorsal arm plates from proximal portion of arm, external (I) and internal (J) views. dorsal muscle fossae; DT, depression for tentacle; K, keel; OL, opening of lateral canal; PR, protrusion; VF, ventral muscle Arrows indicate orientation: d, dorsal side; dis, distal side; pro, proximal side; v, ventral side. Abbreviations: CK, central fossae. Scale bars, 100 μm. knob; DL, dorsal lobe; H, hole; MO, muscular opening; NO, nerve opening; P, perforation; PR, protrusion; PS, parallel striation; TN, tentacle notch; VL, ventral lobe. Scale bars, 100 μm. 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, 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, 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 COI008 (5′-CCDTANGMDATCATDGCRTACATCATTCC-3′) only mentioned in the figure of this reference]. (Okanishi & Fujita, 2013) was used for PCR. The PCR products 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 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 Scientific). Its accession number of DNA Data Bank of Japan calcareous algae (probably Corallina pilulifera) by M. Nakamura (DDBJ) is LC556229. and H. Tamura (third author), 9 April 2020. KMNH IvR 600016: one specimen, Arai Beach, Misaki, Miura, Kanagawa Prefecture, central-eastern Japan, by hand, intertidal zone, collected by H. RESULTS AND DISCUSSION Kohtsuka (last author), 5 July 2011. 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. 6C). On SEM images of microstructure of ossicles for A. kochii, based on observations. In this study, we did not observe ontogenetic series of of oral shield to lateral edge of the disc, approximately 0.25 mm in wide as long, and gradually decrease in size toward the arm tip on dorsal-proximal side elongate, larger, wing-like (Fig. 5A) and internal side, two knobs, composed of more densely meshed two specimens from Misaki, Sagami Bay, Japan. Additionally, examined species and did not dissect mouth ossicles to prevent width and 1.5 mm in length (Fig. 3F). On ventral surface, adoral (Fig. 4C). Lateral arm plates, widely separated by dorsal and plates square, as wide as long, in contact with each other (Fig. 4D). proximal portion of arm, three spiniform arm spines on each lateral those on ventral-proximal side smaller, approximately half width stereom than remaining area, situated at center (Fig. 6D). In the two sequence of mitochondrial Cytochrome c oxidase subunit I (COI) destruction of the precious specimens. Thus, we marked “?” for shields trapezoid, approximately 1.5 times wider than the longest ventral arm plates proximally (Figs 3F; 4A), in contact with each On middle portion of arm, the plates pentagonal, slightly longer arm plate; the dorsal most one slightly flattened and longest, of the dorsal-distal ones (Fig. 5B). In proximal portion of arm, central knobs, a single perforation next to a more distal one and gene including DNA barcoding region from A. kochii is provided oral papillae sensu lato which we are not sure of their origin (Fig. length, in contact with each other (Fig. 3E). Oral plates triangular, other after middle portion of the arms on both ventral and dorsal than wide and in contact each other (Fig. 4E). On distal portion of approximately 1.5 times longer than the corresponding arm “T”-shaped groove on dorsal side (Fig. 5C). Longitudinal groove tentacle notch that extends to distal ventral side (Fig. 6D). Three for future phylogenetic analysis. 3E). Systematics follows O’Hara et al. (2018) and Christodoulou et approximately the same width as length inner side, the length sides (Fig. 4B, C, E, F). On proximal portion of arm, ventral arm arm, the plates triangular, as wide as long, separated (Fig. 4F). On segment; the other two spines almost the same length of the on ventral side of proximal portion of arm and on both dorsal and spine articulations on distal side, with nearly horizontal and parallel

48 Masanori Okanishi, Mayuko Nakamura, Hana Tamura and Hisanori Kohtsuka Redescription of Amphipholis kochii from Sagami Bay 49

ventral and dorsal lobes which are almost in similar size (Fig. 6C, revealed that their parallel striations on their external surfaces (Fig. for their providing critical comments. This work was partly Kroh, A. and Thuy, B. 2013. A new Philippine ophiuroid symbiotic O’Hara, T. D., Stöhr, S., Hugall, A. F., Thuy, B. and Martynov, A. Stöhr, S. 2011. New records and new species of Ophiuroidea E). Surrounds of the articulations slightly sunken but no areoles of 6C) are thicker than those of other species of Amphiuridae (see also supported by KAKENHI Grant Number JP17K07549. on a cassiduloid echinoid species. Zoologischer Anzeiger, 2018. Morphological diagnoses of higher taxa in Ophiuroidea (Echinodermata) from Lifou, Loyalty Islands, New Cale- denser stereom (Fig. 6C, E). Muscle and nerve openings clearly Thuy and Stöhr, 2011; lateral arm plates of Amphiura chiajei 252 (3): 279–288. (Echinodermata) in support of a new classification. European donia. Zootaxa, 3089: 1–50. separated by stereom protuberances (Fig. 6E). Forbes, 1843, fig. 12 (2a, 3a) and Amphiura sundevalli (Müller and Lee, T. and Shin, S. 2019. A new record of the brittle star, Journal of Taxonomy, 416: 1–35. Stöhr S. and Muths D. 2010. Morphological diagnosis of the two On distal portion of arm, lateral arm plates slightly longer Troshcel, 1842), fig. 12 (8a, 9a)), indicating that it possibly be a REFERENCES Amphistigma minuta (Ophiuroidea: Amphilepidida: Okanishi, M. and Fujita, T. 2013. Molecular phylogeny based on genetic lineages of Acrocnida brachiata (Echinodermata: than high, proximal edge slightly convex, external and internal new diagnostic character of genus level for the Amphiuridae. The Amphiuridae), from Jeju Island, Korea. Animal Systematics, increased number of species and genes revealed more robust Ophiuroidea) with description of a new species. Journal of the sides basically smooth (Fig. 6F, G). Two central knobs on internal microstructure of the lateral arm plate is unknown for most species, Alitto, R. A. dos S., Amaral, A. C. Z., de Oliveira, L. D., Serrano, Evolution and Diversity, 35 (2): 84–90. DOI: 10.5635/ family-level systematics of the order Euryalida (Echinoder- Marine Biology Association U.K., 90: 831–843. side and a single perforation next to a more distal knob (Fig. 6G). so further examination of the microstructure of it is necessary for a H., Seger, K. R., Guilhem, P. D. B., Di Domenico, M., ASED.2019.35.2.011 mata: Ophiuroidea). Molecular Phylogenetics and Evolution, Stöhr, S., O’Hara, T. D. and Thuy, B. 2012. Global diversity of On distal side, two spine articulations, each of which was future revision. Christensen, A. B., Lourenço, K. R., Tavares, M. and Borges, Liao, Y. 2004. Fauna Sinica. Vol. 40. Echinodermata: Ophiuroidea. 69 (3): 566–580. brittle stars (Echinodermata: Ophiuroidea). PLoS ONE, 7 (3): represented as a simple hole (Fig. 6H). 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Lateral arm plate morphology in portion of arm, and slightly longer than wide on distal portion of region for Amphopholis kochii (KMNH IvR 600015; LC556229) collection of the Museum of Comparative Zoology. Memoirs Slangestjernerne beskrevne – Med nogle Bemaerkninger om Okanishi, M., Senatoku, A., Martynov, A. and Fujita, T. 2018. A brittle stars (Echinodermata: Ophiuroidea): new perspectives ·· arm (Fig. 7C–F). Intersections of mesh structure of stereom of the and so far, any DNA sequence of A. kochii has not been registered of the Museum of Comparative Zoology at Harvard College, Selvdelingen hos Straaldyrene –. Oversigt over det Kongelige new cryptic species of Asteronyx Müller and Troschel, 1842 for ophiuroid micropalaeontology and classification. Zootaxa, ventral arm plate protruding on external side and smooth, not in GenBank. A BLAST search of the sequence (LC556229) at the 25 (4): 165–376. Danske Videnskabernes Selska bs forhandlinger, 77: 75–158. (Echinodermata: Ophiuroidea), based on molecular phylo- 3013: 1–47. protruding on internal side (Fig. 7C–F). DDBJ website (http://blast.ddbj.nig.ac.jp/blastn?lang=ja) yielded Djakonov, A. M. 1954. Ophiuroids of the USSR Seas. In: Lyman, T. 1875. Zoological Results of the Hassler Expedition. 2. geny and morphology, from off Pacific Coast of Japan. Thuy, B. and Stöhr, S. 2016. A new morphological phylogeny of the On proximal portion of arm, dorsal most arm spine Amphioplus daleus (Lyman, 1882) (accession no. KU895008) as Pavlovskii, E. N. (ed.) Keys to fauna USSR. Wiener Bindery Ophiuridae and Astrophytidae. Illustrated catalogue of the Zoologischer Anzeiger, 274: 14–33. Ophiuroidea (Echinodermata) accords with molecular evidence cylindrical (Fig. 7G), others spiniform (Fig. 7H), on distal portion the closest sequence with 20.8% K2P genetic distance. It falls Ltd., Jerusalem, pp. 1–123. Museum of Comparative Zoology, at Harvard College, 8 (2): Saba, M., Tomida, Y. and Kimoto, T. 1982. Echinoderms fauna of and renders microfossils accessible for cladistics. PLoS ONE, of arm, all arm spines spiniform (Fig. 7I, J). Spines with simple and within the range of interspecific differences (2.2% to 23%) for COI Fell, H. B. 1960. Synoptic key to the genera of Ophiuroidae. 1–34. Ise Bay, and the northern and middle parts of Kumano-nada. 11 (5): e0156140. DOI: 10.1371/journal.pone.0156140 fine stereom with minute thorns which are pointing to external side in ophiuroids (e.g., Okanishi et al., 2018). Thus, it is reasonable to Zoology Publications from Victoria University of Wellington, Lyman, T. 1882. Report on the Opiuroidea. Report on the Scientific Bulletin of the Mie Prefectural Museum, 4: 1–82. on proximal portion of arms (Fig. 7G, H), and are to the lateral side assume that we provide the first DNA sequence of A. kochii 26: 1–44. Results of the Voyage of H.M.S. Challenger during the years Shin, S. and Rho, B.-J. 1996. Illustrated Encyclopedia of Fauna & on distal portion of arm (Fig. 7G–J). including DNA barcoding region. Although DNA barcoding data Fujita, T. and Kohtsuka, H. 2003. Ophiuroids (Echinodermata) 1873-76, Zoology, 5 (1): 1–386. Flora of Korea. Vol. 36. Echinodermata. The Ministry of Distribution. Amphipholis kochii has been recorded from the has been used in various taxa as a powerful tool for species from Notojima Islands and its adjacent waters in the Sea of Martynov, A. 2010. Reassessment of the classification of the Education, Seoul. coastal areas in Japan, from north of Kumamoto Prefecture to identification, it has been noted that without proper taxonomic Japan. Report of the Noto Marine Center, 9: 25–38. (In Ophiuroidea (Echinodermata), based on morphological Stöhr, S. 2001. Amphipholis linopneusti n. sp., a sexually dimor- Rishiri Island, north of Hokkaido (Matsumoto, 1917, 1941; background and knowledge and corroboration with other kinds of Japanese with English Abstract) characters. I. General character evaluation and delineation of phic amphiurid brittle star (Echinodermata: Ophiuroidea), Murakami, 1942, 1944; Irimura, 1969, 1979; Saba et al., 1982; data, this approach may fail to identify species accurately Fujita, T., Ishida, Y., Kato, T. and Irimura, S. 2004. Ophiuroids the families Ophiomyxidae and Ophiacanthidae. Zootaxa, epizoic on a spatangoid sea urchin. In: Barker, M. (ed.) Fujita and Kohtsuka, 2003; Komatsu, et al., 2007; This study), (Sundberg et al., 2010). To avoid this, DNA barcoding sequence (Echinodermata) collected from the Oki Islands in the Sea of 2697: 1–154. Echinoderms 2000. Proceedings of the 10th International Peter the Great Bay and Tatar Strait in Russia (Djakonov, 1954), should accompany morphological data for identification, but these Japan. Bulletin of the National Science Museum. Series A Matsumoto, H. 1917. A monograph of Japanese Ophiuroidea, Echinoderm Conference, Dunedin, New Zealand. AA Balke- the East Sea in Korea (Shin and Rho, 1996), and Dalian, “integrative data” are provided for few species of ophiuroids (e.g., (Zoology), 30 (4): 191–218. arranged according to a new classification. Journal of the ma Publishers, Amsterdam, pp. 317–322. northern-eastern China (Liao, 2004), intertidal to 70 m in depth Stöhr and Muthus, 2010; Okanishi et al., 2018; Alitto et al., 2019; Hendler, G. 2018. Armed to the teeth: A new paradigm for the College of Science, Imperial University of Tokyo, 38: 1–408. (see also Fujita et al., 2004). Lee and Shin, 2019). In this study, we provide the integrative data buccal skeleton of brittle stars (Echinodermata: Ophiuroidea). Matsumoto, H. 1941. Report of the Biological Survey of Mutsu Remarks. Morphologically, the two examined specimens for A. kochii for the first time, and the accumulation of such study Contributions in Science, 526: 189–311. Bay. 36. Ophiuroidea of the Mutsu Bay and Vicinities. fall within the genus Amphipholis by virtue of having smooth and should lead to a more accurate classification of ophiuroid in the Irimura, S. 1969. Supplementary report of Dr. Murakami’s paper Science Reports of the Tôhoku Imperial University, 16 (3): naked scales on disc, a pair of infradental papillae on ventral most future. on the ophiurans of Amakusa, Kyushu. Publications from the 331–344. top of jaw, adoral shield spine twice as wide as long, arm spine Amakusa Marine Biological Laboratory, 2 (1): 37–48. Murakami, S. 1942. Ophiurans of Izu, Japan. Journal of the articulations with two smooth separated, parallel, straight dorsal Irimura, S. 1979. Ophiuroidea of Sado Island, the Sea of Japan. Department of Agriculture, Kyushu Imperial University, 7: and ventral lobes, lateral arm plates which are commonly ACKNOWLEDGEMENTS Annual report of the Sado Marine Biological Station, Niigata 1–36. sickle-shaped with pointed dorsal-proximal tip, inner side of lateral University, 9: 1–6. Murakami, S. 1944. Note on the ophiurans of Amakusa, Kyusyu. arm plates with two central knobs (Fell, 1960; O’Hara et al., 2018). We are most grateful to Mamoru Sekifuji and Michiyo Koehler, R. 1922. Contributions to the biology of the Philippine Journal of the Department of Agriculture, Kyushu Imperial However, according to O’Hara et al. (2017), most of the genera of Kawabata of Misaki Marine Biological Station, for their kind Archipelago and adjacent regions. Ophiurans of the University, 7(8): 259–81. Fig. 7. Amphipholis kochii (KMNH IvR 600015), SEM photographs of ossicles, A, B, dorsal arm plates from distal portion of arm, Amphiuridae, including Amphipholis appeared to be polyphyletic. assistance during sampling. We are also grateful to Dr. Toru Miura Philippine seas and adjacent waters. Smithsonian Institution O’Hara, T. D., Hugall, A. F., Thuy, B., Stöhr, S. and Martynov, A. external (A) and internal (B) views; C–F, ventral arm plates from proximal (C, D) and distal (E, F) portion of arm, external Traditionally, external morphology, such as teeth have been used and Dr. Manabu Yoshida of Misaki Marine Biological Station, The United States National Museum Bulletin, 100 (5): 1–486. V. 2017. Restructuring higher taxonomy using broad-scale (C, E) and internal (D, F) views; G–J, arm spines from proximal (G, H) and distal (I, J) portion of arms, dorsal most (G, I) and as taxonomic characters of the genus in Amphiuridae. However, University of Tokyo for their assistance in photography of the Komatsu, M., Shibata, D., Wakabayashi, K., Kogure, Y., Kano, Y. phylogenomics: the living Ophiuroidea. Molecular Phylo- ventral most (H, J) ones. Arrows indicate orientation: ba, basal side; dis, distal side; ex, external side; pro, proximal side. Scale this molecular evidence indicates that they should be reviewed. specimens with JEOL JSM-5510LV and OLYMPUS SZX7, and and Takahashi, N. 2007. Echinoderms in the littoral region of genetics and Evolution, 107: 415–430. DOI: 10.1016/ bars, 50 μm. Our observation of lateral arm plates on Amphipholis kochii Dr Tim O’Hara of Museum Victoria and an anonymous reviewer Rishiri Island. Rishiri Studies, 26: 1–14. j.ympev.2016.12.006 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, 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, 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 COI008 (5′-CCDTANGMDATCATDGCRTACATCATTCC-3′) only mentioned in the figure of this reference]. (Okanishi & Fujita, 2013) was used for PCR. The PCR products 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 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 Scientific). Its accession number of DNA Data Bank of Japan calcareous algae (probably Corallina pilulifera) by M. Nakamura (DDBJ) is LC556229. and H. Tamura (third author), 9 April 2020. KMNH IvR 600016: one specimen, Arai Beach, Misaki, Miura, Kanagawa Prefecture, central-eastern Japan, by hand, intertidal zone, collected by H. RESULTS AND DISCUSSION Kohtsuka (last author), 5 July 2011. 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. 6C). On SEM images of microstructure of ossicles for A. kochii, based on observations. In this study, we did not observe ontogenetic series of of oral shield to lateral edge of the disc, approximately 0.25 mm in wide as long, and gradually decrease in size toward the arm tip on dorsal-proximal side elongate, larger, wing-like (Fig. 5A) and internal side, two knobs, composed of more densely meshed two specimens from Misaki, Sagami Bay, Japan. Additionally, examined species and did not dissect mouth ossicles to prevent width and 1.5 mm in length (Fig. 3F). On ventral surface, adoral (Fig. 4C). Lateral arm plates, widely separated by dorsal and plates square, as wide as long, in contact with each other (Fig. 4D). proximal portion of arm, three spiniform arm spines on each lateral those on ventral-proximal side smaller, approximately half width stereom than remaining area, situated at center (Fig. 6D). In the two sequence of mitochondrial Cytochrome c oxidase subunit I (COI) destruction of the precious specimens. Thus, we marked “?” for shields trapezoid, approximately 1.5 times wider than the longest ventral arm plates proximally (Figs 3F; 4A), in contact with each On middle portion of arm, the plates pentagonal, slightly longer arm plate; the dorsal most one slightly flattened and longest, of the dorsal-distal ones (Fig. 5B). In proximal portion of arm, central knobs, a single perforation next to a more distal one and gene including DNA barcoding region from A. kochii is provided oral papillae sensu lato which we are not sure of their origin (Fig. length, in contact with each other (Fig. 3E). Oral plates triangular, other after middle portion of the arms on both ventral and dorsal than wide and in contact each other (Fig. 4E). On distal portion of approximately 1.5 times longer than the corresponding arm “T”-shaped groove on dorsal side (Fig. 5C). Longitudinal groove tentacle notch that extends to distal ventral side (Fig. 6D). Three for future phylogenetic analysis. 3E). Systematics follows O’Hara et al. (2018) and Christodoulou et approximately the same width as length inner side, the length sides (Fig. 4B, C, E, F). On proximal portion of arm, ventral arm arm, the plates triangular, as wide as long, separated (Fig. 4F). On segment; the other two spines almost the same length of the on ventral side of proximal portion of arm and on both dorsal and spine articulations on distal side, with nearly horizontal and parallel

48 Masanori Okanishi, Mayuko Nakamura, Hana Tamura and Hisanori Kohtsuka Redescription of Amphipholis kochii from Sagami Bay 49

ventral and dorsal lobes which are almost in similar size (Fig. 6C, revealed that their parallel striations on their external surfaces (Fig. for their providing critical comments. This work was partly Kroh, A. and Thuy, B. 2013. A new Philippine ophiuroid symbiotic O’Hara, T. D., Stöhr, S., Hugall, A. F., Thuy, B. and Martynov, A. Stöhr, S. 2011. New records and new species of Ophiuroidea E). Surrounds of the articulations slightly sunken but no areoles of 6C) are thicker than those of other species of Amphiuridae (see also supported by KAKENHI Grant Number JP17K07549. on a cassiduloid echinoid species. Zoologischer Anzeiger, 2018. Morphological diagnoses of higher taxa in Ophiuroidea (Echinodermata) from Lifou, Loyalty Islands, New Cale- denser stereom (Fig. 6C, E). Muscle and nerve openings clearly Thuy and Stöhr, 2011; lateral arm plates of Amphiura chiajei 252 (3): 279–288. (Echinodermata) in support of a new classification. European donia. Zootaxa, 3089: 1–50. separated by stereom protuberances (Fig. 6E). Forbes, 1843, fig. 12 (2a, 3a) and Amphiura sundevalli (Müller and Lee, T. and Shin, S. 2019. A new record of the brittle star, Journal of Taxonomy, 416: 1–35. Stöhr S. and Muths D. 2010. Morphological diagnosis of the two On distal portion of arm, lateral arm plates slightly longer Troshcel, 1842), fig. 12 (8a, 9a)), indicating that it possibly be a REFERENCES Amphistigma minuta (Ophiuroidea: Amphilepidida: Okanishi, M. and Fujita, T. 2013. Molecular phylogeny based on genetic lineages of Acrocnida brachiata (Echinodermata: than high, proximal edge slightly convex, external and internal new diagnostic character of genus level for the Amphiuridae. The Amphiuridae), from Jeju Island, Korea. Animal Systematics, increased number of species and genes revealed more robust Ophiuroidea) with description of a new species. Journal of the sides basically smooth (Fig. 6F, G). Two central knobs on internal microstructure of the lateral arm plate is unknown for most species, Alitto, R. A. dos S., Amaral, A. C. Z., de Oliveira, L. D., Serrano, Evolution and Diversity, 35 (2): 84–90. DOI: 10.5635/ family-level systematics of the order Euryalida (Echinoder- Marine Biology Association U.K., 90: 831–843. side and a single perforation next to a more distal knob (Fig. 6G). so further examination of the microstructure of it is necessary for a H., Seger, K. R., Guilhem, P. D. B., Di Domenico, M., ASED.2019.35.2.011 mata: Ophiuroidea). Molecular Phylogenetics and Evolution, Stöhr, S., O’Hara, T. D. and Thuy, B. 2012. Global diversity of On distal side, two spine articulations, each of which was future revision. Christensen, A. B., Lourenço, K. R., Tavares, M. and Borges, Liao, Y. 2004. Fauna Sinica. Vol. 40. Echinodermata: Ophiuroidea. 69 (3): 566–580. brittle stars (Echinodermata: Ophiuroidea). PLoS ONE, 7 (3): represented as a simple hole (Fig. 6H). 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T. and Strand, M. 2010. DNA barcoding protruding on internal side (Figs 6I, J; 7A, B). corresponding arm segment; two oral papillae sensu lato; two mine field. Current Biology, 29 (22): 3909–3912.e3. DOI: Förhandlingar, Stockholm, 23 (9): 303–336. (Echinodermata Ophiuroidea) from Ryukyu Islands, south- should accompany taxonomy – the case of Cerebratulus spp Ventral arm plates pentagonal with sharp proximal edge and tentacle scales; arm spines with whitish basal portion. 10.1016/j.cub.2019.09.012 Lütken, C. F. 1872. Ophiuridarum novarum vel minus cognitarum western Japan. Proceedings of the Biological Society of (Nemertea). Molecular Ecology Resources, 10: 274–281. slightly pointing distal-lateral edges, as wide as long on proximal In our molecular study, we obtained 825 bp of COI gene Clark, H. L. 1915. Catalogue of recent ophiurans: based on the descriptiones nonnullae. ogle nye eller mindre bekjerdte Washington, 131: 163–174. Thuy, B. and Stöhr, S. 2011. Lateral arm plate morphology in portion of arm, and slightly longer than wide on distal portion of region for Amphopholis kochii (KMNH IvR 600015; LC556229) collection of the Museum of Comparative Zoology. Memoirs Slangestjernerne beskrevne – Med nogle Bemaerkninger om Okanishi, M., Senatoku, A., Martynov, A. and Fujita, T. 2018. A brittle stars (Echinodermata: Ophiuroidea): new perspectives ·· arm (Fig. 7C–F). Intersections of mesh structure of stereom of the and so far, any DNA sequence of A. kochii has not been registered of the Museum of Comparative Zoology at Harvard College, Selvdelingen hos Straaldyrene –. Oversigt over det Kongelige new cryptic species of Asteronyx Müller and Troschel, 1842 for ophiuroid micropalaeontology and classification. Zootaxa, ventral arm plate protruding on external side and smooth, not in GenBank. A BLAST search of the sequence (LC556229) at the 25 (4): 165–376. Danske Videnskabernes Selska bs forhandlinger, 77: 75–158. (Echinodermata: Ophiuroidea), based on molecular phylo- 3013: 1–47. protruding on internal side (Fig. 7C–F). DDBJ website (http://blast.ddbj.nig.ac.jp/blastn?lang=ja) yielded Djakonov, A. M. 1954. Ophiuroids of the USSR Seas. In: Lyman, T. 1875. Zoological Results of the Hassler Expedition. 2. geny and morphology, from off Pacific Coast of Japan. Thuy, B. and Stöhr, S. 2016. A new morphological phylogeny of the On proximal portion of arm, dorsal most arm spine Amphioplus daleus (Lyman, 1882) (accession no. KU895008) as Pavlovskii, E. N. (ed.) Keys to fauna USSR. Wiener Bindery Ophiuridae and Astrophytidae. Illustrated catalogue of the Zoologischer Anzeiger, 274: 14–33. Ophiuroidea (Echinodermata) accords with molecular evidence cylindrical (Fig. 7G), others spiniform (Fig. 7H), on distal portion the closest sequence with 20.8% K2P genetic distance. It falls Ltd., Jerusalem, pp. 1–123. Museum of Comparative Zoology, at Harvard College, 8 (2): Saba, M., Tomida, Y. and Kimoto, T. 1982. Echinoderms fauna of and renders microfossils accessible for cladistics. PLoS ONE, of arm, all arm spines spiniform (Fig. 7I, J). Spines with simple and within the range of interspecific differences (2.2% to 23%) for COI Fell, H. B. 1960. Synoptic key to the genera of Ophiuroidae. 1–34. Ise Bay, and the northern and middle parts of Kumano-nada. 11 (5): e0156140. DOI: 10.1371/journal.pone.0156140 fine stereom with minute thorns which are pointing to external side in ophiuroids (e.g., Okanishi et al., 2018). Thus, it is reasonable to Zoology Publications from Victoria University of Wellington, Lyman, T. 1882. Report on the Opiuroidea. Report on the Scientific Bulletin of the Mie Prefectural Museum, 4: 1–82. on proximal portion of arms (Fig. 7G, H), and are to the lateral side assume that we provide the first DNA sequence of A. kochii 26: 1–44. Results of the Voyage of H.M.S. Challenger during the years Shin, S. and Rho, B.-J. 1996. Illustrated Encyclopedia of Fauna & on distal portion of arm (Fig. 7G–J). including DNA barcoding region. Although DNA barcoding data Fujita, T. and Kohtsuka, H. 2003. Ophiuroids (Echinodermata) 1873-76, Zoology, 5 (1): 1–386. Flora of Korea. Vol. 36. Echinodermata. The Ministry of Distribution. Amphipholis kochii has been recorded from the has been used in various taxa as a powerful tool for species from Notojima Islands and its adjacent waters in the Sea of Martynov, A. 2010. Reassessment of the classification of the Education, Seoul. coastal areas in Japan, from north of Kumamoto Prefecture to identification, it has been noted that without proper taxonomic Japan. Report of the Noto Marine Center, 9: 25–38. (In Ophiuroidea (Echinodermata), based on morphological Stöhr, S. 2001. Amphipholis linopneusti n. sp., a sexually dimor- Rishiri Island, north of Hokkaido (Matsumoto, 1917, 1941; background and knowledge and corroboration with other kinds of Japanese with English Abstract) characters. I. General character evaluation and delineation of phic amphiurid brittle star (Echinodermata: Ophiuroidea), Murakami, 1942, 1944; Irimura, 1969, 1979; Saba et al., 1982; data, this approach may fail to identify species accurately Fujita, T., Ishida, Y., Kato, T. and Irimura, S. 2004. Ophiuroids the families Ophiomyxidae and Ophiacanthidae. Zootaxa, epizoic on a spatangoid sea urchin. In: Barker, M. (ed.) Fujita and Kohtsuka, 2003; Komatsu, et al., 2007; This study), (Sundberg et al., 2010). To avoid this, DNA barcoding sequence (Echinodermata) collected from the Oki Islands in the Sea of 2697: 1–154. Echinoderms 2000. Proceedings of the 10th International Peter the Great Bay and Tatar Strait in Russia (Djakonov, 1954), should accompany morphological data for identification, but these Japan. Bulletin of the National Science Museum. Series A Matsumoto, H. 1917. A monograph of Japanese Ophiuroidea, Echinoderm Conference, Dunedin, New Zealand. AA Balke- the East Sea in Korea (Shin and Rho, 1996), and Dalian, “integrative data” are provided for few species of ophiuroids (e.g., (Zoology), 30 (4): 191–218. arranged according to a new classification. Journal of the ma Publishers, Amsterdam, pp. 317–322. northern-eastern China (Liao, 2004), intertidal to 70 m in depth Stöhr and Muthus, 2010; Okanishi et al., 2018; Alitto et al., 2019; Hendler, G. 2018. Armed to the teeth: A new paradigm for the College of Science, Imperial University of Tokyo, 38: 1–408. (see also Fujita et al., 2004). Lee and Shin, 2019). In this study, we provide the integrative data buccal skeleton of brittle stars (Echinodermata: Ophiuroidea). Matsumoto, H. 1941. Report of the Biological Survey of Mutsu Remarks. Morphologically, the two examined specimens for A. kochii for the first time, and the accumulation of such study Contributions in Science, 526: 189–311. Bay. 36. Ophiuroidea of the Mutsu Bay and Vicinities. fall within the genus Amphipholis by virtue of having smooth and should lead to a more accurate classification of ophiuroid in the Irimura, S. 1969. Supplementary report of Dr. Murakami’s paper Science Reports of the Tôhoku Imperial University, 16 (3): naked scales on disc, a pair of infradental papillae on ventral most future. on the ophiurans of Amakusa, Kyushu. Publications from the 331–344. top of jaw, adoral shield spine twice as wide as long, arm spine Amakusa Marine Biological Laboratory, 2 (1): 37–48. Murakami, S. 1942. Ophiurans of Izu, Japan. Journal of the articulations with two smooth separated, parallel, straight dorsal Irimura, S. 1979. Ophiuroidea of Sado Island, the Sea of Japan. Department of Agriculture, Kyushu Imperial University, 7: and ventral lobes, lateral arm plates which are commonly ACKNOWLEDGEMENTS Annual report of the Sado Marine Biological Station, Niigata 1–36. sickle-shaped with pointed dorsal-proximal tip, inner side of lateral University, 9: 1–6. Murakami, S. 1944. Note on the ophiurans of Amakusa, Kyusyu. arm plates with two central knobs (Fell, 1960; O’Hara et al., 2018). We are most grateful to Mamoru Sekifuji and Michiyo Koehler, R. 1922. Contributions to the biology of the Philippine Journal of the Department of Agriculture, Kyushu Imperial However, according to O’Hara et al. (2017), most of the genera of Kawabata of Misaki Marine Biological Station, for their kind Archipelago and adjacent regions. Ophiurans of the University, 7(8): 259–81. Fig. 7. Amphipholis kochii (KMNH IvR 600015), SEM photographs of ossicles, A, B, dorsal arm plates from distal portion of arm, Amphiuridae, including Amphipholis appeared to be polyphyletic. assistance during sampling. We are also grateful to Dr. Toru Miura Philippine seas and adjacent waters. Smithsonian Institution O’Hara, T. D., Hugall, A. F., Thuy, B., Stöhr, S. and Martynov, A. external (A) and internal (B) views; C–F, ventral arm plates from proximal (C, D) and distal (E, F) portion of arm, external Traditionally, external morphology, such as teeth have been used and Dr. Manabu Yoshida of Misaki Marine Biological Station, The United States National Museum Bulletin, 100 (5): 1–486. V. 2017. Restructuring higher taxonomy using broad-scale (C, E) and internal (D, F) views; G–J, arm spines from proximal (G, H) and distal (I, J) portion of arms, dorsal most (G, I) and as taxonomic characters of the genus in Amphiuridae. However, University of Tokyo for their assistance in photography of the Komatsu, M., Shibata, D., Wakabayashi, K., Kogure, Y., Kano, Y. phylogenomics: the living Ophiuroidea. Molecular Phylo- ventral most (H, J) ones. Arrows indicate orientation: ba, basal side; dis, distal side; ex, external side; pro, proximal side. Scale this molecular evidence indicates that they should be reviewed. specimens with JEOL JSM-5510LV and OLYMPUS SZX7, and and Takahashi, N. 2007. Echinoderms in the littoral region of genetics and Evolution, 107: 415–430. DOI: 10.1016/ bars, 50 μm. Our observation of lateral arm plates on Amphipholis kochii Dr Tim O’Hara of Museum Victoria and an anonymous reviewer Rishiri Island. Rishiri Studies, 26: 1–14. j.ympev.2016.12.006 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, 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, 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 COI008 (5′-CCDTANGMDATCATDGCRTACATCATTCC-3′) only mentioned in the figure of this reference]. (Okanishi & Fujita, 2013) was used for PCR. The PCR products 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 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 Scientific). Its accession number of DNA Data Bank of Japan calcareous algae (probably Corallina pilulifera) by M. Nakamura (DDBJ) is LC556229. and H. Tamura (third author), 9 April 2020. KMNH IvR 600016: one specimen, Arai Beach, Misaki, Miura, Kanagawa Prefecture, central-eastern Japan, by hand, intertidal zone, collected by H. RESULTS AND DISCUSSION Kohtsuka (last author), 5 July 2011. 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. 6C). On SEM images of microstructure of ossicles for A. kochii, based on observations. In this study, we did not observe ontogenetic series of of oral shield to lateral edge of the disc, approximately 0.25 mm in wide as long, and gradually decrease in size toward the arm tip on dorsal-proximal side elongate, larger, wing-like (Fig. 5A) and internal side, two knobs, composed of more densely meshed two specimens from Misaki, Sagami Bay, Japan. Additionally, examined species and did not dissect mouth ossicles to prevent width and 1.5 mm in length (Fig. 3F). On ventral surface, adoral (Fig. 4C). Lateral arm plates, widely separated by dorsal and plates square, as wide as long, in contact with each other (Fig. 4D). proximal portion of arm, three spiniform arm spines on each lateral those on ventral-proximal side smaller, approximately half width stereom than remaining area, situated at center (Fig. 6D). In the two sequence of mitochondrial Cytochrome c oxidase subunit I (COI) destruction of the precious specimens. Thus, we marked “?” for shields trapezoid, approximately 1.5 times wider than the longest ventral arm plates proximally (Figs 3F; 4A), in contact with each On middle portion of arm, the plates pentagonal, slightly longer arm plate; the dorsal most one slightly flattened and longest, of the dorsal-distal ones (Fig. 5B). In proximal portion of arm, central knobs, a single perforation next to a more distal one and gene including DNA barcoding region from A. kochii is provided oral papillae sensu lato which we are not sure of their origin (Fig. length, in contact with each other (Fig. 3E). Oral plates triangular, other after middle portion of the arms on both ventral and dorsal than wide and in contact each other (Fig. 4E). On distal portion of approximately 1.5 times longer than the corresponding arm “T”-shaped groove on dorsal side (Fig. 5C). Longitudinal groove tentacle notch that extends to distal ventral side (Fig. 6D). Three for future phylogenetic analysis. 3E). Systematics follows O’Hara et al. (2018) and Christodoulou et approximately the same width as length inner side, the length sides (Fig. 4B, C, E, F). On proximal portion of arm, ventral arm arm, the plates triangular, as wide as long, separated (Fig. 4F). On segment; the other two spines almost the same length of the on ventral side of proximal portion of arm and on both dorsal and spine articulations on distal side, with nearly horizontal and parallel

50 Masanori Okanishi, Mayuko Nakamura, Hana Tamura and Hisanori Kohtsuka Redescription of Amphipholis kochii from Sagami Bay 51 ventral and dorsal lobes which are almost in similar size (Fig. 6C, revealed that their parallel striations on their external surfaces (Fig. for their providing critical comments. This work was partly Kroh, A. and Thuy, B. 2013. A new Philippine ophiuroid symbiotic O’Hara, T. D., Stöhr, S., Hugall, A. F., Thuy, B. and Martynov, A. Stöhr, S. 2011. New records and new species of Ophiuroidea E). Surrounds of the articulations slightly sunken but no areoles of 6C) are thicker than those of other species of Amphiuridae (see also supported by KAKENHI Grant Number JP17K07549. on a cassiduloid echinoid species. Zoologischer Anzeiger, 2018. Morphological diagnoses of higher taxa in Ophiuroidea (Echinodermata) from Lifou, Loyalty Islands, New Cale- denser stereom (Fig. 6C, E). Muscle and nerve openings clearly Thuy and Stöhr, 2011; lateral arm plates of Amphiura chiajei 252 (3): 279–288. (Echinodermata) in support of a new classification. European donia. Zootaxa, 3089: 1–50. separated by stereom protuberances (Fig. 6E). Forbes, 1843, fig. 12 (2a, 3a) and Amphiura sundevalli (Müller and Lee, T. and Shin, S. 2019. A new record of the brittle star, Journal of Taxonomy, 416: 1–35. Stöhr S. and Muths D. 2010. Morphological diagnosis of the two On distal portion of arm, lateral arm plates slightly longer Troshcel, 1842), fig. 12 (8a, 9a)), indicating that it possibly be a REFERENCES Amphistigma minuta (Ophiuroidea: Amphilepidida: Okanishi, M. and Fujita, T. 2013. Molecular phylogeny based on genetic lineages of Acrocnida brachiata (Echinodermata: than high, proximal edge slightly convex, external and internal new diagnostic character of genus level for the Amphiuridae. The Amphiuridae), from Jeju Island, Korea. 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Öfversigt af Kongliga Vetenskaps- Akademiens Förhand- with descriptions of new species of Ophiolepis and Database. Available at http://www.marinespecies.org/ with sharpen proximal edge, as wide as long on distal portion of densely scales; radial shields separate each other, approximately DOI: 10.1371/journal.pone.0210331 lingar, Stockholm, 23 (6): 163–166. Ophiozonella (Echinodermata: Ophiuroidea: Amphilepidida). ophiuroidea on 2020-06-10. DOI:10.14284/358 (accessed on arm (Fig. 7A, B). Intersections of mesh structure of stereom of the three times longer than wide; arm spines up to three, the longest Christodoulou, M., O’Hara, T. D., Hugall, A. F. and Arbizu, P. M. Ljungman, A. V. 1867. Ophiuroidea viventia huc usque cognita Zootaxa, 4377 (1): 1–20. 10 June 2020) dorsal arm plate protruding on external side and smooth, not one in proximal portion of arm flatten, 1.5 times longer than the 2019. Dark ophiuroid biodiversity in a prospective abyssal enumerat. Öfversigt af Kongliga Vetenskaps-Akademiens Okanishi, M. and Fujita, Y. 2018b. A new species of Ophioconis Sundberg, P., Vodoti, E. T. and Strand, M. 2010. DNA barcoding protruding on internal side (Figs 6I, J; 7A, B). corresponding arm segment; two oral papillae sensu lato; two mine field. Current Biology, 29 (22): 3909–3912.e3. DOI: Förhandlingar, Stockholm, 23 (9): 303–336. (Echinodermata Ophiuroidea) from Ryukyu Islands, south- should accompany taxonomy – the case of Cerebratulus spp Ventral arm plates pentagonal with sharp proximal edge and tentacle scales; arm spines with whitish basal portion. 10.1016/j.cub.2019.09.012 Lütken, C. F. 1872. Ophiuridarum novarum vel minus cognitarum western Japan. Proceedings of the Biological Society of (Nemertea). Molecular Ecology Resources, 10: 274–281. slightly pointing distal-lateral edges, as wide as long on proximal In our molecular study, we obtained 825 bp of COI gene Clark, H. L. 1915. Catalogue of recent ophiurans: based on the descriptiones nonnullae. ogle nye eller mindre bekjerdte Washington, 131: 163–174. Thuy, B. and Stöhr, S. 2011. Lateral arm plate morphology in portion of arm, and slightly longer than wide on distal portion of region for Amphopholis kochii (KMNH IvR 600015; LC556229) collection of the Museum of Comparative Zoology. Memoirs Slangestjernerne beskrevne – Med nogle Bemaerkninger om Okanishi, M., Senatoku, A., Martynov, A. and Fujita, T. 2018. A brittle stars (Echinodermata: Ophiuroidea): new perspectives ·· arm (Fig. 7C–F). Intersections of mesh structure of stereom of the and so far, any DNA sequence of A. kochii has not been registered of the Museum of Comparative Zoology at Harvard College, Selvdelingen hos Straaldyrene –. Oversigt over det Kongelige new cryptic species of Asteronyx Müller and Troschel, 1842 for ophiuroid micropalaeontology and classification. Zootaxa, ventral arm plate protruding on external side and smooth, not in GenBank. A BLAST search of the sequence (LC556229) at the 25 (4): 165–376. Danske Videnskabernes Selska bs forhandlinger, 77: 75–158. (Echinodermata: Ophiuroidea), based on molecular phylo- 3013: 1–47. protruding on internal side (Fig. 7C–F). DDBJ website (http://blast.ddbj.nig.ac.jp/blastn?lang=ja) yielded Djakonov, A. M. 1954. Ophiuroids of the USSR Seas. In: Lyman, T. 1875. Zoological Results of the Hassler Expedition. 2. geny and morphology, from off Pacific Coast of Japan. Thuy, B. and Stöhr, S. 2016. A new morphological phylogeny of the On proximal portion of arm, dorsal most arm spine Amphioplus daleus (Lyman, 1882) (accession no. KU895008) as Pavlovskii, E. N. (ed.) Keys to fauna USSR. Wiener Bindery Ophiuridae and Astrophytidae. Illustrated catalogue of the Zoologischer Anzeiger, 274: 14–33. Ophiuroidea (Echinodermata) accords with molecular evidence cylindrical (Fig. 7G), others spiniform (Fig. 7H), on distal portion the closest sequence with 20.8% K2P genetic distance. It falls Ltd., Jerusalem, pp. 1–123. Museum of Comparative Zoology, at Harvard College, 8 (2): Saba, M., Tomida, Y. and Kimoto, T. 1982. Echinoderms fauna of and renders microfossils accessible for cladistics. PLoS ONE, of arm, all arm spines spiniform (Fig. 7I, J). Spines with simple and within the range of interspecific differences (2.2% to 23%) for COI Fell, H. B. 1960. Synoptic key to the genera of Ophiuroidae. 1–34. Ise Bay, and the northern and middle parts of Kumano-nada. 11 (5): e0156140. DOI: 10.1371/journal.pone.0156140 fine stereom with minute thorns which are pointing to external side in ophiuroids (e.g., Okanishi et al., 2018). Thus, it is reasonable to Zoology Publications from Victoria University of Wellington, Lyman, T. 1882. Report on the Opiuroidea. Report on the Scientific Bulletin of the Mie Prefectural Museum, 4: 1–82. on proximal portion of arms (Fig. 7G, H), and are to the lateral side assume that we provide the first DNA sequence of A. kochii 26: 1–44. Results of the Voyage of H.M.S. Challenger during the years Shin, S. and Rho, B.-J. 1996. Illustrated Encyclopedia of Fauna & on distal portion of arm (Fig. 7G–J). including DNA barcoding region. Although DNA barcoding data Fujita, T. and Kohtsuka, H. 2003. Ophiuroids (Echinodermata) 1873-76, Zoology, 5 (1): 1–386. Flora of Korea. Vol. 36. Echinodermata. The Ministry of Distribution. Amphipholis kochii has been recorded from the has been used in various taxa as a powerful tool for species from Notojima Islands and its adjacent waters in the Sea of Martynov, A. 2010. Reassessment of the classification of the Education, Seoul. coastal areas in Japan, from north of Kumamoto Prefecture to identification, it has been noted that without proper taxonomic Japan. Report of the Noto Marine Center, 9: 25–38. (In Ophiuroidea (Echinodermata), based on morphological Stöhr, S. 2001. Amphipholis linopneusti n. sp., a sexually dimor- Rishiri Island, north of Hokkaido (Matsumoto, 1917, 1941; background and knowledge and corroboration with other kinds of Japanese with English Abstract) characters. I. General character evaluation and delineation of phic amphiurid brittle star (Echinodermata: Ophiuroidea), Murakami, 1942, 1944; Irimura, 1969, 1979; Saba et al., 1982; data, this approach may fail to identify species accurately Fujita, T., Ishida, Y., Kato, T. and Irimura, S. 2004. Ophiuroids the families Ophiomyxidae and Ophiacanthidae. Zootaxa, epizoic on a spatangoid sea urchin. In: Barker, M. (ed.) Fujita and Kohtsuka, 2003; Komatsu, et al., 2007; This study), (Sundberg et al., 2010). To avoid this, DNA barcoding sequence (Echinodermata) collected from the Oki Islands in the Sea of 2697: 1–154. Echinoderms 2000. Proceedings of the 10th International Peter the Great Bay and Tatar Strait in Russia (Djakonov, 1954), should accompany morphological data for identification, but these Japan. Bulletin of the National Science Museum. Series A Matsumoto, H. 1917. A monograph of Japanese Ophiuroidea, Echinoderm Conference, Dunedin, New Zealand. AA Balke- the East Sea in Korea (Shin and Rho, 1996), and Dalian, “integrative data” are provided for few species of ophiuroids (e.g., (Zoology), 30 (4): 191–218. arranged according to a new classification. Journal of the ma Publishers, Amsterdam, pp. 317–322. northern-eastern China (Liao, 2004), intertidal to 70 m in depth Stöhr and Muthus, 2010; Okanishi et al., 2018; Alitto et al., 2019; Hendler, G. 2018. Armed to the teeth: A new paradigm for the College of Science, Imperial University of Tokyo, 38: 1–408. (see also Fujita et al., 2004). Lee and Shin, 2019). In this study, we provide the integrative data buccal skeleton of brittle stars (Echinodermata: Ophiuroidea). Matsumoto, H. 1941. Report of the Biological Survey of Mutsu Remarks. Morphologically, the two examined specimens for A. kochii for the first time, and the accumulation of such study Contributions in Science, 526: 189–311. Bay. 36. Ophiuroidea of the Mutsu Bay and Vicinities. fall within the genus Amphipholis by virtue of having smooth and should lead to a more accurate classification of ophiuroid in the Irimura, S. 1969. Supplementary report of Dr. Murakami’s paper Science Reports of the Tôhoku Imperial University, 16 (3): naked scales on disc, a pair of infradental papillae on ventral most future. on the ophiurans of Amakusa, Kyushu. Publications from the 331–344. top of jaw, adoral shield spine twice as wide as long, arm spine Amakusa Marine Biological Laboratory, 2 (1): 37–48. Murakami, S. 1942. Ophiurans of Izu, Japan. Journal of the articulations with two smooth separated, parallel, straight dorsal Irimura, S. 1979. Ophiuroidea of Sado Island, the Sea of Japan. Department of Agriculture, Kyushu Imperial University, 7: and ventral lobes, lateral arm plates which are commonly ACKNOWLEDGEMENTS Annual report of the Sado Marine Biological Station, Niigata 1–36. sickle-shaped with pointed dorsal-proximal tip, inner side of lateral University, 9: 1–6. Murakami, S. 1944. Note on the ophiurans of Amakusa, Kyusyu. arm plates with two central knobs (Fell, 1960; O’Hara et al., 2018). We are most grateful to Mamoru Sekifuji and Michiyo Koehler, R. 1922. Contributions to the biology of the Philippine Journal of the Department of Agriculture, Kyushu Imperial However, according to O’Hara et al. (2017), most of the genera of Kawabata of Misaki Marine Biological Station, for their kind Archipelago and adjacent regions. Ophiurans of the University, 7(8): 259–81. Amphiuridae, including Amphipholis appeared to be polyphyletic. assistance during sampling. We are also grateful to Dr. Toru Miura Philippine seas and adjacent waters. Smithsonian Institution O’Hara, T. D., Hugall, A. F., Thuy, B., Stöhr, S. and Martynov, A. Traditionally, external morphology, such as teeth have been used and Dr. Manabu Yoshida of Misaki Marine Biological Station, The United States National Museum Bulletin, 100 (5): 1–486. V. 2017. Restructuring higher taxonomy using broad-scale as taxonomic characters of the genus in Amphiuridae. However, University of Tokyo for their assistance in photography of the Komatsu, M., Shibata, D., Wakabayashi, K., Kogure, Y., Kano, Y. phylogenomics: the living Ophiuroidea. Molecular Phylo- this molecular evidence indicates that they should be reviewed. specimens with JEOL JSM-5510LV and OLYMPUS SZX7, and and Takahashi, N. 2007. Echinoderms in the littoral region of genetics and Evolution, 107: 415–430. DOI: 10.1016/ Our observation of lateral arm plates on Amphipholis kochii Dr Tim O’Hara of Museum Victoria and an anonymous reviewer Rishiri Island. Rishiri Studies, 26: 1–14. j.ympev.2016.12.006 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, 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, 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 COI008 (5′-CCDTANGMDATCATDGCRTACATCATTCC-3′) only mentioned in the figure of this reference]. (Okanishi & Fujita, 2013) was used for PCR. The PCR products 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 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 Scientific). Its accession number of DNA Data Bank of Japan calcareous algae (probably Corallina pilulifera) by M. Nakamura (DDBJ) is LC556229. and H. Tamura (third author), 9 April 2020. KMNH IvR 600016: one specimen, Arai Beach, Misaki, Miura, Kanagawa Prefecture, central-eastern Japan, by hand, intertidal zone, collected by H. RESULTS AND DISCUSSION Kohtsuka (last author), 5 July 2011. 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. 6C). On SEM images of microstructure of ossicles for A. kochii, based on observations. In this study, we did not observe ontogenetic series of of oral shield to lateral edge of the disc, approximately 0.25 mm in wide as long, and gradually decrease in size toward the arm tip on dorsal-proximal side elongate, larger, wing-like (Fig. 5A) and internal side, two knobs, composed of more densely meshed two specimens from Misaki, Sagami Bay, Japan. Additionally, examined species and did not dissect mouth ossicles to prevent width and 1.5 mm in length (Fig. 3F). On ventral surface, adoral (Fig. 4C). Lateral arm plates, widely separated by dorsal and plates square, as wide as long, in contact with each other (Fig. 4D). proximal portion of arm, three spiniform arm spines on each lateral those on ventral-proximal side smaller, approximately half width stereom than remaining area, situated at center (Fig. 6D). In the two sequence of mitochondrial Cytochrome c oxidase subunit I (COI) destruction of the precious specimens. Thus, we marked “?” for shields trapezoid, approximately 1.5 times wider than the longest ventral arm plates proximally (Figs 3F; 4A), in contact with each On middle portion of arm, the plates pentagonal, slightly longer arm plate; the dorsal most one slightly flattened and longest, of the dorsal-distal ones (Fig. 5B). In proximal portion of arm, central knobs, a single perforation next to a more distal one and gene including DNA barcoding region from A. kochii is provided oral papillae sensu lato which we are not sure of their origin (Fig. length, in contact with each other (Fig. 3E). Oral plates triangular, other after middle portion of the arms on both ventral and dorsal than wide and in contact each other (Fig. 4E). On distal portion of approximately 1.5 times longer than the corresponding arm “T”-shaped groove on dorsal side (Fig. 5C). Longitudinal groove tentacle notch that extends to distal ventral side (Fig. 6D). Three for future phylogenetic analysis. 3E). Systematics follows O’Hara et al. (2018) and Christodoulou et approximately the same width as length inner side, the length sides (Fig. 4B, C, E, F). On proximal portion of arm, ventral arm arm, the plates triangular, as wide as long, separated (Fig. 4F). On segment; the other two spines almost the same length of the on ventral side of proximal portion of arm and on both dorsal and spine articulations on distal side, with nearly horizontal and parallel