﻿Description of a new nematode species, Chromadorinacommunis sp. nov. (Nematoda, Chromadoridae), from Changdao Island, China and phylogenetic analysis of Chromadorida based on small subunit rRNA gene sequences

﻿Abstract Chromadorinacommunissp. nov. is described from Changdao Island at the confluence of the Yellow and the Bohai seas. The new species is characterized by its medium-sized body; finely striated cuticle with homogeneous punctations; absence of ocelli; buccal cavity with three equal-sized, solid teeth; four cephalic setae; oval amphidial fovea which is positioned between cephalic setae; curved spicules with tapered distal ends; simple, boat-shaped gubernaculums; five or six cup-shaped precloacal supplements; and conical tail with a very short spinneret. A phylogenetic analysis of small subunit rRNA gene sequences using maximum-likelihood and Bayesin inference confirmed the taxonomic position of Chromadorinacommunissp. nov. within Chromadorinae. Tree topology in Chromadorida shows six morphological families clustered into a monophyletic clade and verifies the taxonomic position of the family Neotonchidae based on morphological and molecular analysis.


Introduction
Nematodes are one of the most widely distributed and diverse groups of the animal phyla, and half a million to 10 million nematode species have been estimated (Hodda 2022). However, a large number of species remain undescribed. Phylogenetic analyses based on molecular sequences, especially the small subunit rRNA gene (SSU), combined with morphological characters has updated the classification of Nematoda (De Blaxter 2002, 2004), revealed relationships among nematode groups (Holterman et al. 2006;Meldal et al. 2007), and elucidated the transition between marine, freshwater, and terrestrial different habitats (Holterman et al. 2008(Holterman et al. , 2019. However, due to the scarcity of nematode sequences, especially those of marine nematodes, relationships among nematodes are far from understood. Species of the order Chromadorida Chitwood, 1993 are mainly marine species, and freshwater and limnetic-terrestrial species are also present among them. The Chromadorida are characterized by the combined characters of a punctate cuticle and a female reproductive system with reflexed ovaries (Lorenzen 1981), which distinguish this order from other nematode orders. Lorenzen (1994) summarized the superfamily Chromadoroidea (corresponding to Chromadorida) systematically and gave a phylogenetic analysis of this group based on morphological features. Tchesunov (2014) reviewed the six families of Chromadorida and provided diagnoses of the families and genera; he listed Chromadoridae with five subfamilies and 38 genera, Cyatholaimidae with four subfamilies and 21 genera, Achromadoridae with a single genus (Achromadora), Ethmolaimidae with three genera, Neotonchidae with six genera, and Selachinematidae with two subfamilies and 11 genera. Venekey et al. (2019) reviewed the family Chromadoridae, provided polytomous identification keys for subfamilies and genera, listed valid species of each genus, and provided a phylogenetic analysis based SSU rRNA gene sequences of 11 genera and large subunit (LSU) rRNA gene sequences of eight genera. Phylogenetic relationships within Chromadorida based on full-length sequences of the SSU rRNA gene has been analyzed for 4 families by Holterman et al. (2008) and six families (22 genera) by Leduc et al. (2017). As more Chromadorida SSU rRNA gene sequences have been deposited in the GenBank, an updated and more detailed phylogenetic analysis is needed.
Until now, 29 species of Chromadorina Filipjev, 1918 have been described, among which 25 species are marine and four are limnetic (C. astacicola W. Schneider, 1932, C. bercziki Andrássy, 1962, C. bioculata (Schultze, 1857) Wieser, 1954, and C. viridis (Linstow, 1876) Wieser, 1954. A new species, C. communis sp. nov., is described here and a molecular phylogeny based on SSU rRNA gene sequences within Chromadora is analyzed. The new species was found during a study of the diversity of free-living marine nematodes of Changdao Island. This is the first new species of Chromadorina from the China Sea.

Sample collection and nematode identification
From Changdao Island in June 2022, undisturbed samples were collected from rock surfaces with Ulva lactuca and U. prolifera (Fig. 1). Samples used for morphological analysis were fixed with 10% formalin in seawater, and samples used for molecular analysis were fixed with 95% alcohol. In the laboratory, sediment samples fixed with formalin were washed through two sieves (mesh sizes 500 μm and 45 μm) to separate meiofauna from macrofauna larger than 500 μm. Meiofauna were transferred into a grid-lined Petri dish and sorted under a stereoscopic microscope. Nematodes were transferred into a mixture of ethanol (50%) and glycerin (ratio 1:9 by volume), the ethanol was allowed to slowly evaporate (McIntyre and Warwick 1984), and the specimens 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).

DNA extraction, PCR amplification, and phylogenetic analysis
Samples used for molecular analysis were washed and separated as with formalin-fixed samples. Species of C. communis sp. nov. were selected and confirmed on the temporary slides based mainly on buccal cavity, spicules, and numbers of precloacal supplements. Genomic DNA of seven nematode specimens were extracted with DNeasy Blood & Tissue kit (Qiagen, Germany) and used as templates of nearly full-length SSU rRNA gene amplification with two sets of primers, 1096F (5' -GGT AAT TCT GGA GCT AAT AC -3') / 1912R (5' -TTT ACG GTC AGA ACT AGG G -3') and 1813F (5' -CTG CGT GAG AGG TGA AAT -3') / 2646R (5' -GCT ACC TTG TTA CGA CTT TT -3') (Holterman et al. 2006). 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 new SSU sequence was deposited in GenBank under accession number OP680597. Near full-length SSU rRNA sequences of Chromadorida and Desmodorida (genus Prodesmodora Micoletzky, 1923) were retrieved from GenBank with BLAST; other sequences in GenBank from Chromadorida and longer than 600 bp were also used in the phylogenetic analysis.
Sequences were aligned with the Clustal W algorithm, and the final alignment consisted of 76 sequences from 34 genera. Substitution models of (GTR (general timereversible) + G (gamma distribution) + I (proportion of invariable sites)) were selected as the best-fit model for SSU alignments. A ML analysis was performed with Mega X with 1000 bootstrap replicates. A BI analysis was constructed with CIPRES (http:// www.phylo.org/) and MrBayes on XSEDE v. 3.2.7a was used; the trees were run with chain length of 10,000,000, burn-in frac = 0.25, and the analysis was rooted with Enoplolaimus sp. (accession number KR265034). The topology of the resulting tree was viewed in FigTree v. 1.4.3 and edited with PowerPoint.  Table 1 Diagnosis. Chromadorina communis sp. nov. is characterized by its medium-sized body, finely striate cuticle with punctuations, buccal cavity with three equally sized teeth, absent ocelli, four cephalic setae 6-8 μm long, oval amphidial fovea level with cephalic setae, curved spicules with tapered distal end, boat-shaped gubernaculums, five or six cup-shaped precloacal supplements, and a conical tail with a short spinneret.
Measurements. Detailed measurements information of individual specimens are shown in the Table 1.
Reproductive system monorchic, with anterior testis outstretched, located to right of intestine. Spicules paired, curved, 1.2-1.3 cloacal body diameters long; proximal end slightly cephalate and distal end tapered. Gubernaculum simple, boat-shaped, parallel to the distal end of spicules. Five or six cup-shaped precloacal supplements, 12 μm from cloacal opening and 12-16 μm apart. Tail conical, with short spinneret, with three caudal glands in line.
Eleven sequences of Chromadorina are included in the phylogenetic analysis, but only three species (C. bioculata, C. germanica, and C. communis sp. nov.) are identified to species, and C. bioculata was found in freshwater. Chromadorina communis sp. nov. shows a close relationship with Chromadorina sp. (KJ636255), but it differs by 3.1% (52 in 1678 bp, including three gaps). Chromadorina communis sp. nov. is supported as a new species by both phylogenetic and morphological analyses.

Molecular phylogenetic relationships and analysis
The ML topology obtained with the SSU rRNA gene sequence is in accordance with the BI topology, and only the BI tree is shown (Fig. 4). The main Chromadorida clade is split into two major clades: Clade A composed of Selachinematidae and Chromadoridae and Clade B composed of Cyatholaimidae, Achromadoridae, Neotonchidae, and Prodesmodora (100% posterior probability and 74% bootstrap value). Ethmolaimidae (with one species only, Ethmolaimus pratensis) splits early from the main Chromadorida clade. Six Chromadorida families constitute well-supported monophylectic clades.
In clade A1, nine genera of Selachinematidae form a well-supported monophyletic group (100% posterior probability and 83% bootstrap value), but Selachinematinae and Choniolaiminae are not shown as monophyletic clades at the subfamily level. In Clade A2, Chromadoridae forms a well-supported monophyletic group (100% posterior probability and 96% bootstrap value). In the subfamily level, only Chromadorinae constitutes a monophyletic clade based on the BI analysis (93% posterior probability); however, it is weakly supported by the ML analysis (39% bootstrap value). Hypodontolaiminae and the genus Endeolophos (Euchromadorinae) constituted a monophyletic clade (97% posterior probability and 85% bootstrap value). Endeolophos shows a close relationship with the genus Chromadorita, which was highly supported by the BI analysis (100% posterior possibility), and it clustered with Hypodontolaiminae. The morphological characters of Endeolophos-cuticle homogeneous, amphidial fovea at the level of the cephalic setae, and gubernaculum without telamons-conform to the subfamily Hypodontolaiminae, while other characters-outer labial sensillar setiform, cuticular with complex structure, pharyngeal bulb absent, and precloacal supplements absent-conform to the Euchromadorinae (Tchesunov 2014). We prefer to keep Endeolophos in Euchromadorinae despite the molecular evidence.
Chromadora shows a close relationship with Chromadorina in clade A2, and Venekey et al. (2019) also noted this and explained this relationship as a result of a misidentification; there are great morphological similarities between Chromadora and Chromadorina (Venekey et al. 2019).
In clade B, Gomphionema parvum (clade B1) of family Neotonchidae split early, but it is weakly supported (65% posterior probability and 40% bootstrap value). The position of Neotonchidae has been uncertain for a long time. Platt (1982) united Neotonchinae and Ethmolaiminae into Ethmolaimidae with the holapomorphy of "cupshaped precloacal supplements with an external articulated flange", but this character was doubted by Lorenzen (1994), as it also occurs in Dichromadora. Tchesunov (2014) also considered the validity of the family Neotonchidae due to its mixed characters of Chromadoridae, Cyatholaimidae, and Microlaimidae. According to our BI and ML analyses, Neotonchinae (clade B1) is a sister taxon to Achromadoridae (clade B2), Prodesmodora (clade B3), and Cyatholaimidae (clade B4), but there is no close relationship with Ethmolaiminae. This is in accordance with the topology tree by Leduc et al. (2017), but it is contrary to Bezerra et al.'s (2013) results based on morphological characters. We prefer to retain Neotonchidae until more molecular evidence is available. In clade B3, two freshwater genera, Achromadora and Prodesmodora, form a highly supported clade (100% posterior probability and 100% bootstrap value). Lorenzen (1981Lorenzen ( , 1994 concluded that Achromadora is a holophylectic based on the the position of the ovary and prevailing parthenogenetic reproduction, and holapomorphy was presented in Prodesmodora. Taxonomic position of Prodesmodora should be reconsidered.

Conclusions
Chromadorina communis sp. nov. is described based on morphological characteristics and distinguished from allied species by the absence of ocelli, a buccal cavity with three equally sized, solid teeth, curved spicules with a tapered distal end, simple and boatshaped gubernaculums, and a conical tail with a very short spinneret. A phylogenetic analysis also supports the validity of the new species. With the description of C. communis sp. nov., 30 species of Chromadorina have been identified.
The phylogenetic analysis of Chromadorida show six families clustered into a monophyletic clade, and this conforms to the morphological taxonomy at the family level. The genus Endeolophos should be kept in Euchromadorinae based on morphological characters, and the position of family Neotonchidae should be considered as valid until further data are available.