The family Coeloplanidae of comb jellies has a greatly compressed oral-aboral axis, and comprises the genera Coeloplana (24 species) and Vallicula (one species) (Mills, 1998). Reports of two platyctenid ctenophore species in Korean waters have been published, including the sessile Lyrocteis imperatoris (Song and Hwang, 2009) and a creeping form, C. anthostella (Song and Hwang, 2010). We report the identification of C. bocki associated with encrusting dendronephthyas,which is a new record for Korea. In addition to a morphological comparison of C. bocki and C. anthostella, this study assessed the nucleotide sequences of nuclear and mitochondrial gene fragments (18S rDNA, internal transcribed spacer 1 [ITS1], and cox1) as molecular markers for the identification of these two Korean Coeloplana species.

Samples were collected by SCUBA from an approximately 20-32 m depth off Munseom Island (33°13′32.45′′N, 126°34′06.84′′E), offshore of Seogwipo-si, Jeju-do, Korea. A single individual of C. bocki was collected from the host, Dendronephthya spinulosa on 16 August 2009, together with C. anthostella from the same host at the same location. Several individuals of each of the two Coeloplana species were collected from D. aff. dendritica in this locality on 20 June 2010. The specimens were the creeping forms with undulating margins, and were attached firmly by the flat oral plane to branches and polyp masses of the rigida group of divaricate dendronephthyas (Fig.1 A, B).

For species identification, the morphology was examined using a stereomicroscope (Stemi SV-6; Carl Zeiss, Jena, Germany) and optical microscope (Olympus BH2; Olympus, Tokyo, Japan), and measurements were made with an ocular micrometer before anesthetizing the animals with a MgCl₂ solution and fixing in 5% neutral formalin.

The morphology of C. bocki and C. anthostella is described in detail, with figures. The fragile specimens, which were attached to the branches and polyp masses of the host dendronephthyas, were deposited at the Natural History Museum. Some were also deposited in the Korean Coral Resource

[Table 1.] PCR primers used in this study

PCR primers used in this study

Bank, Ewha Womans University (Seoul, Korea).

After species identification by microscopy, the samples for molecular analysis were fixed in 100% ethanol before total genomic DNA extraction. The total genomic DNA was extracted using a DNA extraction kit (Qiagen Co., Valencia, CA,USA) according to the manufacturer’s protocol. The target fragments from the three genes (18S, ITS1 and cox1) were amplified by PCR using universal primer sets (Table 1). The PCR reactions were carried out in a 50 μL reaction volume consisting of 10 units of Taq polymerase (Roche, Mannheim, Germany), a 2.5 mM mixture of dNTPs, 2.5mM MgCl₂,and 20 pmol of each primer. The amplification conditions involved an initial denaturation step at 94℃ for 1 min followed by 35 cycles of denaturation at 94℃ for 30 sec, primer annealing at 45-55℃ (45℃ for mtDNA cox1; 48℃ for ITS1; 55℃ for 30 sec for 18S), elongation at 72℃ for 1-2 min, and extension at 72℃ for 10 min. The amplified PCR products were subject to gel electrophoresis, and extracted using the TOPO gel purification reagents supplied with the TOPO XL cloning kit (Invitrogen Co., Carlsbad, CA, USA). After gel purification, each of the PCR products was ligated using a TOPO cloning kit, and transformed into competent E. coli. The cycle sequencing reactions for each of the PCR products were performed in both directions.

Phylum Ctenophora Eschscholtz, 1829

Class Tentaculata Eschscholtz, 1825

Subclass Typhlocoela Ospovat, 1985

Order Platyctenida Bourne, 1900

Genus Coeloplana Kowalevsky, 1880

Coeloplana anthostella Song and Hwang, 2010: 217.

[Table 2.] Symbiotic relationship between one polyp mass of host dendronephthya species and the number of symbionts Coeloplana spp.

Symbiotic relationship between one polyp mass of host dendronephthya species and the number of symbionts Coeloplana spp.

Many inds., Isl. Munseom, 26 Nov 2006, Hwang SJ; many inds., Isl. Munseom, 30 Nov 2006, Hwang SJ; many inds., Isl. Munseom, 2 Dec 2006, Hwang SJ; many inds., Isl. Munseom, 16 Aug 2009, Kim BI, 26-32 m deep by SCUBA; 2 inds., Isl. Munseom, 20 Jun 2010, Hwang SJ, 20-32 m deep by SCUBA.

The GenBank accession numbers: HQ43-5810 (18S rDNA), HQ435812 (ITS1), and HQ435811 (cox1).

This species is distinguished from C. bocki (see Komai, 1920, 1922, 1942, 1955) and C. komaii (see Utinomi,1963) by its smaller size and deep scarlet star-shaped markings, which are similar in color to the host dendronephthya. The creeping flattened ctenophores are oval-shaped, have an undulating margin, and are 0.88×0.53-3.50×2.88 (1.99±0.87×1.38±0.55, n=31) mm (tentacular axis×sagittal axis; length×width) in size. The polar plate is not visible on the aboral pole, but is raised above its periphery or lobed. A sense organ (0.3×0.4 mm in diameter) containing a statocyst (0.08×0.08 mm in diameter) can be observed in the center by optical microscopy. The statolith forms two masses, each of which has more than 20 granules located centrally in the cavity. All individuals collected since November 2006 were attached commensally to the branches and polyp masses of the encrusting alcyonaceans D. spinulosa, D. aff. dendritica and other dendronephthyas at 20-32 m depth off Munseom Island. Each polyp mass (size range=10-25 mm; mean=16.1

[Fig. 1.] Host dendronephthyas. A Dendronephthya spinulosa with numerous individuals of Coeloplana anthostella and one ind. of C. bocki; B D. aff. dendritica with few inds. of C. anthostella and numerous inds. of C. bocki; C D. aff. gracillima with C. anthostella; D D. aff. decussatospinosa with C. anthostella. Scale bars: A-D=10 cm.

mm; n=35) had 1-18 aggregated individuals (mean=7.1; n=250) (Table 2).

Korea (Isl. Jejudo).

Coeloplana bocki Komai, 1920: 576; 1922: 1, Pls. 1-7; 1942: 214; 1955: 13; 1975: 305, fig. 12; Tokioka, 1958: 178, Pl.89, fig. 4; 1968: 210; Nishimura and Noda, 1992: 173, Pl.42, figs. 6, 7; Mills, 1998: 9.

1 ind., Isl. Munseom, 16 Aug 2009, Kim BI, 26-32 m deep by SCUBA; many inds., Isl. Munseom, 20 Jun 2010, Hwang SJ, 20-32 m deep by SCUBA, attached on polyp mass of D. spinulosa and D. aff. dendrocata (Table 2).

Creeping flattened ctenophore represents somewhat hump-like elevation in its central parts of broad and thin basal expansion (Fig.2 B-D). Oval shape with undulating margin, 2.2×1.9-5.0×4.4 (n=7) mm (tentacular axis×sagittal axis, length×width) at individuals with orange stripes (Fig.2 B), and 3.7×2.6-11.0×5.7 (n=4) mm at ones with milky white stripes (Fig.2 C, D).

Two pinnate tentacles having colloblasts in their surface with one side branches (Figs. 2B, D, 3B) entirely retracted into each tentacle sheath at both ends of tentacular axis. Tip of tentacles globular (0.02×0.02-0.04×0.04 mm in dia.), their side and main branches slender, 0.02-0.05 mm and 0.06-0.09 mm in dia. respectively. Tentacle sheath flask-shaped in aboral view, two tentacle bases separated each other at aboral pole and placed in transverse plane (Fig.2 D). Under stereomicroscope, ramification of gastrovascular system not clearly visible (Fig.2 C, D), but visible under light microscope (Fig.3 A). However, milky white stripes branched and anastomosing not related to each mesh consisting of thin reticulated gastrovascular canals (Fig.3 A).

On aboral pole, polar plate visible, with two lobe-like processes at its peripheral margin, and also raised above its periphery (Fig.3 C). Any other dorsal papillae undeveloped on its dorsal surface. At its center of small individual with

[Fig. 2.] Coeloplana bocki. A Polyp mass of D. aff. dendritica attaching numerous inds. of C. bocki; B C. bocki with orange stripes; C A small and a large inds. of C. bocki with milky white stripes; D C. bocki showing two tentacle bases (arrows) and tentacles. Scale bars: A-D=1 mm.

orange stripes, sense organ (0.15×0.15 mm in dia.) with statocyst (0.04×0.04 mm in dia.) in its middle part clearly seen under light microscope (Fig.3 D). Statolith forming one mass of almost 40 granules in its central position of cavity. On oral side, one large mouth (0.6×0.7 mm in dia.) with many pharyngeal folds opened at center of ventral side.

In color, milky white, semi-transparent, with 7-10 blue and 4-6 orange colored branching and anastomosing stripes encircled aboral sense organ towards margin, and reddish orange colored radial stripes at both tentacle bases from one individual on August 16, 2009 (Fig.2 B, D). And also, milky white, semi-transparent, with 7-12 white branching and anastomosing stripes at small individuals and numerous stripes at large ones encircled aboral sense organ towards margin, and also orange colored radial stripes at both tentacle bases from lots of individuals on June 20, 2010 (Fig.2 B-D). However, all of blue and orange colored stripes disappeared in formalin, except for yellowish tentacle bases.

The single individual of this species together with many C. anthostella was found from D. spinulosa on August 16, 2009, and many individuals of the species together with C. anthostella were obtained from the host D. aff. dendricata on June 20, 2010 (Table 2). Of the number of symbionts, Coeloplana spp. was influenced by the another species attaching to the single host dendronephthya.

The GenBank accession numbers: HQ43-5813 (18S rDNA), HQ435814 (ITS1), and HQ435815 (cox1).

The nucleotide sequences of C. bocki differ from C. anthostella by 0.2%, 17.9%, and 11.7% for 18S, ITS1 (including inferred gaps), and cox1, respectively (Table 3). The high level of sequence divergence detected between the two Coeloplana species, particularly both from ITS1 and cox1 indicates that they represent well-established distinct species.

This species can be distinguished from C. antho-

[Table 3.] Nucleotide sequence differences between Coeloplana anthostella and C. bocki in three gene fragments

Nucleotide sequence differences between Coeloplana anthostella and C. bocki in three gene fragments

[Fig. 3.] Coeloplana bocki. A Opening of the tentacle sheath (arrow); B Tentacle expelled from the tentacle sheath; C Aboral sense organ showing polar plates (arrows); D Sense organ (arrow) with a statocyst in the central part of aboral side. Scale bars: A=200 μm B C=100 μm D=50 μm.

stella (see Song and Hwang, 2010) and C. komaii (see Utinomi, 1963) by its unique blue and orange stripes, and/or branching and anastomosing milky-white stripes encircling the aboral sense organ towards the margin (Fig.2 B, D). These specimens were smaller than those in the original description (Komai, 1920, 1922), and have lobe-like processes at the peripheral margin of the polar plate.

Korea (Isl. Jejudo), Japan (Sagami Bay, Tanabe, Misaki).