Introduction
To study the diversity and taxonomy of free-living marine nematodes in Yangma Island, undisturbed sediments were acquired along the coast of the Yellow Sea in July, 2019. Marine nematodes are the dominant meiofauna group.
The genus Microlaimus was erected by de Man in Reference de Man1880 with the type species of M. globiceps de Man, Reference de Man1880 and was the most diverse group in the family Microlaimidae Micoletzky, Reference Micoletzky1922. Gerlach (Reference Gerlach1950) first reviewed the genus, described three new species, redescribed 16 reported species, provided two new names, and subdivided the valid species into six groups mainly based on buccal cavity structure, cuticle, and tail shape. Wieser (Reference Wieser1954) described five new species of Microlaimus and erected the genus Paramicrolaimus Wieser, Reference Wieser1954 in Microlaimidae. Jensen (Reference Jensen1978) reviewed Microlaimidae with more differentiating characters, erected Molgolaimidae Jensen, Reference Jensen1978 to accommodate species with reflexed ovaries and one testis from Microlaimus and transferred eight Microlaimus species to Molgolaimus Ditlevsen, Reference Ditlevsen1921 and regarded M. labradorensis Allgén, Reference Allgén1957, M. tenuicaudatus Allgén, Reference Allgén and Odhner1959 and M. tenuilaimus Allgén, Reference Allgén1932 as species inquirendae for poor description, erected the genus Aponema Jensen, Reference Jensen1978 with type species of A. torosum (Lorenzen, Reference Lorenzen1973) Jensen, Reference Jensen1978 transferred from Microlaimus, four species to Bolbolaimus Cobb, Reference Cobb1920 based on buccal cavity and pharynx structure, three species from Paramicrolaimus. Taxonomic relationships of Microlaimus with the genus Molgolaimus (ovaries reflexed vs ovaries outstretched in Microlaimus) and other genera in Microlaimidae have been deeply discussed. Lorenzen (Reference Lorenzen1981, Reference Lorenzen1994) discussed the phylogenetic position of Microlaimidae and considered Jensen's taxonomic characters variable, downgraded Molgolaimidae as Molgolaiminae Jensen, Reference Jensen1978 in Desmodoridae Filipjev, Reference Filipjev1922 and erected the family Paramicrolaimidae Lorenzen, Reference Lorenzen1981 to accommodate Paramicrolaimus. Kovalyev and Tchesunov (Reference Kovalyev and Tchesunov2005) discussed taxonomic problems of Microlaimus with Molgolaimus, Aponema, and Pseudomicrolaimus Sergeeva, Reference Sergeeva1976 based on characters of ovary structure, tail shape, stoma and copulatory apparatus, described one new species of M. paraconothelis Kovalyev and Tchesunov, Reference Kovalyev and Tchesunov2005, and placed Microlaimus citrus as incertae sedis. Tchesunov (Reference Tchesunov and Schmidt-Rhaesa2014) transferred M. bathyalis (Kovalyev and Miljutina, Reference Kovalyev and Miljutina2009) Tchesunov, Reference Tchesunov and Schmidt-Rhaesa2014, M. martinezi (Miljutin and Miljutina, Reference Miljutin and Miljutina2009) Tchesunov, Reference Tchesunov and Schmidt-Rhaesa2014, M. minutissimus (Kovalyev and Miljutina, Reference Kovalyev and Miljutina2009) Tchesunov, Reference Tchesunov and Schmidt-Rhaesa2014, M. nanus Blome, Reference Blome1982, M. nympha (Bussau and Vopel, Reference Bussau and Vopel1999) Tchesunov, Reference Tchesunov and Schmidt-Rhaesa2014, M. westindicus (Kovalyev and Miljutina, Reference Kovalyev and Miljutina2009) Tchesunov, Reference Tchesunov and Schmidt-Rhaesa2014 from Aponema with absence of gubernaculum apophysis. Leduc (Reference Leduc2016) discussed the differentiation between Microlaimus and Bolbolaimus with the character of a constriction setting off the head from the rest of the body, resurrected M. wieseri (Hopper, Reference Hopper1961) from Bolbolaimus by the presence of set off head, described one new species of M. korari Leduc, Reference Leduc2016, and listed 83 valid species. After Leduc (Reference Leduc2016), M. pecticauda Murphy, Reference Murphy1966 and M. spirifer Warwick, Reference Warwick1970 were transferred to Molgolaimus (Shi and Xu, Reference Shi and Xu2017), by now 87 valid species have been listed in Nemys (Bezerra et al., Reference Bezerra, Decraemer, Eisendle-Flöckner, Hodda, Holovachov, Leduc, Miljutin, Mokievsky, Peña Santiago, Sharma, Smol, Tchesunov, Venekey, Zhao and Vanreusel2023).
The genus Pseudomicrolaimus was first erected by Sergeeva in 1976 with the description of P. murinae Sergeeva, Reference Sergeeva1976 with the character of wide and funnel-shaped buccal cavity with numerous denticles on the subventral side, dorsal tooth much larger than others. Jensen (Reference Jensen1978) regarded Pseudomicrolaimus as a junior synonym of Bolbolaimus for most characters of these two genera agreed well. Genus Pseudomicrolaimus was reconsidered as a valid genus by Kovalyev and Tchesunov (Reference Kovalyev and Tchesunov2005) with presence of numerous denticles on the left subventral side of stoma. By now, three species, P. murinae, P. dentatus (Allgén, Reference Allgén1935) Sergeeva, Reference Sergeeva1976 and P. denticulatus (Gerlach, Reference Gerlach1953) Kovalyev and Tchesunov, Reference Kovalyev and Tchesunov2005 are considered valid.
Materials and methods
In July 2019, undisturbed sediment samples were collected using a syringe (2.6 cm internal diameter) to a depth of 8 cm at the intertidal mud flats of Yangma Island and were vertically subdivided into 0–2 and 2–8 cm depth and fixed with 10% formalin in seawater. In laboratory, samples were stained with rose Bengal for more than 24 h and washed with tap water. Meiofauna was separated from heavier sediment particles by centrifugation in Ludox-TM (Sigma-Aldrich Co., USA) (de Jonge and Bouwman, Reference de Jonge and Bouwman1977), transferred into grid-lined Petri dish and sorted under stereoscopic microscope. Nematodes were transferred into mixture of ethanol (50%) and glycerine (1:9 in volume ratio) with ethanol slowly evaporated, and mounted in glycerine on permanent slides. Descriptions were made using a differential interference contrast microscope (Axiscope-5, Zeiss, Germany). Line drawings were made through an iPad (Apple, USA) and photos were taken with the aid of ZEN software (provided by Zeiss corporation). Type specimens were deposited in the Institute of Oceanology, Chinese Academy of Sciences, Qingdao.
Abbreviations are as follows: a, body length/maximum body diameter; b, body length/pharynx length; c, body length/tail length; c’, tail length/anal body diameter; V%, position of vulva from anterior body end expressed as a percentage of total body length.
Description of new species
Order MICROLAIMIDA Leduc, Verdon and Zhao, Reference Leduc, Verdon and Zhao2017
Superfamily MICROLAIMOIDEA Micoletzky, Reference Micoletzky1922
Family MICROLAIMIDAE Micoletzky, Reference Micoletzky1922
Genus Microlaimus de Man, Reference de Man1880
Diagnosis
Cuticle annulated, in some species also showing punctations or longitudinal bars. Head slightly set off. Spicules usually short and arcuate, seldom long and slender; gubernaculum present, often bent distally but without dorso-caudal apophyses. Papilloid precloacal supplements may be present (based on Leduc, Reference Leduc2016).
Type species
Microlaimus globiceps de Man, Reference de Man1880
Microlaimus paraaffinis sp. nov.
(Figures 1–2, Table 1)
Type material
Four males and three females were measured and studied. Holotype: m#1 on slide 19YMD2-2-65; paratypes: m#2, m#3, m#4 on slides 19YMD2-2-65,19YMD2-2-26, 19YMD2-4-5; f#1 f#2, f#3 on slides 19YMD2-2-46.
Figure 1. Microlaimus paraaffinis sp. nov. (A) Lateral view of male anterior portion, showing buccal cavity and pharynx region (holotype); (B) Lateral view of male anterior end, showing amphids (holotype); (C) Lateral view of female anterior end (19YMD2-2-46); (D) Lateral view of male posterior portion (holotype); (E) Lateral view of spicules and gubernaculum (holotype); (F) Lateral view of female posterior portion (19YMD2-2-46); (G) Lateral view of female body, showing female reproductive structure (19YMD2-2-46). Scale bars: A, B, C = 20 μm; D, F = 30 μm; G = 50 μm.
Figure 2. Microlaimus paraaffinis sp. nov. (A) Lateral view of male body, showing buccal cavity (holotype); (B) Lateral view of male anterior end, showing amphidial fovea (holotype); (C) Lateral view of male posterior region, showing spicules (holotype); (D) Lateral view of male posterior body, showing gubernaculum (holotype); (E) Lateral view of male posterior end, showing male tail (holotype). Scale bars: 20 μm.
Table 1. Individual measurements of Microlaimus paraaffinis sp. nov. (in μm except for ratios)
Type locality and habitat
Specimens were collected from intertidal muddy sediment at Yangma Island, Shandong Province. 37°26´N, 121°36΄E.
Etymology
The species name refers to similarity with species M. affinis.
Measurements
All measurement data are given in Table 1.
Description
Males. Body spindle shaped, 714–785 μm in length. Anterior body end slightly truncated, posterior body end elongated conical. Cuticle striated without dots or punctuations. Cuticle striation from posterior to cephalic setae to tail tip. Head smooth and slightly set off at the cephalic setae region. Six inner labial sensilla and six outer labial sensilla papilliform. Four cephalic setae short, 4 μm in length. Somatic setae absent. Amphidial fovea cryptocircular, 4–6 μm in diameter (33–42% corresponding body diameter), 9–11 μm from anterior body end. Buccal cavity wide, 3 μm in width and 13–16 μm in depth with a large dorsal tooth and two small ventrosublateral teeth, right ventrosublateral tooth situated approximately 3–4 μm anteriorly relative to left one, small longitudinal cuticularized folds present in cheilostoma. Pharynx muscular, 126–140 μm in length, anterior part slightly widened at the buccal cavity region, posterior part widened into bulb (24–32 μm in length). Nerve ring situated 73–80 μm from anterior end. Ventral gland immediately posterior to pharyngeal bulb and excretory pore situated just anterior to nerve ring. Cardia not observed. Tail conico-cylindrical, 95–108 μm (3.7–3.9 cloacal body diameter) in length, with cylindrical portion short (18–19% of tail length). Three caudal glands present and spinneret absent.
Testes outstretched and opposed, anterior and posterior testis to the right of intestine. Spicules paired and L-shaped, slender with equal thickness and distal end tapered, 31–35 μm in length (1.1–1.3 cloacal body diameter). Gubernaculum simple, boat-shaped, 19–21 μm in length and parallel to distal end of spicules. Precloacal supplements absent.
Females. Similar to males in most characters. Reproductive system didelphic-amphidelphic with two ovaries outstretched. Anterior ovary to the left of the intestine (112–122 μm in length), posterior ovary to the right (120–124 μm in length). Vulva located slightly posterior to the mid-body, 393–431 μm from the anterior end. Vagina short and sclerotized.
Differentiation diagnosis and discussion
The new species presents as main characteristics: cuticle striated, four short cephalic setae, buccal cavity with one large dorsal tooth and two small ventrosublateral teeth, amphidial fovea cryptocircular at level of posterior buccal cavity, somatic setae absent, spicules L-shaped and 31–35 μm in length, gubernaculum simple and boat-shaped, precloacal supplements absent, tail conico-cylindrical with cylindrical portion short.
M. paraaffinis sp. nov. similar to M. arenicola Schulz, Reference Schulz1938, M. affinis Gerlach, Reference Gerlach1958, M. borealis Steiner, Reference Steiner1916, M. campiensis Lima, Neres and Esteves, Reference Lima, Neres and Esteves2022, M. papillatus Gerlach, Reference Gerlach1956, M. pinguis Wieser, Reference Wieser1954, M. undulatus Gerlach, Reference Gerlach1953 in body length and amphidial fovea position. M. paraaffinis sp. nov. differs from M. arenicola in the testis number (two testes vs one testis), spicules shape (L-shaped, slender with equal thickness vs crescentic shaped, with proximal end knobbed), and tail shape (conico-cylindrical vs conical). M. paraaffinis sp. nov. differs from M. affinis in de Man c value (6.7–7.5 vs 9.5–10), spicules length and shape (31–35 μm, L-shaped vs 24 μm, slightly curved), gubernaculum length (19–21 μm vs 11 μm), absence of precloacal supplements (6–7 supplements present, redescribed by Kovalyev and Tchesunov, Reference Kovalyev and Tchesunov2005) and tail length and shape (conico-cylindrical, 92–111 μm vs conical, 77 μm). M. paraaffinis sp. nov. differs from M. borealis in the smaller amphidial fovea (33–42% vs 50%, measured according to Steiner, Reference Steiner1916), shorter cephalic setae (0.3 head diameter vs 0.75 head diameter, measured according to Steiner, Reference Steiner1916), and tail length (92–111 μm vs 66–68 μm). M. paraaffinis sp. nov. differs from M. campiensis in the shorter cephalic setae (3–4 μm vs 5–12 μm), spicules shape and length (slender with equal thickness, 31–35 μm vs slender with proximal end enlarged, 38–43μm), absence of precloacal supplements (7 papilliform supplements in M. campiensis) and tail length (3.7–4.4 anal body diameter vs 2.6–3.2 anal body diameter). M. paraaffinis sp. nov. differs from M. papillatus in outer labial sensilla and cephalic setae length (papilliform, 3–4 μm vs 5 μm, 8.5 μm), amphidial fovea diameter (4–5 μm vs 8 μm), spicules length and shape (31–35 μm, equal thickness vs 26 μm, proximal end offset), and absence of precloacal supplements (six precloacal supplements present in M. papillatus). M. paraaffinis sp. nov. differs from M. pinguis in amphidial fovea diameter (4–5 μm vs 10–10.5 μm), spicules shape (L-shaped vs slightly curved), and tail length (3.7–4.4 anal body diameter vs 2.25–2.6 anal body diameter). M. paraaffinis sp. nov. differs from M. undulatus in shorter cephalic setae length (3–4 μm vs 7 μm), amphidial fovea diameter (33–42% vs 53%), spicules length and shape (31–35 μm, L-shaped vs 27 μm, slightly knee-shaped), gubernaculum shape (boat-shaped vs twisted), and shorter tail length (92–111 μm vs 72–83 μm).
Order MICROLAIMIDA Leduc, Verdon and Zhao, Reference Leduc, Verdon and Zhao2017
Superfamily MICROLAIMOIDEA Micoletzky, Reference Micoletzky1922
Family MICROLAIMIDAE Micoletzky, Reference Micoletzky1922
Genus Pseudomicrolaimus Sergeeva, Reference Sergeeva1976
Diagnosis
Cuticle strongly annulated, head truncated and not set off, cephalic setae close to the front end, amphidial fovea unispiral or circular, buccal cavity rather wide and strongly sclerotized, with a large dorsal tooth, and transversal rows of denticles, pharyngeal tissue inflated around the buccal cavity, posterior oval bulb present, copulatory apparatus strongly sclerotized, no dorso-caudal gubernaculum apophyses (based on Tchesunov, Reference Tchesunov and Schmidt-Rhaesa2014).
Type species
Pseudomicrolaimus murinae Sergeeva, Reference Sergeeva1976
Pseudomicrolaimus major sp. nov.
(Figures 3–4, Table 2)
Type material
Three males and three females were measured and studied. Holotype: m#1 on slide 19YMD1-1-4; paratypes: m#2, m#3 on slides 19YMD1-1-9, 19YMD1-1-4; f#1 on slides 19YMD1-1-9; f#2, f#3 on slides 19YMD1-1-4.
Figure 3. Pseudomicrolaimus major sp. nov. (A) Lateral view of male anterior portion, showing buccal cavity and pharynx region (holotype); (B) Lateral view of male anterior end, showing amphids and buccal cavity (holotype); (C) Lateral view of male anterior portion, showing cuticle and amphids (holotype); (D) Lateral view of male posterior portion, showing tail (holotype); (E) Spicules and gubernaculum (holotype); (F) Lateral view of female anterior end (19YMD1-1-9); (G) Lateral view of female posterior portion (19YMD1-1-9); (H) Lateral view of female middle portion, showing female reproductive structure (19YMD1-1-9). Scale bars: A, B, C, D, F = 20 μm; G = 50 μm.
Figure 4. Pseudomicrolaimus major sp. nov. (A) Lateral view of male anterior end, showing buccal cavity (holotype); (B) Lateral view of male anterior end, showing amphidial fovea (holotype); (C) Lateral view of male anterior region, showing denticles and inner labial setae (arrows) (19YMD1-1-9); (D) Lateral view of male pharynx region (19YMD1-1-9); (E) Lateral view of male posterior body, showing spicules and gubernaculum (holotype); (F) Lateral view of female showing vulva (19YMD1-1-9). Scale bars: A, B, C, D, E = 20 μm, F = 50 μm.
Table 2. Individual measurements of Pseudomicrolaimus major sp. nov. (in μm except for ratios)
Type locality and habitat.
Specimens were collected from intertidal muddy sediment at Yangma Island, Shandong Province. 37°26´N, 121°36΄E.
Etymology
The species name refers to large body size of the nematode.
Measurements
All measurement data are given in Table 2.
Description
Males. Body yellow brownish, long, stout and cylindrical, 2586–3258 μm in length. Anterior body end truncated, posterior body end conical. Cuticle annulated. Anterior sensilla in three crowns, six inner labial sensilla papilliform (2 μm in length), six outer labial setae 5–6 μm in length, and four cephalic setae 7–10 μm in length. Eight subcephalic setae present (8–9 μm in length, 11–13 μm from posterior amphidial fovea end). Somatic setae absent. Amphidial fovea cryptospiral at level of buccal cavity, 7–10 μm in diameter (36%–38% corresponding body diameter), 9–12 μm from anterior body end. Buccal cavity wide, 7 μm in width and 16–20 μm in depth with a large cuticularized dorsal tooth and two small ventrosublateral teeth at the same level, small denticles present at left ventrosublateral side of buccal cavity. Pharynx muscular, 188–196 μm in length, anterior part widened at the buccal cavity region, posterior part widened into long oval-shaped bulb. Nerve ring situated 100–115 μm from anterior end. Excretory-secretory system not observed. Cardia not observed. Tail short and conical, 80–95 μm (2.4–2.9 cloacal body diameter) in length. Three caudal glands present and spinneret absent.
Testes outstretched and opposed, the anterior testis to the right and the posterior one to the left of intestine. Spicules paired, curved, 62–65 μm in length (1.9 cloacal body diameter). Proximal third of spicules widened (oval or rectangular-shaped) and the rest portion slender. Gubernaculum simple, boat-shaped, and parallel to distal end of spicules.
Females. Similar to males in most characters. Reproductive system didelphic-amphidelphic with two ovaries outstretched. Anterior ovary to the left of the intestine (279–342 μm in length), posterior ovary to the right (313–336 μm in length). Vulva located slightly posterior to the mid-body, 1779–2236 μm from the anterior end. Vagina short and sclerotized.
Differentiational diagnosis and discussion
The new species presents as main characteristics: body size relatively large, cuticle striated, buccal cavity with one large dorsal tooth and two small subventral teeth at the same level, small denticles present at left subventral side of buccal cavity, amphidial fovea cryptospiral, eight subcephalic setae present, somatic setae absent, spicules curved 62–65 μm in length with proximal portion widened and other portion slender, gubernaculum simple and boat-shaped, tail conical with three caudal glands.
P. major sp. nov. differs from P. denticulatus (Cobb, Reference Cobb1920; Gerlach, Reference Gerlach1953) in the longer body (2586–3403 μm vs 2025–2139 μm), subcephalic setae position (11–14 μm from posterior end of amphidial fovea vs level with posterior end of amphidial fovea), and spicules length and shape (62–65 μm, proximal portion widened vs 33 μm, proximal end offset). P. major sp. nov. differs from P. murinae (Sergeeva, Reference Sergeeva1976) in the longer body (2586–3403 μm vs 841–1167 μm), buccal cavity tooth number (one dorsal tooth vs two dorsal teeth), presence of subcephalic setae (absence in P. murinae), spicules length and shape (62–65 μm, proximal portion widened vs 32.5–37.5 μm, proximal portion slightly cephalated). P. major sp.nov. differs from P. dentatus (Allgén, Reference Allgén1935) in the longer body (2586–3403 μm vs 950 μm), presence of subcephalic setae (absence in P. dentatus), spicules length and shape (62–65 μm, proximal portion widened vs 30 μm, proximal end cephalated).
Dichotomous key for species of genus Pseudomicrolaimus Sergeeva, Reference Sergeeva1976
1. subcephalic setae present2
• subcephalic setae absent3
2. Spicules proximal portion widened, subcephalic setae posterior to posterior amphidial endP. major sp. nov.
– Spicules proximal end offset, subcephalic setae level with posterior amphidial endP. denticulatus
3. Two dorsal teeth presentP. murinae
– One dorsal tooth presentP. dentatus
Acknowledgements
We are greatly thankful to two anonymous reviewers for their kind reviews and valuable suggestions, and Dr Huang Yong for sample collecting.
Authors’ contributions
Wen Guo: sample collection, preparation of Microlaimus paraaffinis sp. nov. and draft preparation; Mengna Wang: preparation of pseduomicrolaimus major sp. nov.; Chunming Wang: picture drawing, manuscript editing.
Financial support
This work was supported by A Project of Shandong Province Higher Educational Science and Technology Program (No. J18KA152) and Open Project of Liaocheng University Animal Husbandry Discipline (Grant no. 319312101).
Competing interest
None.
Data availability statements
The authors confirm that the data supporting the findings of this study are available within the article.
