Morpho-Molecular and Ultrastructural Characterization of Discocriconemella Parasinensis n. Sp. from Zhejiang Province, China

Abstract During a recent inventory survey of the nematofauna of tea plantation at Zhejiang Province, China, Discocriconemella parasinensis n. sp. was detected in the rhizosphere of Camellia sinensis. The new species can be characterized by having the uninterrupted rounded labial disc, en face view showing rectangular-rounded labial plate without submedian lobes, R = 82.6 (80–86), Rex = 22 (21–23), stylet length of 68.3 (59–76) μm, excretory pore located 1–2 annuli posterior to the esophageal bulb, vulva open, postvulval body elongated conoid, and tail conoid with bilobed terminus. Morphologically, the species shares the same lip-type with D. discolabia, D. mauritiensis, D. mineira, D. perseae, and D. sinensis. Phylogenetic relationships of the new species based on D2–D3 expansion segments of 28S, ITS, and 18S rRNA genes revealed that D. parasinensis n. sp. formed a separated clade from other criconematid species, thereby supporting its status as a new species of the genus. The new species showed close phylogenetic relationships with Criconemoides geraerti.

Zhejiang, located in southeast coast of China, is a well-renowned tea producing province and pivotal to the Chinese tea industry in terms of the highest acreage and economic returns (FAO and CAAS, 2021). Over the last 10 years, tea cultivation has been expanded to meet the increased tea consumption and to benefit small-scale growers (Liang et al., 2021).
To maintain high standards of tea cultivation, planted fields are regularly surveyed to examine the presence of pest species. The latter involves the collection of soil samples and determining the associated plant-parasitic nematodes. Recent reports have described the incidence of ring nematodes from tea cultivated areas of Zhejiang (Maria et al., 2018a(Maria et al., , 2019. Therefore, surveys have been carried out to study the nematofauna of tea plantations in Zhejiang. During our recent survey, we isolated a population of Discocriconemella sp. that exhibited uninterrupted labial disc, robust stylet, and conoid elongate posterior body.
Discocriconemella (De Grisse and Loof, 1965) includes 29 species, and so far only three species [D. hengsungica (Choi and Geraert, 1975); D. limitanea (Luc, 1959;De Grisse and Loof, 1965); and D. sinensis (Maria et al., 2019)] have been reported from China (Geraert, 2010;Maria et al., 2018aMaria et al., , 2019. To determine the precise identity of recently isolated specimens, a detailed morphological study on lip region, vulva structure, and tail lobes was conducted using scanning electron microscopy (SEM) and light microscopy. The qualitative and quantitative characteristics of this population were compared with related species, and we found that this species possesses unique characters that support its status as a new species.
Hence, it is described herein as D. parasinensis n. sp. with the following objectives: (i) to provide an integrative morphological and molecular characterization of D. parasinensis n. sp.; (ii) to elucidate important morphological details through SEM observations; and (iii) to study the phylogenetic relationships of this newly discovered Discocriconemella sp. with other criconematid species using 18S, D2-D3 of 28S, and ITS rRNA gene sequences.

Materials and methods
Nematode population sampling, extraction, and morphological identification Nematodes were extracted from soil samples using the Cobb sieving and flotation-centrifugation method (Jenkins, 1964). For morphometric studies, the nematodes were killed and fixed with hot formalin and processed to glycerin (Seinhorst, 1959) as modified by De Grisse (1969). The measurements and light micrographs of nematodes were made with a Nikon Eclipse Ni-U 931845 compound microscope. The drawings were made using a drawing tube attached to the microscope and were redrawn using Corel DRAW software version 16 (Corel). For the SEM examination, 40-50 handpicked nematodes were fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde, washed three times in 0.1M cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated in a series of ethanol solutions, and critical point-dried with CO 2 . After mounting on stubs, the samples were coated with gold at 6-nm to 10-nm thickness and the micro-graphs were made at 3-5 kV operating system of Hitachi SU8010 (Maria et al., 2018a).

Phylogenetic analyses
Newly obtained sequences of 18S, D2-D3 expansion segments of 28S and ITS gene sequences, and available sequences of other nematodes obtained from GenBank were used for phylogenetic reconstructions of criconematid species. The selection of outgroup taxa for each dataset was based on previously published studies (Maria et al., 2018a(Maria et al., , 2019. Multiple sequence alignments of the different genes were completed using the FFT-NS-2 algorithm of MAFFT v7.450 (Katoh et al., 2019). The BioEdit program v7.2.5 (Hall, 1999) was used for sequence alignment visualization and edited using Gblocks v0.91b (Castresana, 2000) using options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences + 1; maximum JOURNAL OF NEMATOLOGY number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half). Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes v3. 1.2 (Ronquist and Huelsenbeck, 2003). The best-fit model of DNA evolution was achieved using JModelTest v2.1.7 (Darriba et al., 2012) with the Akaike information criterion (AIC). The best-fit model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses. The transition model with invariable sites and a gamma-shaped distribution of invariable sites (TIM3+I +G, TIM1+I+G) for the D2-D3 segments of the 28S rRNA and 18S, respectively, and the general time-reversible model with invariable sites and a gamma-shaped distribution (GTR+I+G) for the ITS rRNA gene and were run with four chains for 4 × 10 6 generations, respectively. A combined analysis of the two ribosomal genes was not undertaken because several sequences are not available for all species. The sampling for Markov chains was carried out at intervals of 100 generations. For each analysis, two runs were conducted. After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for more in-depth analyses. The topologies were used to generate a 50% majority-rule consensus tree. On each appropriate clade, posterior probabilities (PP) were given. FigTree software v1.4.3 (Rambaut, 2016) was used for visualization of trees from all analyses.

Description
Female Body habitus C-shape after heat relaxation. Cuticle finely annulated, annuli retrorse with posterior margins smooth. A few anastomoses present at mid or posterior body. The first cephalic annulus enlarged disc like. En face view showed a circular uninterrupted disc without submedian lobes. Lip pattern matching that of group 1 proposed for Discocriconemella species by Vovlas (1992). Labial plate rounded to rectangular in shape slightly raised, with slit-like oral apertures. Stylet rigid and robust, with anchor shaped stylet knobs. Dorsal esophageal gland opening posterior to stylet base esophageal components and general morphology is criconematid type. Esophageal lumen looped in median esophageal bulb, which has medium-sized refractive valvular apparatus. Isthmus narrow, short, encircled by nerve ring. Basal esophageal bulb elongated. Excretory pore conspicuous, situated 1-2 annuli posterior to esophageal bulb. Vulva open (SEM observation), without vulval flaps, the anterior lip slightly overhangs and covers the preceding annuli. Vagina straight, extending for less than half of body diam. Spermatheca rounded to oval, filled with few sperm cells. Gonad single, prodelphic. Oocytes arranged in single file except for a short region of multiplication near anterior end. Anus small, indistinct located 2-3 annuli posterior to vulva. Postvulval body elongated conoid. Tail short, conoid, with a bilobed terminus.

Male and juvenile
Not studied.

Type Host and Locality
The new species was detected in association with Camellia sinensis (L.) Kuntze, 1887 from Zijingang Campus of Zhejiang University, Hangzhou, Zhejiang Province, P.R. China. The geographical position of the sampling site is 120°448˝E, 30°18´36˝N.

Etymology
The specific epithet D. parasinensis formed from the Latin word para = beside or near, and the species epithet sinensis, thereby reflecting its close morphological similarity to D. sinensis. Based on the lip pattern scheme proposed by Vovlas (1992), this new species belongs to group 1 in having a rounded uninterrupted labial disc. It shares the same lip type with D. discolabia (Diab and Jenkins, 1966;De Grisse, 1967), D. mauritiensis (Williams, 1960;De Grisse and Loof, 1965), D. mineira (Vovlas et al., 1989), D. perseae (Cid del Prado Vera and Loof, 1984), and D. sinensis. Moreover, the new species is also compared with D. limitanea (Luc, 1959;De Grisse and Loof, 1965), which is a widespread and common species of the genus.

Molecular profiles and phylogenetic status
The new Discocriconemella species was molecularly characterized using 18S, D2-D3 expansion segments of 28S and ITS sequences of rRNA genes. The newly obtained sequences were deposited in the GenBank. Phylogenetic relationships among criconematid species were inferred using the aforementioned gene sequences using BI and are given in Figures 4-6, respectively.
The 18S tree (Fig. 4) was constructed from 34 criconematid taxa with Paratylenchus straeleni (AY284631) and P. microdorus (AY284633) as outgroup species. The tree has two well supported major clades. The new species is in the first clade and shows sister relationship with Criconemoides geraerti (MN738712-MN738713) and D. sinensis (MZ470425). The cosmopolitan species D. limitanea is in the basal position of the first clade and grouped with species of Mesocriconema, while D. hengsungica grouped with Xenocriconemella macrodora in the second clade. In this tree, all four Discocriconemella species arranged distantly from each other and showed paraphyletic relationships except D. sinensis.
The D2-D3 of the 28S tree (Fig. 5) was constructed from 35 criconematid taxa with P. dianthus (KF242228) and P. hamatus (KF242218) as outgroup species. The tree has four well-supported clades    The ITS tree (Fig. 6) was constructed from 32 criconematid taxa with P. dianthus (KF242271) and P. hamatus (KF242247) as outgroup species. The tree has three well supported clades, the new species grouped in the second clade with C. geraerti (MN738718-MN738719), C. informis (KF908246), and D. sinensis (MK253546) whereas D. hengsungica grouped with Ogma decalineatus and again occupied the basal position in the tree.
Phylogenetic analyses conducted in this study indicated that the D. parasinensis n. sp. closely grouped with C. geraerti and C. informis instead of its own genus members, that is, D. hengsungica, D. limitanea, and D. sinensis. In Discocriconemella, the presence of disc-like lip annulus is the salient character, none of the Criconemoides species reported to have such labial morphology. In this context, the close phylogenetic affinity of D. parasinensis n. sp., with Criconemoides species, is unresolved and at this point remains a subject to further research.

Discussion
Discocriconemella species are generally reported from warmer areas of the world. The distribution of reported species of Discocriconemella is as follows: the majority of the species were reported from Asia (12 spp.), followed by Africa (6 spp.) and South America (6 spp.), very few species were described from Oceania (3 spp.) and North America (2 spp.) (Geraert, 2010). The host associations of Discocriconemella species are also not well documented, the majority of species were reported in association with grasses or woody plants (Siddiqi, 2000).
The labial arrangement of Discocriconemella is the main generic character distinguishing it from other genera of Family Criconematidae. However, this common morphological character of the genus does not seem to reflect in the phylogenetic positioning. In our phylogenetic analyses, D. parasinensis n. sp., grouped next to Criconemoides geraerti instead of other Discocriconemella species. Morphologically, both species are entirely different, their close phylogenetic affinity indicates the presence of divergent lineages in genus Discocriconemella. Only D. hengsungica, D. limitanea, and D. sinensis were described with sequence-based data, however, in all our phylogenetic analyses these species also grouped independently and distant from D. parasinensis n. sp. except for D. sinensis. These paraphyletic relationships were also observed in our previous studies (Maria et al., 2018a(Maria et al., , 2019, however, due to the scarcity of Discocriconemella sequences such phylogenetic positioning for D. parasinensis n. sp. and other members cannot be completely clarified. A similar taxonomic puzzle was observed by Qiao et al. (2018) while describing a new species of genus Labrys-the authors reported that L. fujianensis is sister to Miculenchus and is never closely related to other Labrys species. Disconcordance of 18S and 28S rDNA phylogenies have been observed by Qing et al. (2018) while studying the phylogeny of Tylenchidae taxa and reported that the phylogenetic position of Miculenchus is not straightforward for example in 28S rRNA phylogeny, it has a sister relationship to Malenchus whereas, in the 18S rRNA phylogeny, Miculenchus is sister to Lelenchus and thus divergent from Malenchus. Consequently, divergent positioning of species of the same genus is not unexpected and can be elucidated in detail, once the molecular information of all the member species becomes available.
Since our phylogenetic analyses are not very conclusive, based on species' morphology, we recognize D. parasinensis n. sp. in group 1 of Discocriconemella species. The Discocriconemella grouping was proposed by Vovlas (1992) and divided the genus into four different groups based on the labial disc morphology. The brief definition of each group is as follows: labial disc round with uninterrupted margins (group 1); disc with ventral and dorsal deep indentations (group 2); disc indentations giving a four-lobed appearance (group 3); and rounded disc with paired dorsal and ventral projections (group 4). The Discocriconemella species included in our phylogenetic analyses comes in three different groups (such as D. parasinensis n. sp. and D. sinensis -group 1, D. limitanea -group 2, D. hengsungica -group 4) and hold a unique phylogenetic position in our trees. Therefore, we agree with other nematologists that morphological grouping was only established for the convenience of identification and does not always reflect the evolutionary history (Gutiérrez-Gutiérrez et al., 2013;Tzortzakakis et al., 2014;Handoo et al., 2016;Maria et al., 2018b).
Despite the frequent abundance of criconematids in the rhizosphere of natural vegetation, their life histories and feeding habits are not well studied (Maria et al., 2018a(Maria et al., , 2019. This could be due to the reason that the majority of criconematids were reported from the grasslands, they have never been associated with agricultural production areas. Based on criconematids robust and long stylets they are either considered as epidermal or cortical feeders of higher plants (Siddiqi, 2000). In the present study, D. parasinensis n. sp. was recovered in lower density which suggests that as of yet, it is a mild parasitic species and do not behave as potential pest. In addition to that, the presence of D. parasinensis n. sp. in the rhizosphere of Tea plants (Camellia sinensis) indicated that the known diversity of Discocriconemella has increased and this type of vegetation harboring much more nematofauna that has been expected to be discovered.