Journal of Oceanology and Limnology   2021, Vol. 39 issue(4): 1547-1558     PDF       
http://dx.doi.org/10.1007/s00343-020-0195-2
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

SUN Jing, HUANG Mian, HUANG Yong
Four new species of free-living marine nematode from the sea areas of China
Journal of Oceanology and Limnology, 39(4): 1547-1558
http://dx.doi.org/10.1007/s00343-020-0195-2

Article History

Received May. 12, 2020
accepted in principle Jun. 17, 2020
accepted for publication Aug. 13, 2020
Four new species of free-living marine nematode from the sea areas of China
Jing SUN1, Mian HUANG2,3, Yong HUANG1     
1 College of Life Sciences, Liaocheng University, Liaocheng 252059, China;
2 Department of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
3 University of Chinese Academy of Sciences, Beijing 100049, China
Abstract: Two new species of genus Diplopeltoides Gerlach,1962 and two new species of genus Minolaimus Vitiello,1970 were described based on recently collected specimens from marine sediments of the South China Sea and the East China Sea,respectively. Diplopeltoides conoicaudatus sp. nov. is characterized by annulated cuticle; tiny terminal buccal cavity; short cephalic setae about 2-μm long; loop-shaped amphideal fovea without sclerotized plate; slender and arcuate spicules; triangular gubernaculum possessing a small dorso-caudal apophysis; female with two opposed and reflexed ovaries. Diplopeltoides longifoveatus sp. nov. is characterized by its papilliform cephalic sensilla; long and narrow amphideal fovea with fine transverse striations; inverse S-shaped spicules with a dorsally bent manubrium; gubernaculum with dorso-caudal apophysis; cylindrical tail with round terminal end. Minolaimus multisupplementatus sp. nov. is characterized by cuticle with lateral differentiation of three longitudinal rows of large dots,spiral amphideal fovea with seven turns,pharynx cylindrical without posterior bulb,spicules arcuated with central lamella,gubernaculum arcuate and parallel to spicule,without dorso-caudal apophysis,a papilliform and 29 cup-shaped precloacal supplement; tail conical with long flagelliform portion. Minolaimus apicalis sp. nov. is characterized by cuticle with lateral differentiation of three longitudinal rows of larger dots from the middle of pharynx to the conical portion of tail and a longitudinal row of pores in the pharyngeal region and precloacal region; spiral amphidial fovea with four turns and close to anterior end; pharynx cylindrical with oval posterior bulb,spicules arcuated and proximal end bent dorsally,gubernaculum with a broad curved dorsal apophysis; two sucked-like and six cup-shaped cuticularized precloacal supplements; tail conico-cylindrical with slightly enlarged tip. Updated keys to species of Diplopeltoides and Minolaimus were provided.
Keywords: Diplopeltoides conoicaudatus sp. nov.    Diplopeltoides longifoveatus sp. nov.    Minolaimus multisupplementatus sp. nov.    Minolaimus apicalis sp. nov.    East China Sea    South China Sea    
1 INTRODUCTION

This paper is a part of the author's serial work on free-living marine nematodes from the sea area of China. Over the past few years, more than 300 nominal species in the East China Sea (Sun et al., 2019; Zhai et al., 2020) and more than 290 nominal species in the South China Sea (Fu et al., 2019; Qiao et al., 2020) have been identified from the intertidal and sublittoral sediments. Among these species, four Diplopeltoides species including Diplopeltoides bulbosus (Vitiello, 1972) Holovachov & Boström, 2017, Diplopeltoides nudus (Gerlach, 1956) Tchesunov, 2006 and two new species, and two new Minolaimus species were identified by the authors in this taxonomic research. The four new species are described herein as Diplopeltoides conicaudatus sp. nov., Diplopeltoides longifoveatus sp. nov., Minolaimus multisupplementatus sp. nov. and Minolaimus apicalis sp. nov.

The genus Diplopeltoides was proposed by Gerlach (1962), with the transference of Diplopeltis ornatus (Gerlach, 1950) for a new genus. Holovachov and Boström (2017) made a comprehensive taxonomic revision of Diplopeltoides based on the description of eight species including three new ones from Sweden. Meanwhile, taxonomic changes of the genus Diplopeltula were proposed, and six Diplopeltula species were transferred to the genus Diplopeltoides. The genus Diplopeltoides now contains 19 valid species recorded from all over the world (Holovachov and Boström, 2017; Bezerra et al., 2020). Genus Diplopeltoides is characterized by annulated cuticle; loop-shaped amphideal fovea (inverted U-shaped), in some species lies on strongly cuticularized lateral subcuticular plates; small, funnel-shaped buccal cavity; pharynx subdivided by breaks in muscular tissue into anterior corpus and postcorpus, postcorpus consisting of anterior narrower isthmus and basal bulb; female reproductive system didelphicamphidelphic with two opposed, reflexed ovaries; male reproductive system diorchic, both testes outstretched and opposed; spicules symmetrical, arcuate; gubernaculum usually present; supplements absent; three caudal glands open via three separate openings (based on Holovachov, 2014; Holovachov and Boström, 2017). The genus Minolaimus was established by Vitiello (1970) based on the type species of Minolaimus lineatus Vitiello, 1970. The species of Minolaimus is not common. To date, only two species, Minolaimus lineatus Vitiello, 1970 and Minolaimus cervoides Vitiello, 1970 have been recorded around the world (Gerlach and Riemann, 1973; Bezerra et al., 2020). Furthermore, Minolaimus cervoides Vitiello, 1970 was described based on only females. As yet, the male of the species has not been recorded. Thus, it was the insufficiently described species. Minolaimus species are characterized by cuticle with transverse rows of dots and lateral differentiation of longitudinal rows of larger dots; setiform outer labial and cephalic sensilla; multispiral amphids with at least four turns; buccal cavity small without tooth; posterior end of pharynx usually enlarged, forming bulb or not; precloacal supplements papilliform or cup-shaped; tail conical cylindrical, usually with the elongated posterior cylindrical portion (emended from Platt and Warwick, 1988; Fonseca and Bezerra, 2014; Tchesunov, 2014).

2 MATERIAL AND METHOD

Seabed sediment samples were obtained using a 0.1-m2 improved Gray-O'Hara box in the East China Sea in October 2012 and Qiongzhou Strait of the South China Sea in February 2017. The meiofauna sampling was taken using a syringe with a 2.6-cm inner diameter. The syringe was pushed into the sediment down to 8-cm depth and samples were divided by 0–2, 2–5, and 5–8 cm, then fixed with equivalent 10% formalin in seawater. In the laboratory, sorting and mounting of nematodes were performed as previously described (Huang et al., 2019; Qiao et al., 2020). The descriptions were made from glycerin mounts using a differential interference contrast microscope (Leica DM 2500). Line drawings were made with the aid of a camera lucida. All measurements were taken using Leica LAS X version 3.3.3, and all curved structures were measured along the arc or median line. Type specimens were deposited in the Marine Biological Museum of the Chinese Academy of Sciences, Qingdao.

3 RESULT 3.1 Diplopeltoides conoicaudatus sp. nov. (Figs. 12)
Fig.1 Drawing of D. conoicaudatus sp. nov. a. lateral view of male anterior end, showing buccal cavity, cephalic seta, and amphideal fovea; b. lateral view of male posterior end, showing spicule, gubernaculum, and caudal glands; c. view of entire holotype male, showing two testes; d. view of entire female, showing vulva, ovaries and spermathecae.
Fig.2 Microscopic images of D. conoicaudatus sp. nov. a. anterior end of male, showing amphidial fovea and terminal bulb; b. anterior end of male, showing amphidial fovea and cephalic setae; c. posterior end of male, showing spicule and tail; d. vulva region of female, showing valve, spermatheca and oogonia; e. cloaca region of male, showing spicule and gubernaculum (scale bars: a, c, d, e: 20 μm; b: 10 μm).

Class Chromadorea Inglis, 1983

Order Plectida Gadea, 1973

Family Diplopeltoididae Tchesunov, 1990

Genus Diplopeltoides Gerlach, 1962

3.1.1 Type material

Holotype: ♂1 on slide qzhx 17-1-0-2-6, paratypes: ♂2, ♂3, ♀1 on slides qzhx 17-1-2-5-9, qzhx 17-1-0- 2-4, qzhx 17-1-0-2-3, respectively and ♂4 on slide DH3-3-3-1.

3.1.2 Type locality and habitat

Seafloor muddy sediment at Station NHqzhx1 of Qiongzhou Strait in the South China Sea (20°3′53″N, 110°21′5″E; water depth 13 m) and seafloor muddy sediment at Station DH-3-3 in the East China Sea (30°0′3″N, 123°29′57″E; water depth 69 m).

3.1.3 Etymology

The species name is composed of the Latin adjectives conoidalis and caudatus, referring to the conical tail.

3.1.4 Measurement

All measurement data are given in Table 1.

Table 1 Individual measurements of D. conoicaudatus sp. nov. and D. longifoveatus sp. nov. (in μm except a, b, c, c′, number and V %)
3.1.5 Description

Males. Body stout and attenuating towards both ends, 581–625-μm long. Cuticle annulated, each annule about 2-μm wide along the body, longitudinal striation on annule not observed. Inner and outer labial sensilla not observed. Four cephalic setae, each about 2-μm long. Amphideal fovea ventrally wound and long loop in outline, 16–18-μm long and 7-μm wide; anterior border situated at the level of cephalic setae base. Buccal cavity tiny. Pharynx with a terminal bulb. The cardia well developed, conoid. Nerve ring situated at 60–65 μm from body anterior end (53%– 55% of pharyngeal length). Secretory-excretory system not observed. Tail conical, 3.1–3.5 anal body diameters long. Three caudal gland cells, opening with separate terminal outlets.

Reproductive system diorchic, both testes opposed and outstretched. The anterior testis situated to the left of intestine, 61–82 μm below the pharyngeal bulb; the posterior one situated to the right, 186 μm below the pharyngeal bulb. Sperm cells oval. Spicules slender and arcuate, 41–46-μm along arc. Gubernaculum very small, triangular with a distinctly sclerotized dorso-caudal apophysis, 3-μm long.

Female. Similar to males in most features except the body slightly shorter and thicker. Reproductive system didelphic, two ovaries opposed and reflexed. Anterior ovary situated to the left of intestine, posterior ovary situated to the right of intestine. Oviduct a wide tube. Uterus a short tube. Vagina straight and broad with thick walls. Two oval sac-like spermathecae, each located on side of each gonoduct. Vulva located in the mid-body (51.6% from the anterior end).

3.1.6 Differential diagnosis and discussion

Diplopeltoides conoicaudatus sp. nov. is characterized by short cephalic setae 2-μm long; amphideal fovea without striations and sclerotized plate; conical tail 3.1–3.5 anal body diameters long; relatively long spicules slender and curved (41–46- μm along arc); small triangular gubernaculum with a dorsal apophysis; testes outstretched and opposed.

Diplopeltoides conoicaudatus sp. nov. differs from all known congeners except D. pumilus (Vincx & Gourbault, 1992) Holovachov & Boström, 2017, D. linkei Jensen, 1991 and D. ornatus (Gerlach, 1950) Gerlach, 1962 by the conical tail (vs cylindro-conical or cylindrical). The new species highly resembles D. pumilus, D. linkei, and D. ornatus in the small body size, terminal mouth and conical tail. However, D. conoicaudatus differs from D. pumilus by its longer and slender spicules (41–46 μm vs 19–27 μm with proximal manubrium), triangular gubernaculum with small dorsal apophysis (vs plate-like without apophysis) and slightly shorter tail (c′=3.1–3.5 vs 3.5–4.5). It is differentiated from D. linkei by the lack of sclerotized amphid plaque (vs with sclerotized amphid plate in the latter species), the longer spicules (41–46 μm vs 29–36 μm), and the presence of gubernaculum apophysis (vs absence). Moreover, the present species distinguishes from D. ornatus by the lack of cutiular amphid plaque (vs with sclerotized amphid plate in the latter species) and the much longer spicules (41–46 μm vs 22 μm). Wieser (1956) described a speciemens of D. ornatus which mouth situated dorsally. The difference between the new species and other congeners can be inferred from the key below.

3.2 Diplopeltoides longifoveatus sp. nov. (Figs. 34)
Fig.3 Drawing of D. longifoveatus sp. nov. a. lateral view of male anterior end, showing amphideal fovea, terminal bulb, and excretory system; b. lateral view of male posterior end, showing spicule, gubernaculum, and caudal glands; c. view of entire holotype male, showing two testes.
Fig.4 Microscopic images of D. longifoveatus sp. nov. a, b. lateral view of male anterior end, showing amphidial fovea; c, d. lateral view of male posterior end, showing spicule and gubernaculum; e. cloaca region of male, showing spicule (scale bars: a, b, e: 10 μm; c, d: 20 μm).
3.2.1 Type material

Holotype: ♂1 on slide DH-5-5-8, paratype: ♂2 on slide DH-8-2-4.

3.2.2 Type locality and habitat

Seafloor muddy sediment at Station DH-5-5 in the East China Sea (27°32′42″N, 123°36′12″E; water depth 100 m) and seafloor muddy sediment at Station DH-8-2 in the East China Sea (26°35′18″N, 121°7′58″E; water depth 71 m).

3.2.3 Etymology

The species name is composed of the Latin adjectives longus and foveatus (pitted), referring to the relatively long amphideal fovea, a main feature of the species.

3.2.4 Measurement

All measurement data are given in Table 1.

3.2.5 Description

Males. Body thread, 715–886-μm long. Cuticle fine striated along the body. Inner and outer labial sensilla not observed, cephalic sensilla papilliform. Amphideal fovea long and narrow, loop shaped with distinctly fine transverse striation, 32–33-μm long and 5-μm wide. Anterior border situated at the position of 10–12 μm from the anterior end. Amphids sclerotized plate absent. Buccal cavity tiny. Pharynx cylindrical with a terminal bulb. Nerve ring situated in the mid of pharyngeal length (i.e. 70 μm from the front end). Secretory-excretory system consisting of unicellular gland located at the level of anterior part of intestine, secretory-excretory duct with distinct ampulla and opening just in front of the pharyngeal terminal bulb (i.e. located 102 μm from the front end). Tail cylindrical, 3.5–3.7 anal body diameters long, with round terminal end. Three caudal gland cells with separate subterminal outlets.

Reproductive system diorchic, both testes opposed and outstretched. Spicules paired and symmetrical, inverse S-shaped with proximal dorsally curved manubrium, 26–29-μm long along arc. Gubernaculum small, with sclerotized dorso-caudal apophyses, 3-μm long.

Female not found.

3.2.6 Differential diagnosis and discussion

Only two male specimens are available for the identification. Generally, this is insufficient for establishment of a new nematode species without female. Nonetheless, the male specimens are rather distinct and match with the main diagnostic features of Diplopeltoides, particularly in having annulated cuticle, tiny buccal cavity, long-loop shaped amphids and presence of terminal bulb. Diplopeltoides longifoveatus sp. nov. is characterized by papilliform cephalic sensilla, long and narrow amphideal fovea with fine transverse striation, inverse S-shaped spicules with a dorsally curved manubrium, and small gubernaculum with dorso-caudal apophyses.

Diplopeltoides longifoveatus sp. nov. is most similar to D. anatolii Voronov, 1982 in having short cephalic sensilla, relatively long and narrow amphideal fovea and cylindrical tail. However, the new species differs from D. anatolii by its papilliform cephalic sensilla (vs setiform and 2-μm long), amphideal fovea with transvers striation (vs without transverse striation), shorter spicules (26–29 μm vs 40 μm), inverse S-shaped with a dorsally curved manubrium (vs strongly curved spicules with a straight manubrium), and greater value of "a" (31.1– 42.2 vs 21–30.4). Moreover, the new species is similar to D. bulbosus (Vitiello, 1972) Holovachov & Boström, 2017 in amphideal fovea with fine transvers striation and structure of spicules and tail shape. However, D. bulbosus is much longer (1.12– 1.58 mm), and possesses a stubby amphideal fovea (21–28-μm long and 7–11-μm wide), long and strongly curved spicules (34–50-μm long).

The updated key to species of Diplopeltoides (after Holovachov and Boström, 2017)

1. Body shorter than 0.7 mm; tail conical...............2

– Body longer than 0.8 mm; tail cylindrical, conicocylindrical or elongated conical.................................5

2. Cuticularised plate present; annulation starts at the posterior one fourth of amphid..............................3

– Cuticularised plate absent; annulation starts at the anterior one fourth of amphid..............................4

3. Spicules 22-μm long; gubernaculum triangular............................................D. ornatus (Gerlach, 1950)

– Spicules 29–36-μm long; gubernaculum platelike.........................................D. linkei (Jensen, 1991)

4. Spicules 19–27-μm long; gubernaculum platelike without apophysis............................D. pumilus (Vincx & Gourbault, 1992)

– Spicules 41–46-μm long; gubernaculum triangular with apophysis…D. conoicaudatus sp. nov.

5. Cuticlar annules with londitudinal striation distinctly visible under the light microscope ……....6

– Cuticular annules visually smooth under light microscope, longitudinal striation not visible…........10

6. Cuticularised plate present...............………......7

– Cuticularised plate absent.............................….8

7. Amphid hook-shaped.................................................................D. axayacatli (Holovachov et al., 2009)

– Amphid an inverted U-shaped.........…D. grandis (Holovachov & Sven Boström, 2017)

8. Interamphideal shield crenate.......D. santaclarae (Holovachov et al., 2009)

– Interamphideal shield smooth............................9

9. Body 1.7-mm long; spicules 17-μm long.....................................................D. striatus (Gerlach, 1956)

– Body 1.0–1.2-mm long; spicules 25-μm long..............................D. pulcher (Vincx & Gourbault, 1992)

10. Amphidial fovea with fine transverse striation...................................................................................11

– Amphidial fovea without fine transverse striation.................................................................................18

11. Body 1.12–1.58-mm long; amphid stubby................................................D. bulbosus (Vitiello, 1972)

– Body 0.72–0.89-mm long; amphid slender…… …………………………….D. longifoveatus sp. nov.

12. Cuticular plate present..................................13

– Cuticular plate absent......................................14

13. Cephalic setae 4–6-μm long; amphid 14–21-μm long; precloacal ridge absent...................................... D. suecicus (Holovachov & Sven Boström, 2017)

– Cephalic setae 13-μm long; amphid 6-μm long; precloacal ridge present…............. D. longicaudatus (Holovachov & Sven Boström, 2017)

14. Amphid with wide interamphideal area.........15

– Amphid with narrow interamphideal area.........16

15. Interamphideal area smooth, without ornamentation.........................................................16

– Interamphideal area punctated, areolated or crenated..................................................................17

16. Cephalic setae 17-μm long...........................................................................D. botulus (Wieser, 1959)

– Cephalic setae 5-μm long.............................................................................D. sundensis (Jensen, 1978)

17. Cephalic setae 12–18-μm long; amphid 21–26-μm long......................................................D. paramastigia (Holovachov et al., 2009)

– Cephalic setae 6.5–8.5-μm long; amphid 40–45-μm long............................D. mastigia (Tchesunov, 1990)

18. Cephalic setae longer than 10 μm......................................D. lucanicus (Boucher & Helléouët, 1977)

– Cephalic setae shorter than 10 μm...................19

19. Cephalic setae 3.5–7-μm long.....................................................................D. asetosus (Juario, 1974)

– Cephalic setae less than 1–2-μm long.............20

20. Spicules with dorsally bent manubrium; amphid 17–28-μm long....... D. nudus (Gerlach, 1956)

– Spicules with straight manubrium; amphid 26– 33-μm long..................…D. anatolii (Voronov, 1982)

3.3 Minolaimus multisupplementatus sp. nov. (Figs. 57)
Fig.5 Drawing of M. multisupplementatus sp. nov. a. anterior end of male, showing amphid, and lateral differentiation; b. pharyngeal region of male; c. lateral view of male posterior end; d. lateral view of male cloacal region, showing spicule, gubernaculum, and precloacal supplements.
Fig.6 Microscopic images of M. multisupplementatus sp. nov. a. lateral view of male anterior end, showing cephalic setae; b. sublateral view of male anterior end, showing amphidial fovea and lateral differentiation; c. lateral view of male posterior end, showing precloacal supplements; d. lateral view of male cloacal region, showing spicule and gubernaculum (scale bars: a, b: 10 μm; c, d: 30 μm).
Fig.7 Microscopic images of M. multisupplementatus sp. nov. a. lateral view of male anterior end, showing cephalic setae and amphid; b. lateral view of male posterior region, showing lateral differentiation of cuticle with three rows of larger dots (scale bars: a: 10 μm; b: 30 μm).

Order Araeolaimida De Coninck & Schuurmans Stekhoven, 1933

Family Comesomatidae Filipjev, 1918

Genus Minolaimus Vitiello, 1970

3.3.1 Type material

Only one male was obtained and measured. Holotype male on slide DH4-6-1-7.

3.3.2 Type locality and habitat

Undersea muddy sediment at Station DH4-6 in the East China Sea (28°25′0″N, 125°2′24″E, water depth 101 m).

3.3.3 Etymology

The species name refers to its male having many precloacal supplements.

3.3.4 Measurement

All measurement data were given in Table 2.

Table 2 Individual measurements of M. apicalis sp. nov. and M. multisupplementatus sp. nov. (in μm except a, b, c, c′, number and V %)
3.3.5 Description

Male. Body spindle-shaped with tapered anterior end and long filiform tail. Cuticle marked by transverse rows of punctuations, with lateral differentiation consisting of three longitudinal rows of larger dots from posterior of amphideal fovea to tail conical portion. Width of lateral differentiation 5–6 μm. Cuticular pores not observed. Head tapered or slightly rounded. Buccal cavity minute, without tooth. Six inner labial sensilla papilliform; six outer labial sensilla setiform, 3-μm long; four cephalic setae 4-μm long. Spiral amphideal fovea looking like oval-shaped, with seven turns, 90% of corresponding body diameter in width. Pharynx cylindrical, not enlarged at its base. Cardia conical. Nerve ring surrounding the pharynx at about 37% of its length from the anterior end. The excretory pore not observed. Tail 15 times as long as body diameter at cloaca, conical with distal three quarters filiform portion, without terminal seta. Terminal spinneret present.

Reproductive system diorchic, testes opposed, outstretched. Spicules 1.5 times as long as body diameter at cloaca, arcuate with central lamella; gubernaculum arcuate and parallel to spicule, without dorso-caudal apophysis; a small papilliform and 29 cup-shaped cuticularized precloacal supplements. Posterior ones more closely spaced and gradually increasing in distance apart anteriorly. Posteriormost one and anteriormost one 35 μm and 520 μm in front of cloaca, respectively.

Female not found.

3.3.6 Differential diagnosis and discussion

Only one male specimens is available for the identification. Generally, this is insufficient for establishment of a new nematode species. Nevertheless, the characters of the male specimens match with the diagnostic features of the genus Minolaimus. Minolaimus multisupplementatus sp. nov. is characterized by cuticle with lateral differentiation of three longitudinal rows of large dots; spiral amphideal fovea with seven turns; pharynx cylindrical, not enlarged at base; spicules arcuate with central lamella; gubernaculum arcuate without apophysis; a papilliform and 29 cup-shaped precloacal supplement; tail conical with long flagelliform portion. The new species differs from Minolaimus lineatus Vitiello, 1970 by larger body size (3 305 μm vs 1 752–1 833 μm), much larger number of precloacal supplements (30 vs 20), different shape of precloacal supplements and absence of pharyngeal posterior bulb. In the latter species, pharynx having a long posterior bulb (vs absence of bulb), spicules 38 μm, i.e. 1.3 anal body diameter long (vs 47.5 μm, ie. 1.5 anal body diameter). The new species differs from Minolaimus cervoides Vitiello, 1970 by much larger body size (3 305 μm vs 854–943 μm) and proximal position of amphideal fovea. The latter species was described from females only. Its tail relatively short, 9.6–10 anal body diameter long (vs 15 anal body diameter long in the new species).

3.4 Minolaimus apicalis sp. nov. (Figs. 810)
Fig.8 Drawing of M. apicalis sp. nov. a. view of entire male; b. lateral view of male cloacal region; c. view of entire female; d. lateral view of male pharyngeal region; e. anterior end of male.
Fig.9 Microscopic images of M. apicalis sp. nov. a. lateral view of male anterior end, showing buccal cavity and terminal bulb; b. lateral view of male anterior end, showing cuticle dots and lateral pores; c. lateral view of male tail end; d. cloacal region of male, showing lateral differentiations; e. spicule, gubernacular apophysis and precloacal supplements; f. cloacal region of male, showing gubernacular apophysis, precloacal seta and precloacal supplements (scale bars: a, c: 20 μm; b, d, e, f: 10 μm).
Fig.10 Microscopic images of M. apicalis sp. nov. a. lateral view of female anterior end, showing amphid and lateral differentiations; b. lateral view of entire female; c. vulva region of female, showing vulva, spematheca (anterior arrow) and egg (posterior arrow) (scale bars: a: 10 μm; b: 30 μm; c: 20 μm).
3.4.1 Type material

One male and one female were obtained and measured. Holotype male on slide DH 5-3-2-3, paratype female on slide qzhx 17-1-0-2-2.

3.4.2 Type locality and habitat

Holotype male in the seafloor muddy sediment at Station DH 5-3 in the East China Sea (28°2′24″N, 122°49′36″E, water depth 74 m). Paratype female in the seafloor muddy sediment at Station NHqzhx1 of the Qiongzhou Strait in the South China Sea (20°3′53″N, 110°21′5″E; water depth 13 m).

3.4.3 Etymology

The species name is derived from the Latin word apicalis, refers to the position of amphids close to the head apex.

3.4.4 Measurement

All measurement data were given in Table 2.

3.4.5 Description

Male. Body slender and small. Cuticle marked by transverse rows of larger dots at cervical region and smaller dots at the rest of body. Lateral differentiation consisting of three longitudinal rows of larger dots and a longitudinal row of pores from the middle of pharynx to the conical portion of tail. Head tapered or slightly rounded. Buccal cavity small, cup-shaped, with a slightly cuticularized projection. Inner labial sensilla not observed. Short six outer labial setae and four cephalic setae almost equal in length (2-μm long), and they situated in one circle. Spiral amphidial fovea with four turns, close to the head apex. Pharynx cylindrical with an ovoid posterior bulb. Cardia not discernible Nerve ring surrounding the pharynx at mid-length from the anterior end. The excretory pore not observed. Tail conico-cylindrical with a slightly enlarged tip, 6.5 times as long as body diameter at cloaca. Short caudal setae scattered in conical portion. Terminal spinneret present.

Reproductive system diorchic, testes outstretched. Anterior testis 235 μm from head end. Posterior testis 298 μm from head end. Spicules 1.8 cloacal body diameter long, slender and arcuate, proximal end bent dorsally. Gubernaculum small with a broad curved dorsal apophysis. Two sucked-like and six cup-shaped cuticularized precloacal supplements. The cuticle strongly annulated between the supplements, forming a ring-shaped wall around each, apparently with cuticle ridges between them. Posteriormost one and anteriormost one 15-μm and 107-μm in front of cloaca, respectively. A precloacal seta present at halfway between cloaca and posteriormost supplement, 3-μm long.

Female. Similar to male in most morphological characters except with slightly longer tail. Reproductive system didelphic, two opposed and reflexed ovaries. Anterior ovary located to the right of intestine, posterior ovary to the left of intestine. Two oval, sac-like spermathecae located on each side of each (anterior and posterior) gonoduct. Spermathecae filled with oval spermatozoa. Vagina wide and straight, cuticularized, 0.25 times as long as corresponding body diameters. Vulva located at anterior portion of the body (i.e. at 36.5% of the body length from the anterior end).

3.4.6 Differential diagnosis and discussion

Minolaimus apicalis sp. nov. is characterized by cuticle with lateral differentiation consisting of three longitudinal rows of larger dots and a longitudinal row of pores. Spiral amphidial fovea with four turns and close to anterior end. Pharynx cylindrical with an oval posterior bulb. Spicules slender and arcuate, proximal end bent dorsally. Gubernaculum with curved dorsal apophysis. Two sucked-like and six cup-shaped cuticularized precloacal supplements. Tail conicocylindrical with a slightly swollen tip. The new species differs from other three species in the genus by having relatively short tail, amphideal fovea near the head top, cuticle with two lateral longitudinal rows of pores in the pharyngeal region and precloacal region, precloacal supplements containing two sucked-like ones and six cup-shaped ones.

To date, only two species in genus Minolaimus, M. lineatus Vitiello, 1970 and M. cervoides Vitiello, 1970, both inhabiting muddy bottoms near Marseilles (Gulf of Lion) of France have been recorded around the world (Bezerra et al., 2020). As yet, the male of Minolaimus cervoides Vitiello, 1970 has not been recorded. The comparative table between these two species and the two new species is given in Table 3.

Table 3 Main diagnostic characters of four species within the genus Minolaimus

Position of Minolaimus in the nematode system is not firmly established. Vitiello (1970) initially assigned Minolaimus to the family Cyatholaimidae that was later accepted by Lorenzen(1981, 1994) without comments. Hope and Zhang (1995) transferred Minolaimus from Cyatholaimidae to Comesomatidae based on the pattern of the cuticle, amphid, anterior sensilla, buccal cavity, and the posterior end of pharynx. Hence in the Handbook of Zoology (2014), Minolaimus is present simultaneously as member of two different families and orders, in two chapters written by Fonseca & Bezerra (Araeolaimida) and Tchesunov (Chromadorida). On our opinion, Minolaimus more inclines to Comesomatidae. However, cuticular pores might be present, females have two reflexed ovaries, rather than outstretched ovaries like other species in the Comesomatidae.

The identification key to species of Minolaimus

1. Tail short, not filiform; amphid close to head apex………………..…...........…..M. apicalis sp. nov.

–Tail long, filiform; amphid poster to head apex….….……………………………………..…...…..…..2

2. Body longer than 3 000 μm, with 29 cup-shaped precloacal supplements…...............................………..................................M. multisupplementatus sp. nov.

–Body shorter than 1 900 μm …………………….3

3. Body 1 752–1 833-μm long; tail with 1/5 conical portion; male with 20 papilliform precloacal supplements ……………M. lineatus (Vitiello, 1970)

–Body 854–943-μm long; tail with 1/3 conical portion…………....……M. cervoides (Vitiello, 1970)

4 DATA AVAILABILITY STATEMENT

The authors declare that the data supporting the findings of this study are available within the article. The data will be available on request from the corresponding author.

ACKNOWLEDGMENT: The authors are very thankful to all crew members of R/V Kexue 3 for their kind help in sample collection. We are sincerely grateful to the anonymous reviewers for providing valuable criticisms and improving the manuscript.
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