Two species of Thoracostomopsidae (Nematoda: Enoplida) from Jeju Island, South Korea

During a survey of intertidal zones at beaches on Jeju Island, two species belonging to the family Thoracostomopsidae were discovered. One new species, Enoploides koreanus sp. nov. and one known species, Epacanthion hirsutum Shi & Xu, 2016 are reported. Along with morphological analysis, mitochondrial cytochrome oxidase c subunit 1 (mtCOI) sequences and 18S rRNA sequences of the species were also obtained and used to check relative p-distance and phylogenetic positions. While most species of Enoploides have long spicules, the new species belongs to a group of Enoploides with short spicules < 150 µm). Of the seven species with short spicules, the new species is most closely related to E. disparilis Sergeeva, 1974. They both have similar body length, fairly similar sized and shaped spicules with small gubernaculum running parallel to distal end of spicule, and an index value of b. The new species can be distinguished from E. disparilis by having pre-anal supplementary organ with short conical tail, while E. disparilis lacks pre-anal supplementary organ and has a long conico-cylindrical tail. Along with the description of the new species, the genus Enoploides Ssaweljev, 1912 is bibliographically reviewed and revised. Of 45 species described to date, 27 are now considered valid, 16 species inquirendae due to inadequate descriptions and ambiguity of the material examined, along with two cases of nomen nudum. With this review, we provide an updated diagnosis and list of valid species, a tabular key comparing diagnostic characters of all valid species, and a new complete key to species. One known species, Epacanthion hirsutum Shi & Xu, 2016, is reported in Korea for the first time. The morphology agrees well with the original description provided by Shi & Xu, 2016. As they had already reviewed the genus at the time of reporting four Epacanthion species, we provide only a description, depiction, and measurements for comparison purposes.


INTRODUCTION
The family Thoracostomopsidae Filipjev, 1927 consists of three subfamilies: Thoracostomopsinae Filipjev, 1927 (two genera), Trileptiinae Gerlach &Riemann, 1974 (one genus), andEnoplolaiminae De Coninck, 1965 (19 genera). They are distinguished by the presence or absence of mandibles (Enoplolaiminae or Trileptiinae respectively) or by the presence of a long and eversible spear (Thoracostomopsinae). Enoploides Ssaweljev, 1912, belonging to Enoplolaiminae, was first erected with type species Enoploides typicus Ssaweljev, 1912 from Russia. The genus is characterized by its high lips with striation; Y-shaped mandibles consisting of two lateral bars converging into one solid bar with a claw-like distal end, curving inwards to the lumen; onchia that are usually shorter than the mandibles; and spicules that are typically long armed with gubernaculum. Multiple revisions and updates of the genus have taken place. The most notable revision was made by , who questioned validity of many existing species. They argued that classification in this genus is only possible according to male genital armature (gubernaculum), and that all description based only on females and juveniles be considered species inquirenda. Aside from this, species have continuously been transferred from and to closely related genera such as Enoplus Dujardin, 1845;Enoplolaimus De Man, 1893and Epacanthion Wieser, 1953. Most recently, Smol, Muthumbi & Sharma (2014 listed 28 valid species including three transferred from Epacanthion by Greenslade & Nicholas (1991). By the number, it is clear that Smol, Muthumbi & Sharma (2014) considered and applied the list of species inquirendae provided by , but a full list of species was not supplied. A species list provided by NeMys (Bezerra et al., 2019) still lists some of the species considered invalid by  as valid species; contains erroneous species such as ' 'Enoploides uniformis (Pavljuk, 1984)" (discussed further in results); and is missing two of three species transferred from Epacanthion by Greenslade & Nicholas (1991). Our most recent list of valid species consists of 27 species, including the new species being reported. The last report on Enoploides species dates back to 1993, with a new report on Enoploides stewarti (Nicholas, 1993) from a freshwater lake in South Australia. Most species of the genus are from marine habitats with the exception of two freshwater species (Enoploides fluviatilis Mikoletzky, 1923 and E. stewarti). Of the 27 valid species, 63% (17) were initially reported from Europe; 14.8% (4) from North America; 7.4% (2) from Asia (including the new species); with 3.7% (1) each from South America, Africa, Australia and the Arctic.
The aim of this study was to review and revise the genus Enoploides while reporting a new species, Enoploides koreanus sp. nov., found from Jeju Island, Korea. Epacanthion hirsutum originally reported from East China Sea, is also reported in Korea for the first time. Their respective 18S rRNA and COI genes were sequenced and used to check p-distances and phylogenetic positions. We also agree that all future description of the genus be from a sound male as first proposed by .

Sampling and morphological study
Three seemingly natural and undisturbed beaches of Jeju Island were sampled in September 11, 2018. Two sub-samples of sediment from the intertidal zone were obtained qualitatively using a mini-shovel. Of the subsamples, one was fixed in 5% neutralized formalin solution for morphological analysis and the other was fixed in 70% ethanol for molecular analysis. Samples were brought back to the laboratory and meiofauna were extracted using the Ludox method (Burgess, 2001). Individual specimens were transferred by hand to a Petri dish filled with 10% glycerin. Specimen-containing Petri dishes were placed for a day in a dry oven preset to 40 • C for a day to achieve complete dehydration as described by Seinhorst (1959) with the glycerin-ethanol method. A single specimen was mounted in a drop of glycerin on a slide glass as conferred in the wax-ring method (Hooper, 1986). Specimens were examined and identified using Olympus BX51 and Leica DM2500 microscopes. For scanning electron microscopy, specimens were removed from the slide glass and placed in a drop of glycerin. Drops of distilled water were added gradually to the drop of glycerin to rehydrate the specimen. Hydrated specimens underwent ethanol series for dehydration (20%, 40%, 50%, 70%, 80%, 90%, 95%, 100%, for 10 min each) to be placed in hexamethyldisilazane (HMDS), with slightly altered concentration and duration compared to a method used by Phillips et al. (2016). A pool of HMDS containing the specimen was placed in drying oven to be completely dried overnight. Dried specimens were mounted on a stub to be sputter-coated, then observed with a COXEM EM-30 scanning electron microscope.

DNA extraction and amplification
Each specimen of interest was dissected into head, body, and tail. Heads and tails were retained for morphological analysis and made into permanent slides following Hooper's (1986) wax-ring method. The slides were submitted to the National Institute of Biological Resources (NIBR, Korea). Bodies of each specimen were transferred to a well of distilled water for 20 min to be washed of any remaining ethanol. Washed bodies were moved to individual tubes containing 25 µl of worm lysis buffer, prepared prior to extraction following Williams et al. (1992). The tubes were then placed in PCR-thermo cycler (Takara, Japan) preset to 65 • C for 15 min, 95 • C for 20 min, and 15 • C for 2 min. Two gene loci commonly used for marine nematodes were sequenced: mitochondrial cytochrome oxidase C subunit I (COI) gene and 18S small subunit ribosomal rRNA. All genes were amplified using PCR premix (Bioneer Co., Daejeon, Korea) with 5 µl DNA template, 15 µl distilled water, 1 µl of each primer. COI genes were amplified using primer sets (JB3/JB5) amplifying approximately 300 base pairs (bp) as described by Derycke et al. (2010). PCR cycling conditions were: 94 • C for 5 min, 35 cycles of (94 • C for 30 s; 50 • C for 30 s; 72 • C for 30 s), and 72 • C for 10 min. 18S rRNA was amplified using primer sets (MN18F/22R), amplifying approximately 300 bp. PCR cycling conditions were: 95 • C for 5 min, 37 cycles of (95 • C for 30 s, 56 • C for 1 min, 72 • C for 1 min 30 s), followed by 72 • C for 5 min, as described by Bhadury et al. (2006). Success of amplification was determined by electrophoresis on 1% agarose gel. If amplification was successful, DNA templates were sent to Macrogene (Korea), to be sequenced on an ABI3730XL sequencer.

Molecular data analysis
Sequenced forward and reverse strands were visually checked for signal quality using FinchTV (ver. 1.4.0). Two strands were aligned with ClustalW (Thompson, Higgins & Gibson, 1994) implemented into MEGA (ver. 7.0.26) (Kumar, Stecher & Tamura, 2016) with default parameters. All aligned sequences were confirmed with BLAST search (Altschul et al., 1990) on GenBank to check that the sequences were those of nematodes. Pairwise distances between mtCOI and 18S rRNA sequences were calculated using the K2P model (Kimura, 1980) using MEGA 7. The best fit-model for 18S rRNA datasets were assessed using default parameters implemented in MEGA 7.0 (Kumar, Stecher & Tamura, 2016). Tamura 3-parameter model (Tamura, 1992) with gamma distribution of rates across sites was found to be optimal and used with MEGA 7.0 to build a maximum likelihood (ML) tree with complete deletion and 1,000 bootstrap repetition. Completed tree was exported to FigTree (ver. 1.4.4) (Rambaut, 2009) and visually modified. Phylogenetic tree was not constructed using the obtained mtCOI sequences, as only few mtCOI sequences of Enoploides were available on GenBank.

Bibliographical revision of the genus
The Bremerhaven Checklist of Aquatic Nematodes by Gerlach & Riemann (1974) was initially used to collect original descriptions and references. Original erection of the genus, as well as other previous revisions and diagnosis of the genus was checked (Wieser, 1953;Platt & Warwick, 1983;Smol, Muthumbi & Sharma, 2014). Upon collecting all required references: (1) validity and synonymy of species were examined and determined; (2) a table comparing diagnostic characters of all valid species was compiled; (3) locality and distribution of original descriptions were determined; (4) a new complete key to the genus was compiled.

Nomenclatural acts
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:6F60918D-9DE1-4B75-A251-C01E0694D01F. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Enoploides koreanus sp. nov.
Figs. 1A, 2, 3, Table 1 urn:lsid:zoobank.org:act:87CC02F7-3A2E-4137-84E5-C8E7A97DA6D2 Description: Males ( Fig. 2; holotype n = 1, paratype n = 2). Cuticle smooth above cephalic capsule, strongly striated below cephalic capsule until tail tip (Figs. 4A, 4B). Three lips high with its border striated heavily with grooves, each lip with two inner labial setae. Six inner labial setae, long and thin (11 µm long), at base of lips in one crown. Six longer outer labial setae (43 µm long) and four shorter cephalic setae (15 µm long) in one crown, situated at anterior portion of cephalic capsule. ∼20 subcephalic/cervical setae immediately after, some near second crown of setae near outer labial and cephalic setae, some near region of cephalic capsule end, in random lengths, some short, some as long as cephalic setae (Fig. 4A). Buccal cavity short and funnel shaped, wide at the anterior end, narrowing gradually towards the base. Buccal cavity armed with three equally sized and shaped ''solid'' mandibles and teeth. Mandible Y-shaped, two lateral bars converging into one solid bar, with distal end of each lateral bars claw-like, curving inwards to the lumen. Three onchia of equal size, posterior to each base of mandibles. Amphid not observed. Somatic setae irregularly scattered along the cervical region, in random lengths. Pilosity denser from level of buccal cavity until the nerve ring region. Pharynx long with grooves and sinuous external contours. Cardia inverse triangular shaped seemingly embedded into the intestine. Somatic setae sparsely distributed along the body in singles until tail region.
Metanemes not observed. Testes paired and opposed, anterior testis slightly right of the intestine and posterior testis also to right of the intestine. Precloacal supplementary organ, 8 µm long, 82 µm above cloacal opening, roughly 2.6 anal body diameters above the anus. Spicules paired, simple, thin and curved at an obtuse angle, proximal end with a knob (more distinct in some specimens than others) and distal end blunt and rounded. Spicule width equivalent throughout its length. Gubernaculum simple, rod-like shape, running parallel to distal half of spicule from level of spicule curvature to its distal end. Distal end with slightly rounded head (Fig. 4C). Tail region with some somatic setae in singles with no patterns observed. Tail conical, inconspicuously cylindrical at distal end. Caudal glands just below distal end of the spicules, at level of anus, running until a well-developed spinneret. Several caudal setae observed (Fig. 4B) along the tail with no terminal setae present at tail tip. Female ( Fig. 3; allotype n = 1, paratype n = 5). Female generally longer and larger in size. Short sub-cephalic setae below outer labial and cephalic setae (Fig. 4D). Cervical setae in singles and less frequent compared to male at posterior end of cephalic capsule. Reproductive system didelphic-amphidelphic, both ovaries reflexed, positioned left of intestine (Fig. 3C). Tail region with some caudal setae in singles with no visible patterns. No terminal setae observed at tail tip.   (1993) first acknowledged this group and created a key for Enoploides with short spicules, he included E. polysetosus, but even then it was distinguished for having the longest spicules in the group. We defined this group to consist of those bearing spicules shorter than 150 µm. E. polysetosus is therefore no longer considered to have short spicules. Species such as E. caspersi and E. tyrrhenicus can easily be distinguished from the other by the former's unique post-anal organ and the latter's complex gubernaculum. Of the seven species, the new species is most closely related to E. disparilis in terms of general morphology: (1) they both have simple and short spicules (34-39 µm vs. 35 µm) with a knob on its proximal end; (2) they both have simple and short gubernaculum (12 µm vs. 19 µm) parallel to distal end of the spicule; and (3) they share similar body lengths (2,107-2,307 µm vs. 2,250 µm) and index value of b (4.1-4.4 vs. 4.4). They can be differentiated from one another by the following characteristics: (1)  Group of species with long spicules (>150 µm) can be further divided into subgroups by the morphology of the gubernaculum: (1) S-shaped gubernaculum; (2) complex gubernaculum; (3) gubernacula that are short, simple, small, plated, arcuate, or weak. The term ''S-shaped'' has been used by several authors, including , Pavljuk (1984), to describe certain shapes of the gubernaculum.  conveniently grouped species with this S-shaped gubernaculum (E. cephalophorus, E. gryphus, E. spiculohamatus, E. amphioxi, E. labrostriatus and E. bisulcus) and provided a figure showing different gubernacula of several species within the genus. The S-shape can be extremely general and almost any shape can be regarded as S-shapes given the curving nature of gubernaculum's contours. For instance, gubernaculum of E. spiculohamatus was considered S-shaped by , while gubernaculum of E. vectis was not.  even referred to this group as ''more or less S-shaped'' confirming its ambiguous nature. To reduce ambiguity, any gubernaculum described as complex in the original description or consisting of multiple parts is not considered S-shaped here. There is certainly some uniformity of morphology of these ''complex'' gubernacula, which consist of multiple parts, bluntly shaped, anterior to the spicules. This removes E. spiculohamatus from the S-shaped group initially assigned by , as its gubernaculum consists of multiple parts and more closely resembles other complex-gubernaculum bearing species such as E. brunettii, E. labiatus, E. longispiculosus, and E. vectis. Refer to Table 2 for comparison of diagnostic morphological characters and gubernaculum type of all valid Enoploides species. Etymology: The species name refers to its occurrence in Korea. -index c∼12-16, spicules ∼90 µm long and gubernaculum with weak apophysis. . . E. tyrrhenicus 5. Tail shorter than 100 µm with two post-anal papillae . . . E. fluviatilis -Tail longer than 100 µm . . . 6 6. Spicule ∼100 µm long with plate-like gubernaculum with weak apophysis and three terminal setae at tail tip . . . E. stewarti -Spicule ∼30-40 µm long with rod-like gubernaculum with a rounded head at distal end and no terminal setae at tail tip . . . E. koreanus sp. nov. 7. Gubernaculum S-shaped . . . 16 -Gubernaculum not S-shaped . . . 8 8. Gubernaculum complex with multiple parts . . . 9 -Gubernaculum short/small, weak, arcuate, plate . . . 12 9. Pre-anal supplementary organ less than 1 abd away from cloacal opening . . . E. vectis -Pre-anal supplementary organ 1.5-1.7 abd away from cloacal opening . . . E. spiculohamatus -Pre-anal supplementary organ more than 2 abd away from cloacal opening . . . 10 10. Spicule length less than 200 (<4 abd) . . . E. brunettii -Spicule length greater than 200 (>4 abd) . . . 11 11. Post-anal cuticular element characteristically S-curved . . . E. labiatus -Post-anal cuticular element not s-curved . . . E. longispiculosus 12. Cephalic setae shorter than 10 µm and buccal cavity extremely short (9 µm Table 3 Description: Male ( Fig. 5; n = 1). Cuticle smooth. Lips high with heavy striation and grooves, each lip bearing two inner labial setae. Six inner labial setae, fairly long and thin at base of lips in one crown. Six longer outer labial setae and four shorter cephalic setae in one crown. Cervical setae scattered randomly at posterior end of cephalic capsule, as long as cephalic setae. Buccal cavity funnel shaped, wide at the anterior end, gradually narrowing towards the base. Buccal cavity armed with three equally sized and shaped mandibles and teeth, respectively. Mandibles with two lateral bars diverging away from one another distally. Distal end of each lateral bars ''claw-like'', curving towards the lumen like hooks. Mandibles widening near the base, each armed with fairly weak, narrow looking onchia. Mandibular columns divided by a sheet of cuticle. Pharyngeal glands not readily visible. Pharynx fairly long and muscular, its width consistent throughout its length, except the swollen anterior end. Fairly long somatic setae in singles, randomly distributed along the head. Pilosity intense until level of nerve ring, scarcer throughout. A little below the level of nerve ring, a ring of densely arranged cervical setae. Cardia inverse triangular, seemingly embedded in the intestine. Testes paired and opposed, both ends positioned left of intestine. Spicules slightly curved with small gubernaculum at distal end of spicules. No precloacal supplementary organ observed. Caudal glands after cloacal opening, well-developed. Tail conical-cylindrical, two long sub-terminal setae observed and two terminal setae at tail tip.

Remarks:
The morphology agrees well to the description provided by the original authors, Shi & Xu (2016). Mandibles clearly resemble those seen in Epacanthion species, consisting of two lateral bars (parallel to one another and the space in between not solid) separated by a thin sheet of cuticle. Its distinguishing characteristic, a single row or ring of densely arranged setae below the level of the nerve ring is quite distinct. All measurements are also within the range of the original (Table 3).

Mitochondrial cytochrome oxidase C subunit 1 (mtCOI)
We successfully amplified and sequenced DNA of four Enoploides koreanus sp. nov. and two Epacanthion hirsutum. Despite the JB3/JB5 primers being commonly used in molecular studies of nematodes (Derycke et al., 2005;Derycke et al., 2010;Derycke et al., 2016;Avó et al., 2017) few Enoploides sequences were available on GenBank to produce a meaningful phylogenetic analysis. Instead, the pairwise distance of all available Enoploides mtCOI sequences was calculated using K2P-substitution model using MEGA 7.0. There was no genetic divergence between the new species, while in comparison to other congeners, 19% to 24% divergence was seen (Table 4). This is well within range to genetic divergence seen between congeners using mtCOI sequences (Derycke et al., 2010).

18s rRNA
We successfully amplified and sequenced DNA of four Enoploides koreanus sp. nov. and two Epacanthion hirsutum. To test how our sequences group with existing sequences on GenBank, we rebuilt 18S rRNA ML tree by Pereira et al. (2010) with most Enoploides 18S sequences available on GenBank (listed in Table 5). The reason for selecting their tree was because their study dealt with a number of free-living marine nematodes (especially Thoracostomopsidae) and used the same primer sets utilized in the present study. The ML tree was more or less similar to the one provided by Pereira et al. (2010), with Thoracostomopsidae forming monophyletic clade with 100% bootstrap. Enoploides koreanus and Epacanthion hirsutum sequences obtained from this study both formed a clade with their respective congener species, with 83% and 78% bootstraps respectively (Fig. 6). One 18S rRNA sequence of Epacanthion hirsutum (MG599065) on GenBank was also retrieved to examine similarities. Although this sequence is considerably longer than ours (1,671 bp vs. 310-311 bp), for regions which do overlap, they showed no differences at any of the sites. Sequences obtained in this study have been submitted to GenBank and

DISCUSSION
One matter unresolved from this revision is the synonymy and confusion of species Enoploides spiculohamatus, E. labiatus and E. longispiculosus. Both Wieser & Hopper (1967) and Benwell (1981) suggested abandoning the issue as it cannot be proven. The main problem is that the original descriptions (Bresslau & Schuurmans Stekhoven, 1940;Schuurmans Stekhoven Jr, 1935) are poor and in the case of Schuurmans Stekhoven Jr (1935), it may not even be E. spiculohamatus according to Benwell (1981). As nothing can be done