﻿Two new species of the genus Halichoanolaimus (Nematoda, Selachinematidae) from the intertidal zone of the Yellow Sea, China

﻿Abstract Two new marine nematode species belonging to the genus Halichoanolaimus from the intertidal zone of the Yellow Sea are described. Halichoanolaimussinensissp. nov. is characterized by amphideal fovea with 2.5–3.0 turns, 20–27% of corresponding body diameters; spicules curved, middle portion broad, tapering distally, 1.4–1.5 cloacal body diameters long; gubernaculum slender consisting of two detached lateral pieces tapering distally; 10–13 papilliform precloacal supplements in two groups, the posterior three supplements smaller and closer to each other, the remaining supplements larger and widely spaced; tail conico-cylindrical with a half cylindrical portion. The second new species, Halichoanolaimuszhangisp. nov. is distinct by having lateral differentiation present except in anterior half of pharynx which has even punctations, amphideal fovea with 3.0–3.3 turns, spicules curved, gradually narrowing from proximal to distal end with pointed tip, 7 papilliform precloacal supplements, gradually increasing the spacing distance forward, two rows of subventral conical setae situated at the precloacal region, tail elongated, filiform. An updated key to 30 valid species of Halichoanolaimus is provided.


Introduction
The Yellow Sea is located on the edge of the western Pacific Ocean, between the Chinese mainland and the Korean Peninsula.It is a semi-enclosed inland shallow sea basin.Biodiversity surveys and taxonomical studies on nematodes in the Yellow Sea have been carried out in recent years.More than 350 species of nematodes have been identified, of which 105 species were new to science (Hao et al. 2022;Chu et al. 2023).The new species accounted for 30% of the known species.However, the total number of nematodes in this sea area is unknown, and new species are routinely found.It is, therefore, important to continue investigating the taxonomy of nematodes in the region.
The genus Halichoanolaimus was established by de Man (1886) with the type species of H. robustus (Bastian, 1865).It is a common and diverse genus of predatory nematodes belonging to the family Selachinematidae and found from shallow seas to the abyssal plain (Miljutin et al. 2010;Leduc 2020) The most recent species descriptions were provided by Xiao and Guo (2023).Based on the reviews by Tchesunov (2014), Leduc and Zhao (2016), Leduc (2020) and Huang and Guo (2022), Halichoanolaimus is characterized by a cuticle with lateral differentiation in the form of larger and more widely spaced punctations; all anterior sensilla papilliform; a buccal cavity consisting of two parts separated by a row of teeth; pharynx without bulb; precloacal supplements usually papilliform or setiform and tail conico-cylindrical, elongated with a distal filiform portion, or conical.Within Halichoanolaimus, the species are distinguished by rather few main characters: the number of amphidial turns, number and arrangement of precloacal supplements, structure of spicules and gubernaculum, and shape and length of the tail.A key to the identification of 22 valid species for the genus was given by Zograf et al. (2015).Subsequently, six species, H. anisospermus Leduc & Zhao, 2016, H. stagnalis Gagarin & Long, 2017, H. funestus Leduc, 2020, H. ossilagulus Leduc, 2020, H. pumilus Leduc, 2020and H. sicaoensis Xiao & Guo, 2023, were described.To date, 28 valid species within Halichoanolaimus have been recorded worldwide.

Materials and methods
In order to investigate the diversity of free-living nematodes along the coast of the Yellow Sea, China, sediment samples were collected in several intertidal sites in 2008 and 2022 respectively.The meiofauna samples were obtained from the top sediment layer (0-8 cm deep) using a 2.9 cm diameter sawn-off syringe.The samples were fixed with an equal amount of 10% formalin solution.
In the laboratory, samples were stained with 0.1% Rose Bengal for more than 12 hours (Higgins and Thiel 1988).The stained samples were poured into two layers of sieves (500 and 30 µm mesh sizes) respectively, and washed with tap water to remove silt and separate macrofauna from meiofauna.The heavier sediment particles retained on the 30-micrometer mesh were removed using centrifugation with Ludox-TM colloidal silica (50% colloidal silica suspension in water; Sigma Aldrich Co., USA) with a specific gravity of 1.15 g/ml (de Jonge and Bouwman 1977).Each sample was washed into a Petri dish with distilled water and meiofauna was sorted under a stereoscopic microscope (Olympus SZ 51).Nematodes were transferred into a 9:1 (v/v) solution of 50% alcohol-glycerol in an embryo dish to slowly evaporate to pure glycerol, and then mounted into permanent slides (McIntyre and Warwick 1984).
Finally, the specimens were mounted in glycerin on permanent slides.Observation and measurement were carried out using a differential interference contrast microscope (Leica DM 2500) and Leica software of LAS X version 3.3.3.Line drawings were made with the aid of a camera lucida.Type specimens were deposited in the Marine Biological Museum of the Chinese Academy of Sciences, Qingdao.
Abbreviations are as follows: a, the ratio of body length to maximum body diameter; abd, body diameter at cloaca or anus; b, ratio of body length to pharynx length; c, ratio of body length to tail length; cbd, corresponding body diameter; c′, ratio of tail length to cloacal or anus body diameter; V%, position of vulva from anterior end expressed as a percentage of total body length.
Type locality and habitat.Holotype and all additional specimens were found from intertidal silt sediment at Rizhao coast of the Yellow Sea; 35°26'N, 119°34'E; 0-2 cm and 2-5 cm sediment depth.
Etymology.The specific epithet refers to the country origin, China.
Measurements.All measurement data are given in Table 1.Description.Males.Body cylindrical, tapering slightly towards posterior end.Cuticle with transverse rows of punctations, lateral differentiation consisting of slightly larger and more widely spaced punctations.Cuticle pore not observed.Somatic setae short, 3 µm long, sparsely distributed.Lip region slightly rounded.Six inner labial sensilla papillose; six outer labial sensilla setiform, 3-4 µm long, at same level as four papilliform cephalic sensilla (Fig. 2C).Amphideal fovea multispiral with 3 turns (Fig. 2B), 25-27% of corresponding body diameter in width, located at the level of the middle of buccal cavity, ca 20 µm from anterior end of body.Buccal cavity large, ca 45 µm deep, divided into anterior and posterior portions by two rows of 15-17 denticles.Anterior portion of buccal cavity cup-shaped, with three sets of three cuticularized rhabdions; posterior portion of buccal cavity narrower, cylindrical, surrounded by three Y-shaped pairs of cuticularized rhabdions with swollen bases, 20 µm long.Pharynx cylindrical, anterior end swelling, wrapped the buccal cavity, without posterior bulb.Pharyngeal lumen cuticularized.Nerve ring at ca 44% of pharynx length from anterior end.Secretory-excretory system present.Renette cell small, situated at level of cardia; ampulla large, excretory pore situated slightly posterior to the nerve ring, ca 180 µm from the anterior end.Cardia small, surrounded by intestine.
Reproductive system diorchic with two opposed, outstretched testes.Anterior testis to the right or ventrally to intestine, posterior testis to the left side of intestine.Spicules paired, curved, middle portion broad, tapering distally, 1.4-1.5 cloacal body diameters long, interior of spicules granular in appearance.Gubernaculum slender, consisting of two detached lateral pieces tapering distally, adjoining the dorsal side of spicules.10-13 papilliform precloacal supplements in two groups, the posterior three supplements smaller and closer to each other (Fig. 2E), located 7 µm from the cloaca; 5 µm from each other; remaining supplements larger and widely spaced, ca 25 µm from each other.Each supplement consisting of conical papilla and an internal duct.Tail conico-cylindrical with posterior cylindrical portion comprising about half of total tail length.Caudal setae absent.Three caudal glands located posterior to spicules, spinneret present, 7 µm long.
Females.Similar to males, but with slightly larger body and slightly smaller amphideal fovea (20% of corresponding body diameter in width and with 2.5 turns).Reproductive system didelphic, with two opposed, reflexed ovaries.Anterior ovary to the left of intestine and posterior ovary to the right of intestine.Vulva situated slightly pre-median.Intestine blind, anus not observed.half cylindrical portion.The new species is most similar to H. sonorus Belogurov & Fadeeva, 1980 in body shape and number of precloacal supplements, but differs from the latter species by numbers of amphid turns (2.5-3 vs 4-4.2), different shape and structure of spicules (spicules without capitulum, distal hook and central spacer vs with weak capitulum, distal hook and central spacer).The new species is also similar to H. stagnalis Gagarin & Long, 2017 in the number of the amphidial turns and tail, but can easily be distinguished from the latter by the different arrangement of precloacal supplements (3 posterior supplements smaller and closer vs 5-6 smaller and closer supplements Halichoanolaimus zhangi sp.nov.https://zoobank.org/CDC5A1AD-A09C-4716-A0DB-5D5336EE0B10Figs 3-5, Table 2 Material examined.Two males and one juvenile were obtained.Holotype: ♂1 on slide 22HSB-11-2-1; paratypes: ♂2 and juvenile on slide 22HSB-11-2-2.Type specimens were deposited in the Marine Biological Museum of the Chinese Academy of Sciences, Qingdao.
Type locality and habitat.Holotype and paratypes were found from intertidal muddy sediment at Rizhao coast along the Yellow Sea; 35°18'N, 119°31'E; 0-2 cm sediment depth.Etymology.The specific epithet "zhangi" is in honor of Professor Zhinan Zhang, a Chinese nematologist, in recognition of his contributions to nematode taxonomy.
Measurements.All measurement data are given in Table 2. Description.Males.Body cylindrical, tapering slightly towards posterior end.Cuticle with transverse rows of punctations.Lateral differentiation presents except to anterior half of pharynx with even punctations.Lateral differentiation consisting of slightly larger and more widely spaced punctations.Four longitudinal rows of pore complexes situated at sublateral sides of pharyngeal region (Fig. 4B).Each row with 10-12 pores.Cephalic region slightly rounded.Inner and outer labial sensilla papillose; four cephalic sensilla setiform, 4-5 µm long.Amphideal fovea multispiral with 3-3.25 turns, located at the level of the buccal cavity base, 27-28 µm from anterior end of body.Buccal cavity large, 43-45 µm deep, divided into anterior and posterior portions by two rows of 25 denticles.Anterior portion of buccal cavity cup-shaped, with three sets of three  cuticularized rhabdions, terminating in three sets of paired denticles; posterior portion of buccal cavity narrower, cylindrical, surrounded by three Y-shaped pairs of cuticularized rhabdions with swollen bases, ca 20 µm long.Pharynx cylindrical, muscular, without anterior or posterior bulb; pharyngeal lumen cuticularized.Nerve ring difficult to distinguish.Secretory-excretory system present.
Ventral gland small, situated at level of cardia; ampulla large, excretory pore situated at position of three corresponding body diameters from the anterior end.Cardia small, partially surrounded by intestine.
Reproductive system diorchic with two opposed, outstretched testes.Anterior testis to the right or ventrally to intestine, posterior testis to the left side of intestine.Spicules paired, curved, gradually narrowing from proximal to distal end with pointed tip, ca 2 cloacal body diameters long.Gubernaculum rod-like, adjoining to the dorsal side of spicules.7 papilliform precloacal supplements, the most posterior supplement located 14 µm from the cloaca; remaining supplements gradually increasing the spacing distance forward, from 9 µm to 26 µm to each other.Each supplement consists of conical papilla and an internal duct.Two rows of short conical setae situated at two subventral sides of the precloacal supplements region of body.Tail conical with a long posterior filiform portion, accounts for 92% total tail length.A row of 5 caudal setae distributed at ventral side of tail conical part, 3-4 µm long.Caudal glands and spinneret present, 7 µm long.
Female not found.Juvenile similar to males in body shape, and with a long filiform tail, except body size smaller, intestine with blind end.
Differential diagnosis and discussion.Halichoanolaimus zhangi sp.nov. is distinct by lateral differentiation presenting posterior to the middle of the pharynx, amphideal fovea with 3.0-3.3turns, spicules curved, gradually narrowing from proximal to distal end with pointed tip, 7 papilliform precloacal supplements, gradually increasing the spacing distance forward, two rows of subventral stout setae situated at precloacal region, tail elongated, filiform.The new Differential diagnosis and discussion.Halichoanolaimus sinensis sp.nov. is characterized by amphideal fovea with 2.5-3.0 turns, 20-27% of corresponding body diameter; spicules curved, middle portion broad, tapering distally, 1.4-1.5 cloacal body diameters long; gubernaculum slender consisting of two detached lateral pieces tapering distally; 10-13 papilliform precloacal supplements in two groups, the posterior three supplements smaller and closer to each other, remaining supplements larger and widely spaced; tail conico-cylindrical with

Figure 1 .
Figure 1.Drawings of Halichoanolaimus sinensis sp.nov.A pharyngeal region of male B posterior end of male C entire body of female D entire body of male E anterior end of male.Scale bars: 30 μm (A, B, E); 100 μm (C, D).

Figure 3 .
Figure 3. Drawings of Halichoanolaimus zhangi sp.nov.A pharyngeal region of holotype B anterior end of holotype C cloacal region of holotype D posterior end of male 2. Scale bars: 30 μm (A, B, C); 50 μm (D).

Figure 5 .
Figure 5. Microscopic images of Halichoanolaimus zhangi sp.nov.A anterior end of holotype, showing lateral differentiation and somatic setae (arrow) B cloacal region of holotype, showing lateral differentiation and conical setae situated at two subventral sides of precloacal supplements (arrow) C anterior end of juvenile, showing buccal cavity and cephalic sensilla (arrow) D posterior portion of juvenile, showing the end of intestine (arrow).Scale bars: 20 μm (A, B, C, D).