Oncholaimus tripapillatus sp. nov., a New Free-Living Marine Nematode of the Genus Oncholaimus Dujardin, 1845 (Nematoda: Enoplida: Oncholaimidae) from the Subtidal Sediment of Dokdo Island, East Sea, Korea, with a New Record of O. qingdaoensis Zhang & Platt, 1983

During a survey of the free-living marine nematodes of Korea, two species belonging to the genus Oncholaimus Dujardin, 1845 were discovered. A new species, Oncholaimus tripapillatus sp. nov. and a newly recorded species, Oncholaimus qingdaoensis Zhang & Platt, 1983, are reported. Oncholaimus tripapillatus sp. nov. was collected from the washing of subtidal coarse sediments around Dokdo Island of the East Sea, Korea. Oncholaimus tripapillatus sp. nov. is characterized by a relatively long (4071–4435 µm in males and 4514–4661 µm in females) and slender body, a slightly constricted head region, relatively long cephalic setae (10–12 µm), males having a precloacal sexual protuberance bearing two small cone-shaped supplementary spines, five pairs of long cloacal setae (three pairs of precloacal and two pairs of postcloacal setae in subventral position), and three remarkable papillae near the end of the tail, with two pairs of subventral setae. The Korean specimens of Oncholaimus qingdaoensis Zhang & Platt, 1983 are almost identical to the Chinese specimens of the original description from the intertidal sand of Qingdao, China. However, the Korean specimens differ from the Chinese specimens in the longer body length in males (3379–3715 µm vs. 2380–2640 µm), the larger spicule length (47–52 µm vs. 34–36 µm), and the presence of ventral tail papillae situated around 14–16 µm from the tail tip. Detailed morphological features and illustrations of two Oncholaimus species from Korea were obtained by differential interference contrast microscopy and scanning electron microscopy. A pictorial key to the species group with distinct tail papillae among the genus Oncholaimus is also provided.

During a continuous ecological investigation on the biodiversity of the free-living marine nematodes around Korean waters, we found two Oncholaimus species from the intertidal and subtidal sediments of the eastern, southern, and western coasts of Korea. The present paper deals with morphological descriptions, illustrations, and photomicrographs from differential interference contrast (DIC) microscopy and scanning electron microscopy. Moreover, we also provide a pictorial key and a comparison table of the diagnostic morphological characteristics of the species within the genus Oncholaimus with distinct tail papillae in the tail region.

Sampling of Taxa
The marine nematodes were obtained from the upper surface of the intertidal sediment from the southern and western coasts of Korea using a hand scoop, as well as by means of a Smith-McIntyre grab from the shallow subtidal benthic environment sediment of Dokdo Islands, located in the East Sea in the northwest Pacific Ocean.

Sample Processing and Preparation of the Specimens
The samples were filtered through a sieve with 67 µm mesh in the field after rinsing with freshwater for less than 1 min for osmotic shock and then fixed in 5% formalin [15]. In the laboratory, the nematodes were sorted from the mixed meiobenthos under a LEICA 205 C stereomicroscope (Leica, Wetzlar, Germany). The nematodes were transferred to glycerol and mounted between two cover slips on an HS slide for morphological observations [16]. The specimens were measured, examined, and drawn using Nomarski differential interference contrast (DIC) microscopy with an Olympus BX53 microscope equipped with a drawing tube and an Olympus DP26 digital camera with the corresponding Olympus CellSens imaging software (Olympus, Tokyo, Japan). The specimens selected for scanning electron microscopy (SEM) were fixed in 5% buffered formalin and then rinsed twice with distilled water to remove the buffered formalin. After rinsing for 5 min each, the specimens were freeze-dried in a cooling stage, mounted on an aluminum stub, sputter-coated with gold/palladium in a high-vacuum evaporator, and examined with an SEC SNE-3200M Desktop Mini SEM.

Terminology and Abbreviations
Measurements are in micrometers (µm). The abbreviations used in the text are as follows: a, body length divided by maximum body diameter; b, body length divided by pharynx length; c, body length divided by tail length; V (%), vulva distance from anterior end as percentage of total body length.

Type Locality and Habitat
The locality was the subtidal zone of Dokdo Islands (37 • 14 21.00" N, 131 • 52 4.00" E), Ulleung-gun, Gyeongsangbuk-do, Korea, where the specimens were collected on 22 May 2015 by H. S. Rho. The nematodes were extracted from the subtidal sediments with tiny shell gravels, and detritus was collected at a depth of 5 m.

Etymology
This species' name, tripapillatus, comes from the Latin, meaning crested, in reference to the three pairs of papillae on the tail tip.

Measurements
See Table 1 for measurements and morphometrics.

Diagnosis
The body is relatively long (4071-4435 µm in males and 4514-4661 µm in females) and slender (a = 82-88 in males and 64-72 in females), with a slightly constricted head, relatively long cephalic setae (10-12 µm), a large buccal cavity with a thick cuticular wall and three teeth (the left subventral tooth is larger than the right subventral and dorsal teeth), two pre-cloacal supplementary spines in males, five pairs of long cloacal setae (three precloacal and two postcloacal setae in the subventral position), a ventrally arcuated conoid tail with three tail papillae and two pairs of short setae.

Description
Males: Bodies are comparatively long (4071-4435 µm) and slender, slightly tapering toward both extremities ( Figure 1A). Maximum body diameter at the mid body level is 49-52 µm. The cuticle of the body is smooth. The diameter of the head is 30 µm wide, with a slight constriction posterior to the cephalic seta. The lip region has six conical lips, which are deeply separated, each bearing small rounded inner labial papillae. Six outer labial and four cephalic setae are arranged in a single circle, almost equal in size (10-12 µm) ( Figures 1C, 3A and 4A). The buccal cavity is large (29-31 µm long and 19-20 µm wide), with sclerotized walls and three teeth. The left subventral tooth (22-24 µm long) is larger than the right subventral and dorsal teeth, and the tip is stretched up to the cephalic setae. The amphideal fovea (8-11 µm wide) is pocket-like, with an elliptical opening that is located 20-25 µm from the anterior end ( Figures 1C and 4B). The pharynx (545-563 µm long) is cylindrical and is approximately 12-13% of total body length, with a corresponding body diameter of 42-43 µm. The excretory pore is situated 80-88 µm from the anterior end. The nerve ring is approximately 5.7-6.1% of the total body length from the anterior end. The somatic setae are randomly distributed across the surface of the body ( Figure 1B). Two opposed testes are situated to the right side of the intestine. The spicules are short and equal, slightly ventrally curved, with a pointed distal end and a cephalated proximal end. The spicules (41-44 µm long) are approximately 1.1-1.2 times of the anal body diameter ( Figures 1D and 4E). Gubernacula are absent. Five pairs of long and stout subventral setae (7-8 µm long) are located around the cloacal region, of which three pairs are situated on each side of the cloacal opening, while the other two pairs are located on both sides behind the cloacal region ( Figure 1D,E and Figure 3C). A precloacal sexual protuberance is present just anterior to the cloacal opening, bearing two small cone-shaped supplementary spines situated on the cloacal protuberance ( Figure 4C). The tail is 92-96 µm in length, conoid, slightly curved ventrally, and approximately 2.5-2.7 times the anal body diameter ( Figure 1D,E and Figure 3C). Three remarkable papillae are subventrally situated on the tail tip, of which only the anteriormost papilla consists of a pair. There are two pairs of subventral setae around the tail papillae ( Figures 3D and 4D).
Females: Similar to the males in general appearance, but they differ in tail shape and sexual characteristics (Figure 2A,C and Figure 3B). The conical tail (110-114 µm long) is slightly arcuate with a blunt posterior end but without a tail papilla. The tail is 3.0-3.2 times the anal body diameter, with two pairs of short terminal setae ( Figure 2D). The reproductive system is monodelphic with anterior reproductive branches, lying to the right side of the intestines. They have two intra-uterine eggs, 124-169 µm in length and 56-67 µm in width. The vulva is situated at 66%-67% of the total body length from the anterior end ( Figure 2B). The demanian system is composed of one osmosium connecting the ductus entericus and the intestine, and one uvette, located at 170 µm in front of anus, connecting the ductus uterinus with a main tube ( Figure 2E).

Differential Diagnosis and Relationships
The genus Oncholaimus Dujardin, 1845 is the largest genus of the family Oncholaimidae Filipjev, 1916, containing about 120 species, which have thus far been described from various localities of littoral and deep-sea habitats in oceans all over the world [2,4]. They are distinguished from the other genera of the family by the presence of the largest left ventrosublateral tooth and relatively short spicules, with a monodelphic-prodelphic reproductive system in females [1]. After comparing and compiling the valid species of the genus Oncholaimus on the basis of the taxonomic key characteristics among the congeners, we artificially distinguished a group of 26 species with a conspicuous ventral papilla on the tail, which is the most important diagnostic characteristic of the genus, including the present new species, Oncholaimus tripapillatus sp. nov., and Therefore, we herein provide a pictorial identification key and a comparison table of the diagnostic morphological characteristics for the species group with a conspicuous ventral papilla on the tail, with diagnostic characteristics also including the morphology of the anterior head region and the posterior tail region showing spicule and gubernaculum, which are generally important taxonomic key characters to discriminate closely related nematode species ( Figure 5, Table 2).           Body length  3379  3505  3715  4061  3550  a  82  86  85  75  67  b  7  7  7  7  7  c  46  45  44  56  43  Head diameter at the level of cephalic  setae  28  29  29  32  31   Body diameter at the level of the cardia  36  40  38  44  47  Maximum body diameter  41  41  44  54  53  Cephalic setae length  10  12  11  11  11  Amphideal fovea diameter  10  12  11  10  12  Amphideal fovea length  5  6  7  5  7  Buccal cavity diameter  17  19  18  17  21  Buccal cavity length  32  31  33  35  34  Anterior end to the largest tooth tip  5  5  7  6  8  Anterior end to the other teeth tips  13  12  13  12  14  Anterior end to amphids  22  20  23  22  22  Anterior end to excretory pores  84  79  77  68  81  Anterior end to the nerve ring  229  241  243  265  247  Pharynx length  492  533  555  560  514  Spicule length  47  50  52  --Cloacal setae length  8  8  8  --Anterior end to the vulva  ---2812 2990 Body diameter at level of the vulva ---54 56

Material Examined
The examined material included three males (MABIK NA00156663, KIOST NEM-1-2565, and KIOST NEM-1-2604) and two females (KIOST NEM-1-1719 and KIOST NEM-1-2605), mounted in anhydrous glycerin between two coverslips on an HS slide, sealed with nail polish. One male specimen (MABIK NA00156663) was deposited in the nematode collection at the specimen conservation room of the Marine Biodiversity Institute of Korea (MABIK), and the other specimens were deposited in the Bio-Resources Bank of Marine Nematodes (BRBMN), Korea Institute of Ocean Science and Technology.

Measurements
See Table 3 for measurements and morphometrics.

Description
Males: Bodies are comparatively long (3379-3715 µm) and slender, typically cylindrical in appearance ( Figure 6A). The maximum body diameter at the mid body level is 41-44 µm. The head is constricted immediately posterior to the cephalic setae ( Figures 6C and 8A). The head region (28-29 µm in width) has typically six rounded lips, slightly attenuated toward the end, with a minute rounded inner labial papilla. Six outer labial and four cephalic setae are arranged in one circle, almost equal in size (10-12 µm), inserted at the level of the amphideal fovea. The buccal cavity (31-33 µm deep, 17-19 µm wide) has a thick cuticular wall and three well-developed teeth. The left subventral tooth is the largest (26-28 µm), with its tip stretched up to the cephalic setae, while the right subventral and dorsal teeth are almost equal in size (19-21 µm). The amphideal fovea is pocket-like, 10-12 µm in width (corresponding head diameter of 37%-41%), and 5-7 µm in height, located 20-23 µm from the anterior end ( Figures 6C and 8B). The pharynx (492-555 µm long) is cylindrical, approximately 15% of the total body length, with a corresponding body diameter of 36-40 µm. The excretory pore is situated 77-84 µm from the anterior end. The nerve ring is situated 229-243 µm from the anterior end, with a corresponding body diameter of 35-38 µm, and 44%-47% of the length of the pharynx ( Figure 6B). The somatic setae are sparsely distributed on the surface across the body. Two opposed testes are located on the right side of the intestine. The spicules are short and straight (47-52 µm long), proximally cephalated and pointed distally ( Figure 6D,E and Figure 8E). The gubernacula are absent. Four pairs of long and stout setae are located in the cloacal region in a row ( Figures 6D,E and 8C). Two unapparent pairs of precloacal spines are situated at the cloacal opening ( Figures 6E and 8D). The tail is 74-85 µm long, approximately 2.5-2.7 times the anal body diameter. A conspicuous ventral tail papilla is situated approximately 14-16 µm from the tail tip ( Figure 6D,E and Figure 8E). Two pairs of ventral setae (3-4 µm) are situated in the tail papilla.
Females: Very similar to the males but differ in several dimensions, tail shape, and sexual characteristics. The body is 3550-4061 µm long, with a maximum body diameter of 53-54 µm ( Figure 7A,C). The conical tail (72-83 µm long) is slightly curved ventrally, without a ventral papilla ( Figure 7D), and approximately 1.9-2.1 times the anal body diameter. The reproductive system is monodelphic with anterior reproductive branches, lying to the right side of the intestines. The vulva is situated at 69-84% of the total body length from the anterior end ( Figure 7B). The demanian system is composed of one osmosium connecting the ductus entericus and the intestine, and one uvette, located at 870 µm in front of the anus, connecting the ductus uterinus with a main tube ( Figure 7B).

Differential Diagnosis and Relationships
Oncholaimus qingdaoensis Zhang & Platt, 1983 was first described from intertidal sand in Qingdao, China. Oncholaimus qingdaoensis has been previously reported only in that type of locality. The present Korean specimens of Oncholaimus qingdaoensis were discovered from the intertidal sandy sediments of the southern and western coasts of Korea. Oncholaimus qingdaoensis is easily distinguished from its congeners as having the following characteristic combinations: (1) the presence of a constricted head, (2) relatively long cephalic setae,