﻿Two new species of Hypodontolaiminae (Nematoda, Chromadorida, Chromadoridae) from the Yellow Sea with a phylogenetic analysis in the subfamily

﻿Abstract Two new species of Hypodontolaiminae, Dichromadoramediasp. nov. and Neochromadoraparabilineatasp. nov., were isolated and described from the Yellow Sea, China. Dichromadoramediasp. nov. is characterized by four long cephalic setae, the amphidial fovea transverse oval in the male and slit-shaped in the female, the pharynx with a single posterior bulb, spicules curved and distally bifurcated, gubernaculum jointed, four (1+3) precloacal supplements papilliform, and the tail conical elongated with a short spinneret. Neochromadoraparabilineatasp. nov. is characterized by the buccal cavity with one large hollow dorsal tooth and two small subventral teeth, the pharynx with an obvious posterior bulb, spicules L-shaped and widened medially, gubernaculum boat-shaped, seven cup-shaped and equidistant precloacal supplements, and a long and gradually tapering tail. The phylogenetic analysis of maximum likelihood and Bayesian inference based on rDNA sequences confirmed the taxonomic positions of Neochromadoraparabilineatasp. nov. and Dichromadoramediasp. nov. within Hypodontolaiminae. Tree topology in Hypodontolaiminae shows the genera Neochromadora, Dichromadora, Ptycholaimellus, and Spilophorella as polyphyletic groups, and the genus Chromadorita as a paraphyletic group.


Introduction
Nematodes are the most widely distributed and diverse metazoans on the planet, and a large number of nematode species still remain unidentified (Hodda 2022).Chromadoridae Filipjev, 1917 is one of the largest families of nematodes and shown as a monophyletic group with synapomorphies of male monorchid with an anterior testis; precloacal supplements cup-shaped or absent but never tubular; females with two reflexed ovaries, anterior to the right of the intestine, posterior to the left of the intestine (Tchesunov 2014).Chromadoridae has been reviewed systematically by Wieser (1954a), Gerlach andRiemann (1973/1974), Lorenzen (1981Lorenzen ( , 1994)), Tchesunov (2014), and Venekey et al. (2019), and its phylogenetic relationships have been analyzed based on rDNA sequences by Holterman et al. (2008), Leduc et al. (2017), Venekey et al. (2019), and Guo ZooKeys 1190: 281-302 (2024), DOI: 10.3897/zookeys.1190.113418 Huixin Liang et al.: Two new species of Hypodontolaiminae from the Yellow Sea et al. (2023).The number of rDNA sequences in Chromadoridae have rapidly increased in GenBank: up to now, 250 sequences from 20 genera of Small Subunit (SSU) and 145 sequences from 11 genera of D2-D3 fragment of Large Subunit (LSU) have been deposited in GenBank.In our study of marine nematode taxonomy from the Rizhao coast, at the Yellow China Sea, two new species from the subfamily Hypodontolaiminae are described, Dichromadora media sp.nov.and Neochromadora parabilineata sp.nov.Ribosome DNA sequences from these two new species and three other species, Dichromadora sinica Huang & Zhang, 2010, Dichromadora major Huang & Zhang, 2010, and Dichromadora multisetosa Huang & Zhang, 2010, are acquired for phylogenetic analysis.

Sample collection
In July 2022, undisturbed samples were collected from intertidal sediments in the Rizhao coast, the Yellow China Sea.Sediments samples were vertically collected with a syringe (2.6 cm internal diameter) to a depth of 8 cm and subdivided into 0-2 and 2-8 cm depth parts.Sediments used for morphological analysis were fixed in a 10% formalin solution in seawater and for molecular analysis were preserved in 95% ethanol.Formalin-fixed samples were stained with 0.1% of Rose Bengal for more than 24 hours.Meiofauna were extracted from the sediment through Ludox centrifugation (Higgins and Thiel 1988), washed through two sieves with mesh sizes of 500 μm and 45 μm with tap water to separate meiofauna from macrofauna (larger than 500 μm), transferred to a grid-lined Petri dish, and sorted under a stereoscopic microscope.Nematodes were transferred into a mixture of ethanol (50%) and glycerin in the ratio 1:9 by volume with the ethanol slowly evaporated away (McIntyre and Warwick 1984).Nematodes were mounted in glycerin on permanent slides.Descriptions were made using an Axiscope-5 differential interference contrast microscope (Zeiss, Germany).Line drawings were made with the aid of iPad (Apple, USA), and photographs were taken with the aid of ZEN software (Zeiss).Type specimens were deposited in the Institute of Oceanology, Chinese Academy of Sciences, Qingdao.
Sediments used for molecular analysis were washed and separated as with formalin-fixed samples except without Rose Bengal dying.Seven male specimens of D. media sp.nov., two male specimens of N. parabilineata sp.nov., five male specimens of D. sinica, two male specimens of D. major, and two male specimens of D. multisetosa were separated and confirmed on the temporary slides.

DNA extraction, PCR amplification, and phylogenetic analysis
Genomic DNA was extracted with DNeasy Blood & Tissue kit (Qiagen, Germany) and used as amplification templates for nearly full length SSU rDNA gene, with primers of G18S4F (5' -GCT TGT CTC AAA GAT TAA GCC -3') / 18PR (5' -TGA TCC WMC RGC AGG TTC AC -3') (Blaxter et al. 1998), and D2-D3 fragment of LSU rDNA gene with primers of D2A (5' -ACA AGT ACC GTG AGG GAA AGT TG -3') / D3B (5' -TCG GAA GGA ACC AGC TAC TA -3') (Nunn 1992).PCR was conducted as described by Zhao et al. (2015).The PCR product was sequenced by Genewiz (China).The sequences were assembled in Genious v. Sequences of subfamily Hypodontolaiminae in GenBank were used for phylogenetic analysis.Forty-nine SSU rDNA sequences from seven genera (Table 1) longer than 600 bp were selected and aligned with the Muscle algorithm.Substitution models of (GTR (general time-reversible) + G (gamma distribution) + I (proportion of invariable sites)) were selected as the best-fit model and the analysis was rooted with Latronema whataitai Leduc & Zhao, 2015 (accession number KR048680).Sixteen D2-D3 fragment of LSU rDNA sequences from five genera (Table 2) were selected and aligned with the Muscle algorithm.Substitution models of (GTR (general time-reversible) + G (gamma distribution)) were selected as the best-fit model, and the analysis was rooted with Latronema whataitai (accession number KR04868).
Measurements.All measurement data are given in Table 3. Description.Males.Body cylindrical and medium sized (881-925 μm in length).Cuticle with transverse rows of dots and a differentiation consisting of two longitudinal rows of distinct larger dots starting posterior the amphidial fovea and extending to the tail tip (2 μm in width).Transverse bars connecting the two larger dots beginning from the middle of the pharynx to the middle of tail.Somatic setae present sparsely along the lateral differentiation in two longitudinal rows, short in the head and tail (9 μm in length), long in the middle of the body (12 μm in length).Inner and outer labial sensilla papilliform.Four cephalic sensilla setiform at the level of amphidial fovea (1.20-1.36 head diameter in length).Head blunt.Amphidial fovea oval (4 µm in width and 3 µm in length), small (26.7%-28.6%corresponding body diameter) and situated 0.47-0.57head diameter from the anterior end.Buccal cavity cuticularized with a large, hollow and straight dorsal tooth and two small ventrosublateral teeth.Cheilostoma short with longitudinal cuticularized ribs.Pharynx cylindrical, anterior region surrounding buccal cavity slightly swollen, posterior region swollen into an elongated single bulb with plasmatic interruptions resembling a double bulb (18.8-20.0% of pharynx length).Nerve ring slightly posterior to middle pharynx region (53.6-60.9% of pharynx length).Renette cell of secretory-excretory system situated posterior to pharynx bulb, excretory pore located at anterior buccal cavity (6-8 μm from anterior end).Cardia not observed.
Females.Similar to males in most characteristics.Amphidial fovea slit-like (50.0%corresponding body diameter).Cephalic setae short (10-11 μm in length).Reproductive system didelphic, with opposed and reflexed ovaries.Anterior ovary to right of intestine and posterior ovary to left of intestine.Eggs oval shaped, 8-10 × 10-11 μm.Vulva at the middle of the total body.Vagina short.
Etymology.Species epithet media refers to the medium body size.
Dichromadora media sp.nov.shows a close relationship with D. sinica in the phylogenetic trees (Figs 5,6) based on rDNA sequences and it differs by 2% (39 in 1656 bp, including two gaps) in SSU and 5% (38 in 770 bp, including four  gaps) in LSU D2-D3 fragment, but they can be morphologically differentiated based on pharynx bulb shape, spicule shape, gubernaculum shape and precloacal supplements.

Diagnosis (based on Venekey et al. 2019).
Cuticle ornamentation heterogeneous and complex, with lateral differentiation visible as two or three longitudinal rows of large dots.Six small outer labial setae or papillae and four cephalic setae in separate circles.Inner labial sensilla may be conspicuous in one species (N.munita).Presence of somatic setae in some species.Amphidial fovea transverse slit-like and loop shaped.Buccal cavity with a dorsal tooth and two ventrosublateral teeth, in some species the dorsal one being larger than the others.Denticles can be present.Pharynx anteriorly not swollen or swollen next to the dorsal tooth.Pharynx with a single well-developed posterior bulb.Male usually with numerous precloacal supplements.
Measurements.All measurement data are given in Table 4. Description.Males.Body medium sized (864-943 μm), anterior end truncated and posterior end tapered.Cuticle heterogeneous and complex, five transverse rows of small dots present just posterior to cephalic setae, two or three longitudinal rows of larger dots posterior to the cephalic setae to middle part of body, larger dots changing to rectangular markings from middle body to posterior part of cloaca and rectangular markings changing back to larger dots until tail end.Six inner and six outer labial sensilla papilliform, four setiform cephalic sensilla (0.5-0.7 head diameter in length).Somatic setae present in pharynx and tail region (8 μm in length).Amphidial fovea situated at level of cephalic setae, transverse oval, 6-7 µm in width and 2 µm in length (50-58% corresponding body diameter).Buccal cavity shallow, 10-14 µm in depth.Cheilostoma short with cuticularized longitudinal folds.Pharyngostoma with one large hollow dorsal tooth and two small subventral teeth.Pharynx cylindrical, pos- terior region swollen into an oval bulb (17.5-17.7% of pharynx length).Nerve ring slightly posterior to middle pharynx region (64.2-66.9% of pharynx length).Secretory-excretory system present; renette cell situated posterior to pharynx bulb, excretory pore at level with cephalic setae.Cardia not observed.
Females.Similar to males in most characteristics.Tail slightly longer than in males (5.8-6.8 anal body diameters in length).Reproductive system didelphic, with opposed and reflexed ovaries.Anterior ovary to left of intestine and posterior ovary to right of intestine.Spermatheca present.Vulva situated anterior to middle of body.Vagina short and muscularized.
Etymology.Species epithet parabilineata refers to the new species being similar to Neochromadora bilineata.

Molecular phylogenetic analysis
The ML topology trees which are obtained based on the rDNA gene sequences are mostly in accordance with the BI topology trees, and only the BI trees are shown in Figs 5, 6.Sequences of seven genera of the subfamily Hypodontolaiminae, Chromadorita Filipjev, 1922, Dichromadora, Hypodontolaimus de Man, 1886, Innocuonema Inglis, 1969, Neochromadora, Ptycholaimellus Cobb, 1920and Spilophorella Filipjev, 1917  analysis.These species share a common character of peribuccal cavity tissue with an asymmetrical dorsal swelling and only one posterior pharynx bulb.However, Chromadorita can be morphologically differentiated from them by having the cuticle homogeneous without any lateral differentiation.
Six sequences of genus Dichromadora have been identified to species level, but they are in four different clades in both SSU and LSU analyses and therefore paraphyletic.Among the species of Dichromadora, Dichromadora multisetosa is the only species with the gubernaculum not being boat-shaped but with dorsal caudal apophysis, and clustered with Chromadorita humila (gubernaculum possessing arched dorsal-caudal apophysis) highly supported by the LSU topology tree (posterior probability 85, bootstrap value 51 in SSU; posterior probability 100, bootstrap value 81 in LSU).Dichromadora sinica and D. media sp.nov.are highly clustered based on rDNA sequences (posterior probability 100, bootstrap value 93 in SSU; posterior probability 86, bootstrap value 71 in LSU), and can be distinguished based on the pharyngeal bulb, precloacal supplements, and gubernaculum shape.
Species of genus Neochromadora differ from other genera of Hypodontolaiminae mainly based on the cuticle ornamentation being heterogeneous and complex.However, the cuticle structure is sometimes seen as a variable character (e.g., Leduc et al. 2017), and sequences of Neochromadora present as polyphyletic clades in SSU and LSU topology trees.Neochromadora bilineata (OQ396744) and N. parabilineata sp.nov.have a close relationship in the SSU analysis (posterior probability 94, bootstrap value 68), but the LSU sequence of N. bilineata was missing.Relationships between these two species should be further discussed when more molecular data is available, in combination with morphological characters.
The genera Ptycholaimellus and Spilophorella are paraphyletic clades in both LSU and SSU analyses.Ptycholaimellus, Hypodontolaimus, and Dichromadora all show morphological similarities with each other and clades are weakly supported in the SSU analysis (posterior probability 88 in SSU).Differences between these three genera are slight, and they are clustered within one morphological group based on buccal cavity, peribuccal pharyngeal tissue, and supplements by Venekey et al. (2019).

Figure 3 .
Figure 3. Neochromadora parabilineata sp.nov.A lateral view of male anterior region showing cuticle and amphidial fovea (holotype) B lateral view of male buccal cavity (holotype) C lateral view of male anterior region showing pharyngeal region (holotype) D lateral view of male cuticle at pharynx region (holotype) E lateral view of male cuticle at middle body (holotype) F lateral view of female anterior region showing buccal cavity and pharyngeal region (22HSB11-1-18) G lateral view of spicules and gubernaculum (22HSB11-2-20) H lateral view of male posterior body, showing precloacal supplements and tail (holotype) I lateral view of female whole body (22HSB11-2-18) J lateral view of female posterior body showing tail (22HSB11-2-18).Scale bars: 20 µm (A-H, J); 50 µm (I).

Figure 5 .
Figure 5. Bayesian inference tree of the subfamily Hypodontolaiminae inferred from Small Subunit (SSU) sequences under the general time-reversible (GTR) + gamma distribution (G) + proportion of invariable sites (I) model.Posterior probability (left) and bootstrap values (right) are given on corresponding clades.The sequences obtained in this study are shown in bold.The scales indicate substitutions per site.

Figure 6 .
Figure 6.Bayesian inference tree of the subfamily Hypodontolaiminae inferred from the D2-D3 fragment of Large Subunit (LSU) sequences under the general time-reversible (GTR) + gamma distribution (G) model.Posterior probability (left) and bootstrap values (right) are given on corresponding clades.The sequences obtained in this study are shown in bold.The scales indicate substitutions per site.

Table 1 .
SSU information of samples used for phylogenetic analysis.

Table 2 .
D2-D3 fragment of LSU information of samples used for phylogenetic analysis.

Results Taxonomic account Order Chromadorida Chitwood, 1933 Family Chromadoridae Filipjev, 1917 Subfamily Hypodontolaiminae De Coninck, 1965 Genus Dichromadora Kreis, 1929 Diagnosis (based on Venekey et al. 2019).
Cuticle with homogeneous ornamentation and a pronounced lateral differentiation of two longitudinal rows of enlarged dots.Six outer labial papillae and four cephalic setae in separate circles.Amphideal fovea transverse slit-like and loop shaped.Buccal cavity with a triangular hollow dorsal tooth or a large dorsal tooth and two additional ven- trosublateral ones; denticles can be present.Peribuccal pharyngeal tissue not swollen anteriorly or with an asymmetrical dorsal swelling; a distinct posterior pharyngeal bulb.Precloacal supplements present or absent.Remarks.The genus Dichromadora was erected by Kreis in 1929 with the type species Dichromadora microdonta species are currently considered as valid (based on Venekey et al. 2019):35.Dichromadora weddellensisVermeeren, Vanreusel & Vanhove, 2004 (Antarctic Sea, Halley Bay)