INTRODUCTION

Since Cobb (1920) first described the type species of the genus Belbolla as B. tenuidensCobb, 1920 from the intertidal sand habitat of the Atlantic Ocean, 16 nominal species are currently recognized in the genus BelbollaAndrássy, 1973 (Andrássy 1973;Rho et al. 2020). The genus shows worldwide distribution and occurs in various habitats, from littoral sediment to brackish water and subtidal sediment environments (Huang and Zhang 2005;Nasira et al. 2014;Gagarin and Thanh 2016;Gagarin and Tu 2016;Rho et al. 2020). Of these, ten species of the genus Belbolla have been reported around the Pacific Ocean as follows: B. sundaensis (Micoletzky 1930) from the Sunda Island, Indonesia; B. californica (Allgén 1951) from the California, San Diego, USA; B. intarmaBelogurov and Belogurova, 1980 from the subtidal sediment of the Tatar Strait, Russia; B. zhangiGuo and Warwick, 2001 from the sublittoral sediment of the Bohai Sea, China; B. huanghaiensisHuang and Zhang, 2005, B. stenocephalumHuang and Zhang, 2005 and B. warwickiHuang and Zhang, 2005 from the sublittoral sediment of the Yellow Sea, China; B. gracilis Gagarin and Thanh, 2016 and B. vietnamicaGagarin and Tu, 2016 from the Yen River estuary of the Vietnam; most recently, B. wonkimi Rho, Lee, Lee and Min, 2020 from subtidal sediment of the West Sea, Korea (Micoletzky 1930;Allgén 1951, Belogurov and Belogurova 1980;Guo and Warwick 2001;Huang and Zhang 2005;Gagarin and Thanh 2016;Gagarin and Tu 2016;Rho et al. 2020) (Table 3).

During a continuous faunistic survey on the biodiversity of the free-living marine nematodes around the Korea, two new Belbolla nematodes were recovered from the washings of a shallow subtidal sediment from the western and southern outermost islands of Korea.

This paper proposes and describes two new Belbolla species, B. koreensis sp. nov. and B. hoonsooi sp. nov., using illustrations and differential interference contrast (DIC) photomicrographs, and we discuss its taxonomic relationships with other nominal species of the genus Belbolla. It is the second taxonomic report on the species of the genus Belbolla from the Korean seawaters.

MATERIALS AND METHODS

The marine nematode specimens were gathered from the washings of shallow subtidal sediment, which were collected at a depth of 12 m using scientific Scuba diving technique on the western and southern outermost islands of Korea (Fig. 1). At first, meiobenthos were roughly separated from the sediment by decantation methods using a 67 μm mesh sieve in the survey field after freshwater rinsing for less than a few minutes to reduce the attachment from the sediment (Kristensen 1989), and then fixed in 4% neutralized formalin in sea water. After the initial sampling, meiobenthos were extracted once again from the rough samples using the Ludox flotation method (Burgess 2001) in the laboratory, and the concentrated samples were subsequently fixed in 4% neutralized formalin solution. The marine nematode specimens were picked up under a high magnification of dissecting microscope (LEICA M205 C, Wetzlar, Germany). Specimens for morphological observation under a light microscope were dehydrated through a graded series of glycerin according to Seinhorst (1959) and mounted on a HS slide (Shirayama et al. 1993). The mounted nematodes were examined and photographed using an Olympus BX53 microscope equipped with an Olympus DP26 digital camera (Olympus, Tokyo, Japan). All drawings and measurements were made with the aid of a camera lucida. The terminology used for the description and measurements was followed by Huang and Zhang (2005).

SYSTEMATIC ACCOUNTS

Phylum Nematoda Potts, 1932

Class Enoplea Inglis, 1983

Order Enoplida Filipjev, 1929

Family Enchelidiidae Filipjev, 1918

Genus BelbollaAndrássy, 1973

Type species: Belbolla tenuidens (Cobb, 1920) Andrássy, 1973.

Sixteen valid species of the genus BelbollaAndrássy, 1973

Belbolla koreensis sp. nov. (Figs. 2, 3; Table 1)

Type material. Holotype male (MABIK NA00156662), in glycerin on a HS slide, from Gyeongnyeolbi-yeoldo Is., Taean-gun, Chungcheongnam-do, 18 August 2021 (leg. HS Rho and H Lee) was deposited in the nematode collection at the specimen conservation room of the Marine Biodiversity Institute of Korea (MABIK), Seochun, Korea. One paratype male (KIOST NEM-1-2608), in glycerin on a HS slide, from Gageodo Is., Sinan-gun, Jeollanam-do, 23 June 2020 (leg. HS Rho and H Lee), was deposited in the nematode collection at the specimen conservation room of the Bio-Resources Bank of Marine Nematodes (BRBNM), East Sea Research Institute, Korea Institute of Ocean Science & Technology (KIOST), Korea.

Etymology. The proposed specific name koreensis is named after the type locality of the new species.

Type locality. Shallow subtidal zone of Gyeongnyeolbiyeoldo Is. (36°36ʹ50.04ʺN, 125°34ʹ38.47ʺE), Taean-gun, Chungcheongnam-do, Republic of Korea.

Habitat. Subtidal sediment composed of tiny oyster shell gravels and detritus collected at a depth of 12 m.

Measurements. See Table 1 for measurements and morphometrics.

Diagnosis. Body very long (3,080-3,426 μm long); ocelli absent; eight oesophageal bulbs in the posterior region of pharynx; well-developed two winged precloacal supplements (47-59 μm vs. 43-46 μm long, anterior precloacal supplement slightly longer than posterior one); strongly arcuated, very thick, L-shaped spicule (122-127 μm long, 1.7-1.9 a.b.d. long) with three separated blunt distal tip; well-developed relatively long cylindrical shaped gubernacular apophysis with blunt teeth covering two thirds of the dorsal margin (57-58 μm long, 0.8-0.9 a.b.d. long).

Description. Males (Holotype and paratype). Body slender, 3,080-3,426 μm long; anterior body region outstretched anteriorly and strongly tapered anteriorly from mid oesophagus to cephalic region; posterior body region slightly curved ventrally in fixed specimen (Figs. 2A, C, 3A, C, D). Head diameter 13-15 μm wide, distinctly narrowed. Body at level of end of posterior pharyngeal region 6.0-6.5 times as wide as head region width. Body cuticle, smooth, no lateral differentiation. Anterior parts of head around cephalic setae, separated with slight constriction (Figs. 2B, 3B). Lip region slightly extruded in fixed specimen. Six short, thin labial setae on lip region arranged in one circle. Inner labial setae not observed. Ten cephalic setae (10-11 μm long) inserted at level of amphideal fovea and arranged in one circle. Maximum body width 91-114 μm wide. Buccal cavity wide (7 μm) with thick wall 2.0- 2.9 times as long (14-16 μm) as wide. Buccal cavity with one large right dorsolateral tooth and two less prominent teeth, which are composed of dorsal and left ventrolateral in position (Figs. 2B, 3B). Buccal cavity large, wide, separated into two buccal chambers by a cuticular ring, which is smooth with no denticles. Amphideal fovea located at level of stoma. Ocelli absent. Several cervical setae, 18-23 μm in length. Oesophagus muscular and expands gradually and evenly, 626-762 μm long and posteriorly modified into eight oesophageal bulbs (length of oesophageal bulb 284-338 μm) (Figs. 2A, 3C). Oesophagus corresponding diameter 78-98 μm wide. Nerve ring 291-328 μm from anterior end with 59-67 μm corresponding body diameter (c.d.) and at 43-46.5% of oesophagus length. Cardia short, conoid. Male diorchic, testis opposed and outstretched, located left or right of intestine. Spicules 122-127 μm long (1.7-1.9 times as long as a.b.d.), strongly arcuated, very thick, L-shaped with three separated blunt distal tip; proximal end strongly cephalated. Gubernaculum 57-58 μm long (0.8-0.9 a.b.d. long), well-developed relatively long cylindrical shaped apophysis with blunt teeth covering two thirds of dorsal margin (Figs. 2C, F, 3D, F). Two winged precloacal supplements strongly developed (Figs. 2D, E, 3D, E); anterior precloacal supplement 351-412 μm lies from cloacal opening and posterior precloacal supplement lies 223-298 μm from cloacal opening (Figs. 2C, 3D); anterior precloacal supplement slightly longer than posterior one (47-59 μm vs. 43-46 μm long). Tail long and slender, conico-cylindrical, 226-260 μm long, 3.2-3.9 times a.b.d. long, tapering with distal third cylindrical and with slightly swollen tip (Figs. 2C, 3D).

Female. Unknown.

Differential diagnosis and relationships, see the Discussion.

Belbolla hoonsooi sp. nov. (Figs. 4, 5; Table 1)

Type material. Holotype male (MABIK NA00156661), in glycerin on a HS slide, from Gageodo Is., Sinan-gun, Jeollanam- do, 23 June 2020 (leg. HS Rho and H Lee), was deposited in the nematode collection at the specimen conservation room of the Marine Biodiversity Institute of Korea (MABIK), Seochun, Korea.

Etymology. The scientific name is taken in honor of Professor Hoon Soo Kim, the authoritative scientist on the decapod taxonomic research of Korea.

Type locality. Shallow subtidal zone of Gageodo Is. (34° 03ʹ55.86ʺN, 125°06ʹ32.28ʺE), Sinan-gun, Jeollanam-do, Republic of Korea.

Habitat. Subtidal sediment with oyster shell gravels and detritus collected at a depth of 12 m.

Measurements. See Table 1 for measurements and morphometrics.

Diagnosis. Body relatively long (3,494 μm long); ocelli absent; eight oesophageal bulbs in the posterior region of pharynx; well-developed two winged precloacal supplements (43 μm vs. 36 μm long, anterior precloacal supplement slightly longer than posterior one); strongly arcuated, L-shaped spicule (119 μm long, 1.5 a.b.d. long) with three separated blunt distal tip; well-developed relatively long inverted triangle shaped gubernacular apophysis with blunt teeth covering half of the dorsal margin (45 μm long, 0.6 a.b.d. long).

Description. Male (Holotype). Body slender, 3,494 μm long; anterior body region outstretched anteriorly and strongly tapered anteriorly from mid oesophagus to cephalic region; posterior body region strongly curved ventrally in fixed specimen (Fig. 5A). Head diameter 15 μm wide, distinctly narrowed. Body at level of end of posterior pharyngeal region 6.9 times as wide as head region width. Body cuticle, smooth, no lateral differentiation. Anterior parts of head around cephalic setae, separated with slight constriction (Figs. 4B, 5B). Lip region slightly extruded in fixed specimen. Six short, thin labial setae on lip region arranged in one circle. Inner labial setae not observed. Ten cephalic setae (9 μm long) inserted at level of amphideal fovea and arranged in one circle. Maximum body width 118 μm wide. Buccal cavity wide (6 μm) with thick wall 2.5 times as long (15 μm) as wide. Buccal cavity with one large right dorsolateral tooth and two less prominent teeth, which are composed of dorsal and left ventrolateral in position (Figs. 4B, 5B). Buccal cavity large, wide, separated into two buccal chambers by a cuticular ring, which is smooth with no denticles. Amphideal fovea located at level of stoma. Ocelli absent. Several cervical setae, 22 μm in length. Oesopha- gus muscular and expands gradually and evenly, 741 μm long and posteriorly modified into eight oesophageal bulbs (length of oesophageal bulb 327 μm) (Figs. 4A, 5C). Oesophagus corresponding diameter 103 μm wide. Nerve ring 324 μm from anterior end with 66 μm c.d. and at 43.7% of oesophagus length. Cardia short, conoid. Male diorchic, testis opposed and outstretched, located left or right of intestine. Spicules 119 μm long (1.5 times as long as a.b.d.), strongly arcuated, slightly thick, L-shaped with three separated blunt distal tip; proximal end opened. Gubernaculum 45 μm long (0.6 a.b.d. long), well-developed relatively long inverted triangle shaped apophysis with blunt teeth covering half of dorsal margin (Figs. 4C, F, 5F). Two winged precloacal supplements strongly developed (Figs. 4C, D, E, 5D, E); anterior precloacal supplement 343 μm lies from cloacal opening and posterior precloacal supplement lies 211 μm from cloacal opening (Figs. 4C, 5D); anterior precloacal supplement slightly longer than posterior one (43 μm vs. 36 μm long). Tail long and slender, conico-cylindrical, 276 μm long, 3.5 times a.b.d. long, tapering with distal third cylindrical and with slightly swollen tip (Figs. 4C, 5D).

Female. Unknown.

Differential diagnosis and relationships, see the Discussion.

DISCUSSION

The genus BelbollaAndrássy, 1973 is classified within the family Enchelidiidae Filipjev, 1928 belongs to the order Enoplida Filipjev, 1929. The genus Belbolla has a unique diagnostic morphological character, which is a series of bulb-like muscular oesophageal swellings (4 to 10) in the posterior section of pharynx, compare to the other genera within the family Enchelidiidae Filipjev, 1928. Moreover, the genus Belbolla is also characterized by the diagnostic features as follows: the presence or absence of ocelli, having a large and narrow buccal cavity divided into two parts by a sclerotized transverse band, and having the three teeth within the buccal cavity, of which one ventrosubdorsal is the largest (Smol et al. 2014).

Species level differentiation within the genus Belbolla depends on the following taxonomic characters used in the keys of Belogurov and Belogurova (1980) and Huang and Zhang (2005): (1) the presence/absence of precloacal supplements; (2) the length and shape of precloacal supplements; (3) the number of oesophageal bulbs in the posterior section of pharynx, which is the most remarkable key character within the genus Belbolla; (4) shape and length of the spicules; and (5) shape and length of the gubernacular apophysis (Belogurov and Belogurova 1980;Huang and Zhang 2005;Rho et al. 2020).

Currently, 16 valid species within the genus Belbolla have been previously described from various habitats in oceans all over the world based on the morphological characters shown in Table 2 (Micoletzky 1930;Allgén 1951;Belogurov and Belogurova 1980;Guo and Warwick 2001;Huang and Zhang 2005;Gagarin and Thanh 2016;Gagarin and Tu 2016;Rho et al. 2020). Of these, ten species of the genus Belbolla have been reported around the Pacific Ocean as mentioned in the introduction section of this paper (Table 3). However, only one Belbolla species, B. wonkimi Rho, Lee, Lee and Min, 2020, have so far been reported from a subtidal sediment habitat of the East Sea, Korea.

During a survey of the free-living marine nematofauna of the outermost islands of Korea, two Belbolla species were collected from washings of a shallow subtidal sediment environmental habitat. We identified both species are new to science based on the morphological study, and describe herein as B. koreensis sp. nov. and B. hoonsooi sp. nov.

Belbolla koreensis sp. nov. is characterized by the following combination of characteristics: (1) the longer body length (3,080-3,462 μm long); (2) the absence of ocelli; (3) eight oesophageal bulbs in the posterior region of pharynx; (4) well-developed two winged precloacal supplements (anterior precloacal supplement slightly longer than posterior one: 47-59 μm vs. 43-46 μm long); (5) strongly arcuated, very thick, L-shaped spicule (122-127 μm long, 1.7- 1.9 a.b.d. long) with three separated blunt distal tip; and (6) well-developed relatively long cylindrical shaped gubernacular apophysis with blunt teeth covering two thirds of the dorsal margin (57-58 μm long, 0.8-0.9 a.b.d. long).

Within the genus Belbolla having eight oesophageal bulbs in the posterior region of pharynx, which is the main taxonomic key character of the genus, B. koreensis sp. nov. is most closely related to B. hoonsooi sp. nov. mainly by possessing the longer body and spicules length, the number of the precloacal supplements, and the overall shape of spicules and gubernacular apophysis (Table 2). However, B. koreensis sp. nov. differs from B. hoonsooi sp. nov. by (1) the detailed shape of gubernacular apophysis (a long cylindrical shaped with blunt teeth covering two thirds of the dorsal margin of gubernacular apophysis vs. a long inverted triangle shaped with blunt teeth covering half of the dorsal margin); (2) the relative length of gubernacular apophysis to total body length (1.7-1.9% vs. 1.3%); (3) the relative length of spicules to total body length (3.6-4.1% vs. 3.4%); (4) the longer anterior precloacal supplement (47-59 μm vs. 43 μm) and posterior precloacal supplement (43-46 μm vs. 36 μm); (5) the relative length of the anterior precloacal supplement to total body length (1.5-1.7% vs. 1.2%); (6) the relative length of the posterior precloacal supplement to total body length (1.3-1.4% vs. 1.0%); (7) distance of the posterior precloacal supplement from cloacal opening (223-298 μm vs. 211 μm); (8) distance between two precloacal supplements (114-128 μm vs. 132 μm); and (9) the shorter tail length (226-260 μm vs. 276 μm).

Belbolla koreensis sp. nov. is also morphologically close to B. stenocephalumHuang and Zhang, 2005 in having the eight oesophageal bulbs, but discernible from it by longer body (3,080-3,462 μm long vs. 2,226-2,394 μm long) and different shape of spicules (122-127 μm long, strongly arcuated, very thick, L-shaped with three separated blunt distal tip vs. 80-100 μm long, curved with a swollen distal tip).

Belbolla koreensis sp. nov. is similar to B. zhangiGuo and Warwick, 2001, described from the sublittoral sediment samples in the Bohai sea, in having a long gubernacular apophysis and well-developed two winged precloacal supplements, but it can be easily distinguished from B. zhangi by the number of oesophageal blub (8 bulbs vs. 8 or 9 ones, mostly 9 bulbs), the shape of distal tip of the spicules (three separated blunt distal tip vs. tapered distal tip), and the length of gubernacular apophysis (57-58 μm long, shorter than half the length of spicules vs. 40-45 μm long, longer than half the length of spicules).

Belbolla hoonsooi sp. nov. is characterized by the following combination of characteristics: (1) the longer body length (3,494 μm long); (2) the absence of ocelli; (3) eight oesophageal bulbs in the posterior region of pharynx; (4) well-developed two winged precloacal supplements (anterior precloacal supplement slightly longer than posterior one: 43 μm vs. 36 μm long); (5) strongly arcuated, L-shaped spicule (119 μm long, 1.5 a.b.d. long) with three separated blunt distal tip; and (6) well-developed relatively long inverted triangle shaped gubernacular apophysis with blunt teeth covering half of the dorsal margin (45 μm long, 0.6 a.b.d. long).

Belbolla hoonsooi sp. nov. shares the character combination of a similar long body, the number and shape of the precloacal supplements, and the overall shape of spicules and gubernacular apophysis with B. koreensis sp. nov. and B. zhangi among the congeners. However, B. hoonsooi sp. nov. is easily distinguished from B. koreensis sp. nov. in the morphological taxonomic key characters of the genus as already indicated at the above part. Belbolla hoonsooi sp. nov. can be also easily distinguished from B. zhangiGuo and Warwick, 2001 by the number of oesophageal blub (8 bulbs vs. 8 or 9 ones, mostly 9 bulbs), the shape of distal tip of the spicules (three separated blunt distal tip vs. tapered distal tip), the length of gubernacular apophysis (45 μm long, shorter than half the length of spicules vs. 40-45 μm long, longer than half the length of spicules), and the longer tail length (276 μm vs. 170-210 μm).

The present two new Belbolla species, B. koreensis sp. nov. and B. hoonsooi sp. nov., collected from a shallow subtidal sediment habitat from the western and southern outermost islands of Korea, share a very similar morphological trait that has never been revealed among the members of the genus Belbolla, that is the presence of three separated blunt distal tip of the spicules, and well-developed relatively long gubernacular apophysis with blunt teeth covering a part of the dorsal margin of the gubernacular apophysis.