Bibliographic revision of Mesacanthion Filipjev, 1927 (Nematoda: Thoracostomopsidae) with description of a new species from Jeju Island, South Korea

A new species of the genus Mesacanthion Filipjev, 1927 was discovered during a survey of natural beaches of Jeju Island in South Korea. The new species Mesacanthion jejuensis sp. nov. shares general morphology of the genus such as the outer labial and cephalic setae being situated at the middle of cephalic capsule, well-developed mandibles with two columns united by a curved bar, and three equally sized and shaped teeth shorter than the mandibles. The new species belongs to a group of Mesacanthion species in which spicules are shorter than two anal body diameters. The new species is most closely related to M. pannosum, first discovered in Puget Sound, Washington, in terms of having enlarged cervical setae flap at the end of cephalic capsule, spicules which are shorter than 2 anal body diameter, both supplementary organ and gubernaculum. It can be distinguished from M. pannosum by its stronger inner labial setae, longer outer labial setae, and difference in the index value of b and c’. Along with the description of Mesacanthion jejuensis sp. nov., the genus Mesacanthion Filipjev, 1927 is bibliographically reviewed and revised. Including the new species, a total of 48 species are described within the genus; 39 which are valid; eight which are considered to be species inquirenda due to misplacement of genus and poor description; one which is considered nomen nudum. An updated diagnosis of the genus is provided along with a compiled tabular key comparing different diagnostic morphological characters of all valid species, as well as a pictorial key consisting of 21 species with spicules shorter than two anal body diameters.

Family Thoracostomopsidae was first erected by Filipjev (1927) and it is composed of three subfamilies: Thoracostomopsinae (Filipjev, 1927) (2 genera), Trileptiinae (Gerlach & Riemann, 1974) (one genus), and Enoplolaiminae De Coninck, 1965 (19 genera). The three subfamilies can be differentiated by the presence or absence of mandibles (Enoplolaiminae or Trileptiinae respectively), with Thoracostomopsinae uniquely bearing a long and eversible spear (Smol & Coomans, 2006). Now total of 238 species belonging to 22 genera make up the family to date (Bezerra et al., 2019). The genus Mesacanthion (Filipjev, 1927) was first erected as a subgenus of Enoplolaimus (De Man, 1893) with type species Mesacanthion lucifer (Filipjev, 1927;Gerlach & Riemann, 1974) discovered from Barents Sea. Filipjev (1927) specified the characters of the genus Mesacanthion to be three short equal (seldom slightly different) onchia, cephalic setae placed in the middle or anterior to the cephalic capsule with tapered tail with a short dactyli/claviform terminal part. Many of the species currently belonging to the genus Mesacanthion were those transferred from the genus Enoplolaimus when Mesacanthion had been newly erected as a subgenus by Filipjev (1927). Most species (98%) belonging to this genus are recorded from marine environments with exception to one species (Mesacanthion alexandrinus Nicholas, 1993), which was recorded in freshwater environment. Of the valid species, 40% (16) were described from Europe; 20% (eight) from America (four from North and South); 17.5% (seven) from Asia (mainly from western Asia), 15% (six) from Africa, and 7.5% (three) from Australia. The genus Mesacanthion is the second most diverse genus in the family next to Enoplolaimus (De Man, 1893), with 40 valid species recorded to date.
The aim of this study was to review the genus by compiling information such as species distribution, tabular and pictorial key of the genus while determining the validity of existing species. In addition to the revision, Mesacanthion jejuensis sp. nov. is described from Jeju Island, South Korea. An updated diagnosis of the genus is provided with a compiled tabular key consisting of all valid species as well as a pictorial key consisting of 21 species with spicules shorter than two anal body diameters.

Sampling and morphological study
A series of sampling took place in June 2018, during a survey of natural beaches of Jeju Island, South Korea (Fig. 1). Two sub-samples of the sediments from the intertidal zone were obtained using a 10 cm 2 acryl sampling tube. Sediments were fixed in 5% neutralized formalin solution and brought back to the laboratory. Meiofauna were extracted using the Ludox method (Burgess, 2001), and post-fixed with 70% ethanol dyed with Rose bengal. Nematodes were counted and individual specimens of interest were picked to a Petri dish filled with 10% glycerin. The dish was placed in a drying oven set at 40 • C for a day or two to be completely dehydrated as conferred in the glycerin-ethanol method (Seinhorst,  1959). A single or as many as five specimens (depending on their size) were mounted in a single drop of anhydrous glycerin on a glass slide using the wax-ring method (Hooper, 1986). Mounted specimens were identified under Olympus BX51, Leica DM5000B and DM2500 microscopes. All morphometric measurements were done manually using IC measure v.2.0.0.161 software. For scanning electron microscopy, specimens were placed in a drop of glycerin and gradually mixed with drops of distilled water to be washed from any remnant of glycerin. Hydrated specimen were treated to ethanol series for dehydration (20%, 40%, 50%, 70%, 80%, 90%, 95%, 100%, for 10 min each) and then placed in hexamethyldisilazane (HMDS). Specimens bathed in HMDS were placed in a drying oven to be dried. Once dried, specimens were mounted on a stub to be splutter coated, and observed with COXEM EM-30 microscope.

Revision of the genus
The Bremerhaven Checklist of Aquatic Nematodes by Gerlach & Riemann (1974) was used as primary referral when collecting original descriptions/references and additional information on their distribution. Any updates and changes made to the genus subsequent to 1974 were checked using NeMys, World Database of Nematodes. Once all references had been collected; (1) tabular key consisting of diagnostic characters of all valid species were compiled, (2) distribution of species were determined, (3) validity of each species were determined via comparison and examination, (4) diagnosis of the genus was updated. To construct a pictorial key, original depictions were collected from respective papers and their heads, tails and spicules (if available) were resized and oriented using Adobe Photoshop CS6 for optimum comparison between species. The original drawings were retraced using Wacom Intuous Pro Pen Tablet and Adobe Photoshop CS6.

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: 989DF431-166A-4534-9A37-9AC408194DE7. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.
Notes on generic diagnosis: Mesacanthion, Enoplolaimus De Man, 1893, Paramesacanthion Wieser, 1953, and Oxyonchus Filipjev, 1927 bear mandibles which are arch-shaped, consisting of two rod-like columns while mandibles of Enoploides are solid, two lateral bars fused to form a single rod. Oxyonchus can be distinguished from other genera which bear similar mandibles by its two uniquely large ventrosublateral teeth which extend to anterior end of the mandibles with small dorsal tooth. Mesacanthion, Paramesacanthion, Enoplolaimus, all have teeth shorter than the mandibles, but the latter can be distinguished by the placement of their outer labial and cephalic setae at posterior end of cephalic capsule. Mesacanthion species have their outer labial and cephalic setae at the middle or anterior end of the cephalic capsule, similar to Paramesacanthion species except outer labial and cephalic setae are only in front of anterior end of cephalic capsule. Mesacanthion and Paramesacanthion share the most characters, making them the closest related genera within the family. The two genera can be differentiated from each other however by the following three characteristics: 1. Outer labial and cephalic setae are located at the anterior end of cephalic capsule for Paramesacanthion while outer labial and cephalic setae are located at the middle or anterior end of cephalic capsule for Mesacanthion. Paramesacanthion species have extra ring(s) of subcephalic setae located at the middle of cephalic capsule where outer labial and cephalic setae would be located for Mesacanthion species. This means when compared to Mesacanthion species, Paramesacanthion species may appear to have extra ring(s) of setae at the anterior end of cephalic capsule, in between inner labial setae and cephalic setae/outer labial setae. This seemingly additional ring of setae are the true cephalic setae, while ring of setae at the middle of cephalic capsule are actually the sub-cephalic setae for Paramesacanthion species; 2. Sexual dimorphism is apparent in the pilosity of the head for Paramesacanthion species, while it is not apparent in Mesacanthion species; 3. All Paramesacanthion species have spicules consisting of two portions, distal and proximal, articulating from one another, while only some Mesacanthion species (M. audax, M. ditlevseni, M. infantile and M. jejuensis sp. nov.) have bipartite spicules divided by a transversal seam, but without the obvious articulation or constriction.

Nomen nudum
1. Mesacanthion microsetosus Allgen, 1932 (nomen nudum - Bezerra et al., 2019) [Allgén, 1932: 110-111, fig. 7A Measurements: See Table 1 for detailed measurements and morphometric ratios. Description: Male (Fig. 2). Cuticle smooth above cephalic capsule, finely striated posterior to cephalic capsule until tail tip. Three lips well developed; edges of lips narrowed and distally pointy curving outwards, each lips carrying two inner labial setae. Six inner labial setae, stout and conical 12 µm long. Six longer outer labial setae and four shorter cephalic setae sharing one crown, situated at midlevel of cephalic capsule. Cephalic capsule vaguely set off at mid-level, anterior part narrow, and posterior part gradually thicker. Buccal cavity funnel shaped, wide at anterior end, gradually narrowing to the base. Coffee-bean shaped epidermal glands distributed along dorsal plane from anterior end of body until posterior end. Buccal cavity armed with three mandibles and three teeth. Mandible consisting of two rods distancing from one another anteriorly joined by anterior rod. Lateral edges of each rod with teeth or denticle pointing to the lumen (Fig. 3E). ∼5-6 short cervical setae in singles at level of posterior end of cephalic capsule. Modified cervical setae, a flap, inverse triangular, just posterior to a single lateral outer labial setae at posterior end of cephalic capsule, observable in all four males on both lateral body sides (Fig. 4B). Amphid ambiguously present below the cervical flap, pouch-shaped. Two pores observed diagonally below cervical flap and amphid (Fig. 4B). Cervical somatic setae in 8 groups of 2-3 around pharyngeal region a, roughly two cephalic capsule lengths below level of cephalic capsule end (Figs. 4A and 4B). Some cervical setae partly possessing irregular lateral and terminal processes, resembling penicillus or plumule (Figs. 4A and 4B). Somatic setae scarcely distributed along the body in singles until tail region. Pharynx fairly long and annulated with plasmatic lens-like interlayers and sinuous external contours, cardia triangular and going into the middle of intestine. Metanemes not visible. Testes paired opposed, both ends situated to the right of the intestine. Thick supplement, 18 µm long, 165 µm above from cloacal opening. Spicules paired, bipartite, symmetrical, curved slightly and thick. Each spicule with distinct transverse, oblique seam, dividing it distal and proximal portions (Figs. 5A and 5B). Distal portion shorter than proximal portion. Distal portion slightly curved towards cloacal opening, anterior end with one denticle just above and/away from its round pointy end. Proximal portion rather straight, posterior end with a knob/neck-like constriction. Gubernaculum embracing spicules, shaped like irregular triangle, lateral end which lies lateral to the spicule, almost perpendicular to axis of the anus, even extending beyond distal end of spicule, and the other end arching off at an angle towards the tail. Tail elongated and papilliform. five somatic setae in tail region. Caudal gland protruded anterior to the anus, their nucleus-containing bodies located along the  Corresponding body diameter at vulva n/a n/a 108 ± 31 (80-141) Distance from anterior end to vulva as percentage of total body length n/a n/a 54 ± 1 (54-55) a 46.6 43.1 ± 5.1 (35.6-46.6) 34.9 ± 3.1 (32.8-38.5) b 5 4.8 ± 0.2 (4.5-5) 4.6 ± 0.2 (4.5-4.8) c 12.8 12.4 ± 0.6 (11.7-12.9) 12.9 ± 0.8 (12-13.5) posterior midgut. Spinneret well developed. 1 short caudal (terminal) setae (with porous) just above distal end of tail. Female (Fig. 3). Female generally longer and larger in size. Three lips higher in female, edges of lips noticeably stronger in female, distal end aggressively curved, each lips carrying two inner labial setae. No subtle sexual dimorphism found in setae in the head region, other than shorter length outer labial and cephalic setae compared to male. Short knobs on each anterior end of mandible. Female lacking cervical setae flap on cephalic capsule end. Amphid not observed. Groups of cervical setae found in esophageal region in males are in singles as opposed to doubles/trios (Fig. 5D). Vulva located at 55% of total body length with protruding lips. Reproductive system didelphic amphidelphic, both ends flexed inwards. Both ovaries positioned left of the intestine, antidromously reflexed. Tail conico-cylindrical, three somatic setae in tail region with no apparent caudal setae. Diagnosis: Mesacanthion. Body length 2700-4630 µm. Cuticle finely striated along the body, smooth only in cephalic capsule region, head set off with cephalic capsule. Metanemes not visible. Six inner labial setae 8-15 µm. Six longer outer labial setae 36-59 µm, four shorter cephalic setae 18-34 µm long sharing one crown. Buccal cavity armed with mandible and three teeth. Mandible consisting of two rods distancing from one another anteriorly joined by anterior rod. Lateral edges of each rod with teeth or denticle pointing to the lumen. Buccal cavity 37-61 µm long. 8-9 groups of cervical setae in groups of two to three at stoma region. Cervical setae in single groups in females. Males with testis paired and opposed. Spicule paired, symmetrical, slightly curved, divided into two portions by a seam. Distal portion shorter than proximal. Proximal portion with knob/neck-like end. Gubernaculum paired, shaped like an irregular triangle with caudal apophysis, distal end extending beyond spicules and ventrally towards cloacal opening. Precloacal supplementary organ present. Three to four somatic setae distributed along the tale. Tail conico-cylindrical, c' 4-5.7, cylindrical portion of the tail constituting about 30% of the entire tail length. Differential diagnosis: Total of 23 species of Mesacanthion with spicules shorter than 2 abd were examined. Species such as M. arcuatile, M. conicum and M. cricetoides were omitted from examination due to the fact that only female was ever described. Also, species with asymmetrical spicules (anisomorphic and anisometric) were omitted even if the shorter spicule is shorter than 2 abd, since Gagarin & Klerman (2006) already provided a key for those group of species. M. tenuicaudatum which most likely does have spicules shorter than 2 abd, is also omitted from examination and pictorial key as there are no depiction of the specimen available. Description also lacks information regarding gubernaculum, measurements of anal body diameter and length of all setae, making it not feasible for comparison. Lastly, M. virile is included for analysis, despite its lack of information on abd. Although it cannot be confirmed that it possesses spicules which are shorter than 2 abd, given the length of spicule and other relative body measurements, it is likely that this species belongs to this group.
The new species is most similar to M. pannosum as they both share striking resemblance in overall morphology. They both have spicules shorter than 2 abd with presence of both supplementary organ and gubernaculum. Index value of both species (a and c) are within range to each other as well. Most interesting character shared by the two species is the presence of modified/transformed cervical setae seen as a flap. This flap-like appendage is seen as inverse triangle just below a single outer labial seta (lateral seta), at the end of cephalic capsule. It was first observed by Wieser (1959) in males of M. pannosum and it has been a character unique to the species until it now. Like M. Pannosum, the flap is also observed only in males of the new species, and the morphology is in line with those previously seen in M. pannosum. The only difference concerning the flap between the two species is that M. pannosum had two flaps next to each other, but only a single flap is seen in the new species (Fig. 4B).
The validity of diagnostic value in presence or absence of a seam (bipart spicule), can be questionable considering it is an ambiguous character and could be mistaken from a diffraction caused by a large gubernaculum. Due to this reason, the seam was not given a significant importance in the diagnosis of species within the genus, but the character and species which bear it are still discussed for reference.
Total of four species within the genus Mesacanthion have paired bipartite spicules: M. audax, M. ditlevseni, M. infantile, and M. jejuensis sp. nov. They all have spicules which are shorter than 2 abd, but M. audax is most easily distinguished from the other three species by its lack of supplementary organ. While the three species have supplementary organ, the new species resembles M. ditlevseni the most. They both have stout inner labial setae with presence of both supplementary organ and triangular gubernaculum. Their index value (a, b, and c') are also within range from one another. The new species can be distinguished by its proportionally longer cephalic setae (in which is double the length of cephalic setae observed in M. ditlevseni); presence of cervical flap in new species; dense distribution of cervical setae in groups of doubles/trios below at stoma region in male; differing details to its spicules and gubernaculum. The spicule of the new species is different from M. ditlevseni in that distal portion of the spicule is shorter than proximal portion where vice versa is true for M. ditlevseni. This is peculiar characteristic unique to the new species, as all bipartite spicules found in Mesacanthion species have longer distal portion over proximal portion. We believe that diagnostic value of modified cervical setae flap is greater than bipartite spicule (spicule with seam) in terms of ambiguity. Therefore, the species most similar to the new species is considered to be M. pannosum.
Etymology: The species name jejuensis is given as the species was discovered from coast Jeju Island, South Korea.

Pictorial key to species with spicules shorter than 2 anal body diameter within the genus Mesacanthion and morphometric values for valid species of Mesacanthion
Figs. 6-8, Table 2 Key to species with spicules shorter than 2 anal body diameters within the genus Mesacanthion

DISCUSSION
The genus Mesacanthion currently consist of 39 valid species. Of these valid species, Mesacanthion ungulatum (Wieser, 1953) is most ambiguous in terms of validity. The species was erected from a description based on only two juvenile specimens, with reasoning that the species in question bears extremely long labial setae and high lips. While there is no problem with the validity according to the International Code of Zoological Nomenclature, since a new species can be described at any life stage, it is ambiguous nonetheless in its current state as its distinguishing characters for the species is no longer unique to the species. Labial setae in M. ungulatum is noted to be extremely long, measuring to be 15 µm might have been lengthy for the genus at the time of original description, but currently compared to other species within the genus, it is quite average in length. Mesacanthion arabium (Warwick, 1973) bears labial setae measuring from 23-25 µm, albeit its overall longer body length. Even when comparing proportionally to body length, Mesacanthion fricum (Inglis, 1966) (body length 1650 µm/length of inner labial setae 13 µm) and Mesacanthion hirsutum (Gerlach, 1953(Gerlach, ) (1155(Gerlach, -1982 have longer proportioned inner labial setae than M. ungulatum (2250-2430 µm/15 µm). The only characteristic discerning this species from the rest is then by its high lips, but even that is questionable, considering later described species such as Mesacanthion alexandrinus (Nicholas, 1993), while it does not mention in the description, depict just as high lips as shown in M. ungulatum. It would be appropriate to consider this a synonymization case, and find a species described after M. ungulatum which is most similar to follow the Principle of Priority. Unfortunately, remaining characteristics of M. ungulatum is very generic to the genus and the fact that there is no male to compare its spicules, gubernaculum and supplementary organ which can be unique to each species, no further action can be taken other than to place it as species inquirendum. There is however 18S ribosomal RNA gene, partial sequence of M. ungulatum available on GenBank. It seems that the sample specimen was obtained from Chile, the type locality, so there is high probability that more information can be gathered regarding adult stage of this species. Hopefully someone can review the species with a sound adult specimen to clear this species of its current status. Diagnosis of the genus Mesacanthion provided by Smol, Muthumbi & Sharma (2014), was updated in this study based on our review and findings. The original diagnosis specifically states that spicules are generally short, but if long, gubernaculum with caudal apophysis is to be present. This is true in most cases, but with exceptions in species such as M. brevista, which has one of the longest spicules in the genus (165 µm) with no gubernaculum at all, and M. arabium, which has a pair of short spicules (24 µm) bearing a triangular gubernaculum which resembles a caudal apophysis. In addition, accounts for different types of spicules were added (symmetric/asymmetric, bipartite, etc.), so that later encounters of new species with bipartite spicules is not simple mindedly mistaken as Paramesacanthion as opposed to Mesacanthion, as was the case with us.
Full-size DOI: 10.7717/peerj.8023/ fig-6 and can be bipartite or in whole. The part of it being bipartite can be perplexing when it comes to species identification, as it can lead to wrongful placement of the species to the related genus Paramesacanthion. Diagnosis of Paramesacanthion provided by Smol, Muthumbi & Sharma (2014) specifically mentions its distinguishing characteristic is ''spicules consisting of two portions, distal and proximal, articulating with each other'', while diverse nature of spicules in Mesacanthion is missing in its diagnosis. Even while  Wieser (1953) specifically mentioned this when erecting the genus, not to confuse ''the subcephalic setae of the male with the true cephalic setae since the former occupy that level of the head on which in Mesacanthion the insertion of the cephalic setae takes place'' (p.80). As such, the new species has a paired spicule which are symmetric from one another. They are bipartite with distal portion slightly shorter than proximal part. Distal and proximal is divided by a seam which seems to thicken or ''arm'' around the distal portion of the spicule. Proximal end arcuate and distal end set with pointing spine or a ''barb''. Metanemes are one character which was surprisingly not observed within the new species. While no species belonging to Mesacanthion have yet been described to date with description or depiction of metanemes, orthometaneme of dorsolateral kind was expected to be present within the new species prior to inspection. Diagnosis of the family Thoracostomopsidae (Filipjev, 1927) (according to Smol, Muthumbi & Sharma, 2014 specifically states ''only dorsolateral orthometanemes with a robus scapulus but no caudal filament''. Species belonging to Mesacanthion's most closely related genus, Paramesacanthion abyssorum Bussau 1995, was also recorded with presence of dorsolateral orthometanemes. Not only that, ''coffee bean shaped epidermal glands'' which were sighted alongside dorsolateral orthometanemes in P. abyssorum are very much present within the new species as well (Figs. 2A and 3A). Given that orthometanemes are subtler in their appearance compared to loxometanemes, it is quite possible that even other species of Mesacanthion already Mesacanthion arcuatile Wieser, 1959 No male described or measured Mesacanthion banale Filipjev, 1927;Gerlach & Riemann, 1974 No male described or measured Mesacanthion brachycolle Allgén, 1959 No male described or measured Mesacanthion breviseta Filipjev, 1927;Gerlach & Riemann, 1974 (121) Mesacanthion conicum Filipjev, 1918;Filipjev, 1927 No male described or measured Mesacanthion cricetoides Wieser, 1959 No male described or measured Mesacanthion diplechma Southern, 1914;Filipjev, 1927    described, could have had them present. Despite it being more difficult to spot in older types due to their conditions, it'll be important for future descriptions of any species belonging to the family Thoracostomopsidae to identify their metanemes. After discovering the new species, the type locality was visited twice more in August and November of 2018, to obtain alcohol samples of the specimen for molecular analysis. While the efforts were unfortunately fruitless until now, we are hopeful that we will get the required specimen for additional molecular analysis in the future. It'll be interesting to compare the relationship between close related species by the means of molecular phylogenetic data.

CONCLUSION
The discovery of Mesacanthion jejuensis sp. nov., has led to number of findings: (1) the new species closely related to M. pannosum, in terms of general morphology (bearing precloacal supplementary organ and gubernaculum) and having modified cervical setae flap.
(2) the new species, like three other species within the genus (M. audax, M. ditlevseni, M. infantile), has a pair of bipartite spicule. (3) the diagnosis of the genus Mesacanthion has been updated to account for diverse nature of spicules. (4) the genus Mesacanthion has been reviewed and revised, transferring two species, M. brachycolle and M. ungulatum to species inquirenda, updating the total number of valid species to 39 species. While we were unable to obtain genetic data for the new species, further efforts will be made in order to investigate the phylogenetic relationship and placement of species within the genus Mesacanthion.

Abbreviations a
body length/maximum body diameter abd anal body diameter b body length/pharynx length c body length/tail length calc calculated or measured from published measurements and/or figures c' tail length/anal body diameter