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Research Article
Two new species of Hypodontolaiminae (Nematoda, Chromadorida, Chromadoridae) from the Yellow Sea with a phylogenetic analysis in the subfamily
expand article infoHuixin Liang‡, Wen Guo‡, Chunming Wang‡
‡ Liaocheng University, Liaocheng, China

Corresponding author: Chunming Wang ( wangchunming@lcu.edu.cn )

Academic editor: Nic Smol
© 2024 Huixin Liang, Wen Guo, Chunming Wang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Liang H, Guo W, Wang C (2024) Two new species of Hypodontolaiminae (Nematoda, Chromadorida, Chromadoridae) from the Yellow Sea with a phylogenetic analysis in the subfamily. ZooKeys 1190: 281-302. https://doi.org/10.3897/zookeys.1190.113418
ZooBank: urn:lsid:zoobank.org:pub:EDBA3DBA-08BD-49AC-9ED5-ABFBDCF40207
Open Access

Abstract

Two new species of Hypodontolaiminae, Dichromadora media sp. nov. and Neochromadora parabilineata sp. nov., were isolated and described from the Yellow Sea, China. Dichromadora media sp. 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. Neochromadora parabilineata sp. 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 Neochromadora parabilineata sp. nov. and Dichromadora media sp. 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.

Key words

China, Dichromadora, marine nematode, Neochromadora

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 and Riemann (1973/1974), Lorenzen (1981, 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 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.

Materials and methods

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. 6.1.2. The newly obtained SSU rDNA sequences have the accession numbers as follows: D. media sp. nov. OR479913, N. parabilineata sp. nov. OR126985, D. sinica OR479916, D. major OR479911, and D. multisetosa OR479915; the D2–D3 fragment of LSU rDNA sequence accession numbers are D. media sp. nov. OR479918, N. parabilineata sp. nov. OR135360, D. sinica OR479914, D. major OR479912, and D. multisetosa OR479917. All have been deposited in GenBank.

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).

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

Species GenBank number Reference Locality
Chromadorita aff. leuckarti MF409784.1 Schenk et al. 2018 Germany
Chromadorita cf. leuckarti FJ040473.1 Holterman et al. 2008 –
Chromadorita humila OQ396742.1 Sun et al. 2023 China
Chromadorita leuckarti FJ969119.1 van Megen et al. 2009 –
Chromadorita leuckarti KJ636254.1 Bert et al. 2014 –
Chromadorita spinicauda OK317201.1 Leduc and Zhao 2023 New Zealand
Dichromadora major OR479911.1 Wang et al. 2023 China
Dichromadora media sp. nov. OR479913.1 Wang et al. 2023 China
Dichromadora multisetosa OR479915.1 Wang et al. 2023 China
Dichromadora simplex MG669747.1 Macheriotou et al. 2018 Vietnam
Dichromadora sinica OR479916.1 Wang et al. 2023 China
Dichromadora sp. MN250081.1 Pereira et al. 2019 Beaufort Sea (USA)
Dichromadora sp. FJ040506.1 Holterman et al. 2008 –
Dichromadora sp. MN250085.1 Pereira et al. 2019 Beaufort Sea (USA)
Dichromadora sp. MG669748.1 Macheriotou et al. 2018 Netherlands
Dichromadora sp. MN250044.1 Pereira et al. 2019 Beaufort Sea (USA)
Dichromadora sp. MK626828.1 Tytgat et al. 2019 Vietnam
Dichromadora sp. MG669752.1 Macheriotou et al. 2018 Vietnam
Dichromadora sp. MG669751.1 Macheriotou et al. 2018 Vietnam
cf. Dichromadora sp. KJ636253.1 Bert et al. 2014 –
Hypodontolaimus inaequalis MG669813.1 Macheriotou 2018 Netherlands
Hypodontolaimus inaequalis MG669812.1 Macheriotou et al. 2018 Netherlands
Innocuonema tentabundum AY854208.1 Meldal 2005 Southampton (United Kingdom)
Innocuonema tentabundum JN968213.1 Fonseca et al. 2012 –
Neochromadora bilineata OQ396744.1 Sun et al. 2023 China
Neochromadora parabilineata sp. nov. OR126985.1 Wang et al. 2023 China
Neochromadora poecilosomoides OQ396720.1 Chu et al. 2023 China
Neochromadora sp. AY854210.1 Meldal 2005 Southampton (United Kingdom)
Neochromadora sp. MG669893.1 Macheriotou et al. 2018 Netherlands
Neochromadora sp. KX944147.1 Avo et al. 2017 Mira estuary (Portugal)
Neochromadora sp. MN250121.1 Pereira et al. 2019 Beaufort Sea (USA)
Neochromadora sp. JN968279.1 Fonseca et al. 2012 –
Ptycholaimellus areniculus MG669987.1 Macheriotou et al. 2018 Vietnam
Ptycholaimellus areniculus MG669988.1 Macheriotou et al. 2018 Vietnam
Ptycholaimellus brevisetosus MK626833.1 Tytgat et al. 2019 Vietnam
Ptycholaimellus brevisetosus MK626834.1 Tytgat et al. 2019 Vietnam
Ptycholaimellus brevisetosus MK626809.1 Tytgat et al. 2019 Vietnam
Ptycholaimellus brevisetosus MG669989.1 Macheriotou et al. 2018 Vietnam
Ptycholaimellus ocellatus OQ538290.1 Sun et al. 2023 China
Ptycholaimellus spiculuncus OK317202.1 Leduc and Zhao 2023 New Zealand
Ptycholaimellus sp. FJ040472.1 Holterman et al. 2008 –
Ptycholaimellus sp. KX944158.1 Avo et al. 2017 Mira estuary (Portugal)
Ptycholaimellus sp. JN968285.1 Fonseca et al. 2012 –
Ptycholaimellus sp. MG669992.1 Macheriotou et al. 2018 Vietnam
Ptycholaimellus sp. JN968257.1 Fonseca et al. 2012 –
Spilophorella aberrans MG670031.1 Macheriotou et al. 2018 Vietnam
Spilophorella paradoxa AY854211.1 Meldal 2005 Southampton (United Kingdom)
Spilophorella paradoxa JN968274.1 Fonseca 2012 –
Spilophorella sp. MG670032.1 Macheriotou et al. 2018 Vietnam
Latronema whataitai KR048680.1 Leduc and Zhao 2016 –
Table 2.
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D2–D3 fragment of LSU information of samples used for phylogenetic analysis.

Species GenBank number Reference Locality
Chromadorita humila OQ396736.1 Sun et al. 2023 China
Chromadorita spinicauda OK317226.1 Leduc and Zhao 2023 New Zealand
Dichromadora cucullata GU003894.1 Rodrigues et al. 2010 USA
Dichromadora major OR479912.1 Wang et al. 2023 China
Dichromadora media sp. nov. OR479918.1 Wang et al. 2023 China
Dichromadora multisetosa OR479917.1 Wang et al. 2023 China
Dichromadora sinica OR479914.1 Wang et al. 2023 China
Dichromadora sp. KC755220.1 Vogt et al. 2014 Wilhelmshaven (Germany)
Neochromadora aff. poecilosoma KC755218.1 Vogt et al. 2014 Jadebusen (Germany)
Neochromadora parabilineata sp. nov. OR135360.1 Wang et al. 2023 China
Neochromadora poecilosomoides OQ417520.1 Sun et al. 2023 China
Neochromadora sp. KC755219.1 Vogt et al. 2014 Wilhelmshaven (Germany)
Ptycholaimellus ocellatus OQ466609.1 Sun et al. 2023 China
Ptycholaimellus spiculuncus OK317227.1 Leduc and Zhao 2023 New Zealand
Spilophorella sp. DQ077766.1 De Ley et al. 2009 Mexico
Spilophorella sp. GU003892.1 Rodrigues et al. 2010 USA
Latronema whataitai KR048681.1 Leduc and Zhao 2016 New Zealand

The ML analyses were performed with Mega X with 1000 bootstrap replicates. The BI analyses were constructed with CIPRES (http://www.phylo.org/) and MrBayes on XSEDE v. 3.2.7a were used; the trees were run with chain length of 10,000,000, burn-in frac = 0.25. The topology of the resulting trees was visualized using FigTree v. 1.4.3 and refined with PowerPoint.

Results

Taxonomic account

Order Chromadorida Chitwood, 1933

Family Chromadoridae Filipjev, 1917

Subfamily Hypodontolaiminae De Coninck, 1965

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 ventrosublateral 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 Kreis, 1929 with the genus characters the cuticle with two longitudinal rows of dots, small tooth, pharynx bulb big and round, ovaries paired symmetrical and reflexed, and males with or without precloacal supplements. Six species from the genus Chromadora (C. cephalata Steiner, 1916, C. cricophana Filipjev, 1922, C. geophila de Man, 1876, C. parapoecilosoma Micoletzky, 1922, C. sabulicola Filipjev, 1918, C. setosa Bütschli, 1874) were transferred to Dichromadora by Kreis (1929). Later, D. hyalocheile De Coninck & Schuurmans Stekhoven, 1933, D. tobaensis Schneider, 1937, D. strandi Allgén, 1940, D. punctata Schuurmans Stekhoven, 1950, D. tenuicauda Schuurmans Stekhoven, 1950, and D. abnormis Gerlach, 1953a were described. Wieser (1954a) described D. dissipata Wieser, 1954a, revised the genus characters based mainly on tooth shape and provided new combinations. After Wieser (1954a), D. apapillata Timm, 1961, D. arcospiculum Timm, 1961, D. simplex Timm, 1961, D. islandica Kreis, 1963, D. scandula Lorenzen, 1966, and D. cucullata Lorenzen, 1973 were described and Gerlach and Riemann (1973) presented a list of sixteen species. Later, 12 species (D. amphidiscoides Kito, 1981, D. abyssalis Bussau, 1993, D. gathuai Muthumbi & Vincx, 1998, D. loiseae Muthumbi & Vincx, 1998, D. longicaudata Muthumbi & Vincx, 1998, D. quadripapillata Muthumbi & Vincx, 1998, D. parasimplex Dashchenko, 2002, D. parva Vermeeren, Vanreusel & Vanhove, 2004, D. polaris Vermeeren, Vanreusel & Vanhove, 2004, D. polarsternis Vermeeren, Vanreusel & Vanhove, 2004, D. southernis Vermeeren, Vanreusel & Vanhove, 2004 and D. weddellensis Vermeeren, Vanreusel & Vanhove, 2004) were described. Huang and Zhang (2010) described three species from the China Sea, D. major, D. multisetosa, and D. sinica, and provided a short review of Dichromadora. Dichromadora abyssalis was considered as valid by Holovachov (2020) based on the high quality descriptions and illustrations despite not following the International Code of Zoological Nomenclature (Venekey et al. 2019). With the addition of D. rigida Thanh, Tu & Gagarin, 2016 and D. agilis Long, Gagarin & Tu, 2022, 35 species are currently considered as valid (based on Venekey et al. 2019):

1. Dichromadora abnormis Gerlach, 1953 (Italy, San Rossore and Tirrenia beaches)

2. Dichromadora abyssalis Bussau, 1993 (SE Pacific, Peru Basin)

3. Dichromadora agilis Long, Gagarin & Tu, 2022 (Vietnam, Quảng Ninh)

4. Dichromadora amphidiscoides Kito, 1981 (Japan, Oshoro Bay)

5. Dichromadora antarctica (Cobb, 1914) (Antarctica, Cape Royd; = Spilophora antarctica Cobb, 1914)

6. Dichromadora apapillata Timm, 1961 (Indian Ocean, Bay of Bengal)

7. Dichromadora arcospiculum Timm, 1961 (Indian Ocean, Bay of Bengal)

8. Dichromadora cephalata (Steiner, 1916) (Arctic Ocean, Barents Sea; = Chromadora cephalata Steiner, 1916, Chromadora cricophana Filipjev, 1922)

9. Dichromadora cucullata Lorenzen, 1973 (North Sea, Baltic Sea, Helgoland)

10. Dichromadora dissipata Wieser, 1954 (Chile, Seno de Reloncaví)

11. Dichromadora gathuai Muthumbi & Vincx, 1998 (Indian Ocean, Kenyan coast)

12. Dichromadora geophila (de Man, 1876) (North Sea, Netherlands; = Chromadora canadensis (Cobb, 1914), Chromadora geophila (de Man, 1876), Hypodontolaimus geophilus (de Man, 1876), Spiliphera geophila de Man, 1876, Spiliphera spectabilis Allgén, 1929)

13. Dichromadora gracilis (Kreis, 1929) (France, Trebeurden; = Spilophorella gracilis Kreis, 1929)

14. Dichromadora hyalocheile De Coninck & Schuurmans Stekhoven, 1933 (Belgium, Oostende)

15. Dichromadora islandica Kreis, 1963 (Iceland, Eyjafjörður)

16. Dichromadora loiseae Muthumbi & Vincx, 1998 (Indian Ocean, Kenyan coast)

17. Dichromadora longicaudata Muthumbi & Vincx, 1998 (Indian Ocean, Kenyan coast)

18. Dichromadora major Huang & Zhang, 2010 (China, Yellow Sea)

19. Dichromadora media sp. nov. (China, Yellow Sea)

20. Dichromadora microdonta Kreis, 1929 (France, English Channel)

21. Dichromadora multisetosa Huang & Zhang, 2010 (China, Yellow Sea)

22. Dichromadora parasimplex Dashchenko, 2002 (New Guinea, Astrolabe Bay)

23. Dichromadora parva Vermeeren, Vanreusel & Vanhove, 2004 (Antarctic Sea, Halley Bay)

24. Dichromadora polaris Vermeeren, Vanreusel & Vanhove, 2004 (Antarctic Sea, Halley Bay)

25. Dichromadora polarsternis Vermeeren, Vanreusel & Vanhove, 2004 (Antarctic Sea, Halley Bay)

26. Dichromadora punctata Schuurmans Stekhoven, 1950 (Mediterranean, Villefranche Bay)

27. Dichromadora quadripapillata Muthumbi & Vincx, 1998 (Indian Ocean, Kenyan coast)

28. Dichromadora rigida Thanh, Tu & Gagarin, 2016 (Vietnam, Yen River Estuary)

29. Dichromadora scandula Lorenzen, 1966 (North Sea, Schleswig-Holstein)

30. Dichromadora simplex Timm, 1961 (Indian Ocean, Bay of Bengal)

31. Dichromadora sinica Huang & Zhang, 2010 (China, Yellow Sea)

32. Dichromadora southernis Vermeeren, Vanreusel & Vanhove, 2004 (Antarctic Sea, Halley Bay)

33. Dichromadora strandi Allgén, 1940 (Norway, Knivskjaerodden)

34. Dichromadora tobaensis Schneider, 1937 (Indonesia, Sumatra)

35. Dichromadora weddellensis Vermeeren, Vanreusel & Vanhove, 2004 (Antarctic Sea, Halley Bay)

Dichromadora media sp. nov.

https://zoobank.org/4F7D70E0-54B3-422C-9A87-F496EA14C572
Figs 1, 2, Table 3

Diagnosis

Medium body size, cuticle with transverse rows of dots and a lateral differentiation of two longitudinal larger dots, four long cephalic setae, buccal cavity large with one large, hollow and straight dorsal tooth and two small ventrosublateral teeth, amphidial fovea transverse oval in male and slit-shaped in female, pharynx with single posterior bulb, spicules curved and distally bifurcated, gubernaculum jointed, four (1+3) papilliform precloacal supplements, tail conical elongated with short spinneret.

Figure 1. 

Dichromadora media 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 female anterior region showing buccal cavity, amphidial fovea and pharyngeal region (22ZJT8-2-7) E lateral view of female whole body (22ZJT8-2-7) F lateral view of male posterior body, showing precloacal supplements and tail (holotype); G lateral view of spicules and gubernaculum (22ZJT8-2-5) H lateral view of female posterior body showing tail (22ZJT8-2-7). Scale bars: 20 µm (A–D, F, H); 50 µm (E).

Material examined

Four males and three females were measured and studied. Holotype: ♂ 1 on slide 22ZJT8–1–2; Paratypes: ♂ 2 on 22ZJT8–1–2, ♂ 3 on 22ZJT8–1–2, ♂ 4 on 22ZJT8–2–5, ♀ 1 on 22ZJT8–2–7, ♀ 2 on 22ZJT8–2–7, and ♀ 3 on 22ZJT8–2–6.

Figure 2. 

Dichromadora media sp. nov. A lateral view of male anterior region showing tooth (arrow) (holotype) B lateral view of male anterior region showing cuticle, amphidial fovea (arrow) (holotype) C lateral view of male anterior region showing pharyngeal region (holotype) D lateral view of male posterior body, showing spicules (holotype) E lateral view of male posterior body, showing gubernaculum (22ZJT8-1-2) F lateral view of male posterior body, showing precloacal supplements (arrows) (holotype) G lateral view of female anterior region showing amphidial fovea (arrow) (22ZJT8-1-2) H lateral view of distal end of spicules (22ZJT8-2-5). Scale bars: 20 µm.

Type locality and habitat

Rizhao coast, Shandong Province, China, 35°27′N, 119°35′E, 0–2 cm sediment depth, sandy sediment.

Measurements

All measurement data are given in Table 3.

Table 3.
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Measurements of Dichromadora media sp. nov. (in µm except for ratios).

Characters Holotype Paratypes Paratypes
male males (n = 3) females (n = 3)
Total body length 925 895±14.5(881–910) 812±44.6(761–844)
Maximum body diameter 23 22.3±0.6(22–23) 27.7±0.6(27–28)
Head diameter 15 14.3±0.6(14–15) 16±0(16–16)
Length of cephalic setae 18 18.3±0.6(18–19) 10.7±0.6(10–11)
Amphidial fovea width 4 4±0(4–4) 8±0(8–8)
Amphidial fovea from anterior end 7 7.3±0.6(7–8) 4.7±0.6(4–5)
Body diameter at amphidial fovea 15 14.3±0.6(14–15) 16±0(16–16)
Nerve ring from anterior end 64 64.3±5.1(60–70) 64±4.6(59–68)
Body diameter at nerve ring 18 18±0(18–18) 20.3±1.2(19–21)
Pharynx length 116 114.7±2.5(112–117) 119±4.4(116–124)
Pharynx bulb length 22 22.3±0.6(22–23) 25.3±1.5(24–27)
Body diameter at base of pharynx 19 19±0(19–19) 22.3±1.2(21–23)
Cloacal/anal body diameter 23 22.3±0.6(22–23) 15.7±0.6(15–16)
Spicules length along arc 23 24.3±0.6(24–25) –
Gubernaculum length 23 22±1(21–23) –
Vulva from anterior end – – 399±16.1(381–412)
Body diameter at vulva – – 27.3±0.6(27–28)
V% – – 49.2±0.8(48.6–50.1)
Precloacal supplements 1+3 1+3 –
Tail length 103 107±3.5(105–111) 106.7±6.8(99–112)
a 40.2 40.1±1.2(38.9–41.4) 29.4±1.9(27.2–30.8)
b 8.0 7.8±0.1(7.8–7.9) 6.8±0.3(6.6–7.2)
c 9.0 8.4±0.4(7.9–8.7) 7.6±0.2(7.4–7.7)
c’ 4.5 4.8±0.2(4.6–5.0) 6.8±0.2(6.6–7)

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 0.47–0.57 head 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.

The reproductive system monorchid, with extended testis located to the right of intestine. Spicules equal and slightly curved, 23–25 μm (1.0–1.1 cloacal body diameter) along arc, proximal end slightly widened and distal end bifurcated. Gubernaculum jointed without apophysis. Four (1 + 3) precloacal supplements papilliform, anterior three supplements closely distanced and posteriormost supplement distant from the three anterior ones, distance between the supplements 9–12 μm, 8–9 μm, 16–19 μm, respectively, and posteriormost supplement 7–8 μm from cloaca. Tail elongated conical, gradually tapering, 4.5–5.0 cloacal body diameters. Spinneret very short, 1–2 μm in length.

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.

Remarks

Dichromadora media sp. nov. differs from all other species of the genus Dichromadora by the amphidial fovea shape and jointed gubernaculum and it is similar to D. dissipata, D. quadripapillata, and D. sinica in body length and precloacal supplements number. However, it differs from D. dissipata in cephalic setae length (10–19 μm vs 9–9.5 μm), spicules length (23–25 μm vs 39 μm), gubernaculum shape (double-jointed without apophysis vs not jointed with dorsal apophysis) and precloacal supplements (1+3 vs 5); differs from D. quadripapillata in cephalic setae length (10–19 μm vs 4–5 μm), spicules shape (slightly curved and distally bifurcated vs curved with pointed distal end) and precloacal supplements shape (papilliform vs cup-shaped); differs from D. sinica in cuticle differentiation (lateral differentiation with transverse bars vs lateral differentiation without transverse bars), pharynx bulb shape (single bulb with plastic interruptions vs double bulb), spicules shape (slightly curved and distally bifurcated vs distal end with a hook), gubernaculum shape (jointed vs not jointed), and precloacal supplements arrangement (1+3 vs 3+1).

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.

Neochromadora Micoletzky, 1924

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.

Remarks

The genus Neochromadora was erected by Micoletzky (1924) with the type species Neochromadora poecilosoma (de Man, 1893). And six species, Neochromadora aberrans (Cobb, 1930), Neochromadora craspedota (Steiner, 1916), Neochromadora edentata (Cobb, 1914), Neochromadora izhorica (Filipjev, 1929), Neochromadora poecilosomoides (Filipjev, 1918), Neochromadora sabulicola (Filipjev, 1918) have been added to Neochromadora. Gerlach (1951) described N. tecta Gerlach, 1951, redescribed N. poecilosoma, N. izhorica (Filipjev, 1929), and transferred Spiliphera trichophora (Steiner, 1921) to Neochromadora. Later Gerlach (1952, 1953b) described N. attenuate Gerlach, 1952 and N. complexa Gerlach, 1953b. Wieser (1954a) divided Neochromadora into two subgenera Neochromadorina (Wieser, 1954a) and Trichodorina (Wieser, 1954a) based on tooth structure, cervical and somatic setal length, and pharyngeal bulb and described three new species: N. lateralis Wieser, 1954a, N. calathifera Wieser, 1954a, and N. torquata Wieser, 1954a. Later, Wieser (1954b) described another two species N. amembranata Wieser, 1954b and N. brevisetosa Wieser, 1954b. Afterwards, six species, N. bonita Gerlach, 1956, N. coudenhovei Wieser, 1956, N. notocraspedota Allgén, 1958, N. appiana Wieser, 1959, N. pugilator Wieser, 1959 and N. bicoronata (Wieser, 1959) (synonym Endeolophos spinosus (Gerlach, 1957) were described. Wieser (1959) withdrew the subgenus Trichodorina with a redescription of N. poecilosoma found in Puget Sound. Later, 13 species (N. alatocorpa Hopper, 1961, N. nitida Timm, 1961, N. munita Lorenzen, 1971, N. paratecta Blome, 1974, N. paramunita Boucher, 1976, N. angelica Riemann, 1976, N. bilineata Kito, 1978, N. oshoroana Kito, 1981, N. orientalis Lemzina, 1982, N. papillosa Pastor de Ward, 1985, N. lineata Pastor de Ward, 1985, N. nicolae Vincx, 1986 and N. alejandroi Lo Russo & Pastor de Ward, 2012) were described. Hopper (1963) considered Neochromadora trilineata Schneider, 1943 as incertae sedis due to the unavailability of specimens. Vincx (1986) considered N. paramunita as a synonym of N. munita. Up to now, 33 species are currently considered as valid (based on Venekey et al. 2019):

1. Neochromadora aberrans (Cobb, 1930) (Antarctic, Commonwealth Bay; = Spiliphera aberrans Cobb, 1930)

2. Neochromadora alatocorpa Hopper, 1961 (USA, Alabama)

3. Neochromadora alejandroi Lo Russo & Pastor de Ward, 2012 (Argentina, San Matías gulf)

4. Neochromadora amembranata Wieser, 1954 (Italy, Sampieri)

5. Neochromadora angelica Riemann, 1976b (Germany, Helgoland)

6. Neochromadora appiana Wieser, 1959 (USA, Washington)

7. Neochromadora bilineata Kito, 1978 (Japan, Hokkaido)

8. Neochromadora bonita Gerlach, 1956 (Brazil, Cananeia)

9. Neochromadora brevisetosa Wieser, 1954 (Italy, Sampieri)

10. Neochromadora calathifera Wieser, 1954b (Chile, Seno Reloncavi)

11. Neochromadora complexa Gerlach, 1953a (Chile, Seno Ultima Esperanza)

12. Neochromadora coudenhovei Wieser, 1956b (Greece, Piraeus)

13. Neochromadora craspedota (Steiner, 1916) (Arctic Ocean, Barents Sea; = Chromadora craspedota Steiner, 1916)

14. Neochromadora edentata (Cobb, 1914) (Antarctic, Cape Royds; = Nygmatonchus edentata (Cobb, 1914) Wieser, 1954, Spiliphera edentata Cobb, 1914)

15. Neochromadora izhorica (Filipjev, 1929) (Baltic Sea, Neva Bay; = Chromadorella izhorica Filipjev, 1929)

16. Neochromadora lateralis Wieser, 1954 (Chile, Seno Reloncavi)

17. Neochromadora lineata Pastor de Ward, 1985a (Argentina, Deseado river)

18. Neochromadora munita Lorenzen, 1972 (Germany, Helgoland; = Neochromadora paramunita Boucher, 1976)

19. Neochromadora nicolae Vincx, 1986 (North Sea, Southern Bight)

20. Neochromadora nitida Timm, 1961 (Indian Ocean, Bengal Bay)

21. Neochromadora notocraspedota Allgén, 1958 (Uruguay, Uruguay coast)

22. Neochromadora orientalis Lemzina, 1982 (Kyrgyzstan, Lake Issyk-Kul)

23. Neochromadora oshoroana Kito, 1981 (Japan, Oshoro Bay)

24. Neochromadora papillosa Pastor de Ward, 1985 (Argentina, Deseado River)

25. Neochromadora parabilineata sp. nov. (China, Yellow Sea)

26. Neochromadora paratecta Blome, 1974 (Germany, Sylt)

27. Neochromadora poecilosoma (de Man, 1893) (North Sea, English Channel; = Chromadora poecilosoma de Man, 1893)

28. Neochromadora poecilosomoides (Filipjev, 1918) (Black Sea, Kruglaya Bay and Georgievskii Monastery Bay; = Chromadora poecilosomoides Filipjev, 1918)

29. Neochromadora pugilator Wieser, 1959 (USA, Washington)

30. Neochromadora sabulicola (Filipjev, 1918) (Black Sea, Kruglaya Bay and Georgievskii Monastery Bay; = Chromadora sabulicola Filipjev, 1918)

31. Neochromadora tecta Gerlach, 1951 (Germany, Amrum Island)

32. Neochromadora torquata Wieser, 1954 (Chile, Seno Reloncavi)

33. Neochromadora trichophora (Steiner, 1921a) (Spain, Canary Islands; = Spiliphera trichophora Steiner, 1921, Neochromadora longisetosa Schuurmans-Stekhoven, 1935)

Neochromadora parabilineata sp. nov.

https://zoobank.org/27F8BC81-DA38-470D-AD2B-F4B20F302588
Figs 3, 4, Table 4

Diagnosis

Medium body size, buccal cavity with one large hollow dorsal tooth and two small subventral teeth, spicules curved and L-shaped, gubernaculum boat-shaped, seven precloacal supplements cup-shaped, tail conical and gradually tapering.

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).

Material examined

Four males and three females were measured and studied. Holotype: ♂ 1 on slide 22HSB11–2–20; paratypes: ♂ 2 on 22HSB11–1–21, ♂ 3 on 22HSB11–2–18, ♂ 4 on 22HSB11–2–20, ♀ 1 on 22HSB11–2–18, ♀ 2 on 22HSB11–1–18, and ♀ 3 on 22HSB11–2–18.

Figure 4. 

Neochromadora parabilineata sp. nov. A lateral view of male anterior region showing cuticle (holotype) B lateral view of female anterior region showing amphidial fovea (arrow) (22HSB11-2-18) C lateral view of cuticle at middle body (holotype) D lateral view of male posterior body, showing spicules (22HSB11-2-20) E lateral view of male posterior body, showing gubernaculum (22HSB11-2-20; F lateral view of male posterior body, showing precloacal supplements (arrows) (holotype) G lateral view of male posterior body, showing tail and cuticle (22HSB11-2-20). Scale bars: 20 µm.

Type locality and habitat

Rizhao coast, Shandong Province, China, 35°5′N, 119°20′E, 0–2 cm sediment depth, sandy sediment.

Measurements

All measurement data are given in Table 4.

Table 4.
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Measurements of Neochromadora parabilineata sp. nov. (in µm except for ratios).

Characters Holotype Paratypes Paratypes
male males (n = 3) females (n = 3)
Total body length 878 905.3±39.6(864–943) 913±27.8(881–931)
Maximum body diameter 25 25.7±1.5(24–27) 33.7±5.8(27–37)
Head diameter 12 11.7±1.5(10–13) 11.7±0.6(11–12)
Length of cephalic setae 8 7±0(7–7) 7.3±0.6(7–8)
Buccal cavity depth 10 10.7±3.1(8–14) 7±1(6–8)
Amphidial fovea width 6 6.7±0.6(6–7) 6±0(6–6)
Amphidial fovea from anterior end 3 3±0(3–3) 3±0(3–3)
Body diameter at amphidial fovea 12 11.7±0.6(11–12) 12±0(12–12)
Nerve ring from anterior end 86 80.7±3.8(78–85) 84±6.1(80–91)
Body diameter at nerve ring 23 21.3±0.6(21–22) 23.3±0.6(23–24)
Pharynx length 131 124.7±2.1(123–127) 128±2.6(125–130)
Pharynx bulb length 23 22.7±0.6(22–23) 26±3.6(23–30)
Body diameter at the base of pharynx 24 22.3±1.2(21–23) 26.3±2.9(23–28)
Cloacal/anal body diameter 25 24±0(24–24) 19.3±0.6(19–20)
Spicules length along arc 31 29±1(28–30) –
Gubernaculum length 21 20.3±1.5(19–22) –
Vulva from anterior end – – 380.3±34.1(341–401)
Body diameter at vulva – – 32±4.6(27–36)
Precloacal supplements 7 7 –
V% – – 41.6±2.5(38.7–43.1)
Tail length 113 111.3±5.5(105–115) 121.7±12.6(110–135)
a 35.1 35.4±3(32–37.9) 27.6±4.3(25.1–32.6)
b 6.7 7.3±0.3(7–7.6) 7.1±0.1(7–7.2)
c 7.8 8.2±0.7(7.6–9) 7.6±0.8(6.9–8.5)
c’ 4.5 4.6±0.2(4.4–4.8) 6.3±0.5(5.8–6.8)

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, posterior 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.

Reproductive system with a single, outstretched testis. Spicules curved and L-shaped, widened at the middle part, 28–31 μm (0.81–0.86 cloacal body diameters) along arc. Gubernaculum short and boat-shaped, distal end tapered. Seven precloacal supplements cup-shaped, distance between the anteriormost and cloaca, the posteriormost and cloaca, 100 μm and 18 μm respectively, distance between supplements almost equal-distanced. Tail conical and gradually tapering, 4.4–4.8 cloacal body diameter in length. Spinneret short, 5 μm in length.

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.

Differential diagnosis

Neochromadora parabilineata sp. nov. is similar to N. bilineata, N. izhorica, N. complexa, and N. poecilosoma in precloacal supplements number (7–9). But it differs from N. bilineata in body length (864–943 μm vs 567–852 μm), cephalic setae length (7–8 μm vs 4–6 μm), amphidial fovea width (50–58% vs 45% corresponding body diameter), spicules shape and length (L-shaped and widened in the middle portion, 28–31 μm vs arcuate and gradually narrowing, 23–26 μm), and gubernaculum length (19–22 μm vs 15–18 μm); differs from N. izhorica in cephalic seta length (7–8 μm vs 14 μm), pharynx shape (posterior bulb obvious vs posterior bulb weak), spicules length (28–31 μm vs 31.5–34.5 μm), gubernaculum shape (distal end tapered vs distal end with anterior-laterally curved tip) (Riemann 1966); differs from N. complexa in body length (864–943 μm vs 642 μm), spicules shape (curved and L-shaped with middle portion widened vs L-shaped even in width), gubernaculum shape (boat-shaped vs dorsal part slenderly extended), distance between precloacal supplements (10–15 μm vs 2–5 μm) (calculation based on Gerlach 1953b: fig. 11); differs from N. poecilosoma in body length (864–943 μm vs 1900–2000 μm), cephalic setae length (7–8 μm vs 10–14 μm), spicule length (28–31 μm vs 60–65 μm), gubernaculum shape (boat-shaped vs distal tip with small tooth) (de Man 1893).

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.

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. 

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.

Sequences of seven genera of the subfamily Hypodontolaiminae, Chromadorita Filipjev, 1922, Dichromadora, Hypodontolaimus de Man, 1886, Innocuonema Inglis, 1969, Neochromadora, Ptycholaimellus Cobb, 1920 and Spilophorella Filipjev, 1917 are included in the SSU and LSU rDNA analyses. At genus level, only species of Chromadorita cluster in one clade (posterior probability 70 in SSU and 99 in LSU, bootstrap value 99 in LSU) but Chromadorita is shown as paraphyletic. Dichromadora multisetosa (OR479915), D. major (OR479911), N. poecilosomoides (OQ396720), and Innocuonema tentabunda (JN968213, as Chromadorita tentabunda) clustered with the Chromadorita clade in the SSU 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).

Acknowledgements

We are grateful to Dr. Kenji Kito for his kind advice in the Neochromadora parabilineata sp. nov. identification, Dr. Huang Yong for sample collecting, and Dr. Wang Yanting for improving the manuscript. We are sincerely grateful to two anonymous referees and the Subject Editor Nic Smol for providing valuable improvements to the manuscript.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

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 (319312101).

Author contributions

Liang Huixin: phylogenetica ananlysis and description of N. parabilineata; Guo Wen: description of D. media and sample identification; Wang Chunming: Ms draft

Author ORCIDs

Huixin Liang https://orcid.org/0009-0006-9073-2062

Wen Guo https://orcid.org/0000-0002-4452-0003

Chunming Wang https://orcid.org/0000-0003-3630-0921

Data availability

All of the data that support the findings of this study are available in the main text.

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