Morpho-Molecular and Ultrastructural Characterization of Discocriconemella parasinensis n. sp. from Zhejiang Province, China

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 CO₂. 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).

DNA samples were prepared according to Zheng et al. (2003). Four sets of primers (synthesized by Healthy Creatures, Hangzhou, China) were used in the PCR analyses to amplify the near full-length 18S, D2–D3 expansion segments of 28S, and ITS region of rRNA. Partial 18S region was amplified with two sets of primers. The first set was 18s39F (5′-AAAGATTAAGCCATGCATG-3′) and 18s977R (5′-TTTACGGTTAGAACTAGGGCGG-3′), and the second set was 18s900F (5′-AAGACGGACTACAGCGAAAG-3′) and 18s1713R (5′ TCACCTACAGCTACCTTGTTACG-3′) (Olson et al., 2017). Primers for amplification of ITS were F195 (5′-TCCTCCGCTAAATGATATG-3′) and V5367 (5′-TTGATTACGTCCCTGCCCTTT-3′) (Vrain et al., 1992). Primers for amplification of D2–D3 28S were the forward D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and the reverse D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (De Ley et al., 1999). PCR conditions were maintained as described by Ye et al. (2007) and Powers et al. (2010). PCR products were evaluated on 1% agarose gels and stained with Gelred (Tsingke Biotechnology, TSJ003). PCR products of sufficiently high quality were sent for sequencing to Healthy Creatures. The newly obtained sequences were submitted to the GenBank database under accession numbers indicated on the phylogenetic trees.

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, 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 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 × 10⁶ 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.

Discocriconemella parasinensis n. sp. (Figs. 1–3 and Table 1).

Figure 1

Figure 2

Figure 3

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°4′48″E, 30°18′36″N.

Holotype female and 10 female paratypes (slide numbers ZJU-32-01 to ZJU-32-10) deposited in the nematode collection of Zhejiang University, Hangzhou, P.R. China. Five female paratypes were deposited at the USDA nematode collection, Beltsville, MA, USA. The Zoobank code for the new species is as follows: LSID: zoobank.org:act:49F1E049-BA02-46B1-82EF-304F1F40390B.

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.

Discocriconemella parasinensis n. sp. can be characterized by having the uninterrupted rounded labial disc, en face view showing rectangular-rounded labial plate without submedian lobes. Total number of body annuli (R = 82.6 [80–86]), number of annuli from anterior end to excretory pore (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, tail conoid with bilobed terminus.

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.

It differs from D. discolabia by having longer body length (382–556 μm vs. 190–300 μm), longer stylet (59–76 μm vs. 35–47 μm), less number of annuli on the body (R = 80–86 vs. 155–174), less number of annuli between anterior end to excretory pore (Rex = 21–23 vs. 47–56), less number of annuli between vulva to tail terminus (RV = 9–11 vs. 14–17), less number of annuli between anus to tail terminus (Ran = 6–7 vs. 10–14), crenation on annuli absent (vs. present).

From D. mauritiensis by having longer body length (382–556 μm vs. 280–390 μm), longer stylet (59–76 μm vs. 33–38 μm), less number of annuli on the body (R = 80–86 vs. 140–152), less number of annuli between anterior end to excretory pore (Rex = 21–23 vs. 42–45), more number of annuli between vulva and anus (2–3 vs. 1), less number of annuli between anus to tail terminus (Ran = 6–7 vs. 8–10), V value (88–91 vs. 93–96), crenation on annuli absent (vs. present), absence of rudimentary submedian lobes (vs. present), and vulval lip morphology (anterior lip not enlarged vs. enlarged).

D. limitanea belongs to group 2 in having a labial disc with ventral and dorsal deep indentations. The new species differs from D. limitanea by having longer body length (382–556 μm vs. 160–310 μm), less number of annuli between anterior end to excretory pore (Rex = 21–23 vs. 30–38), anastomoses (few vs. numerous), and tail terminus (bilobed vs. bluntly rounded).

From D. mineira by having longer body length (382–556 μm vs. 250–340 μm), V value (88–91 vs. 93–94), VL/VB ratio (1.1–1.5 vs. 0.8–1.0), absence of rudimentary submedian lobes (vs. present), the position of excretory pore (1–2 annuli posterior to esophageal bulb vs. at or anterior to it), the shape of vagina (straight vs. curved), shape of vulval anterior lip (simple vs. bilobed), and tail terminus (bilobed vs. lobed).

From D. perseae by having longer body length (382–556 μm vs. 220–370 μm), less number of annuli on the body (R = 80–86 vs. 108–126), less number of annuli between anterior end to excretory pore (Rex = 21–23 vs. 32–38), less number of annuli between vulva to tail terminus (RV = 9–11 vs. 14–20), less number of annuli between anus to tail terminus (Ran = 6–7 vs. 9–12), the position of excretory pore (1–2 annuli posterior to esophageal bulb vs. at the base of it), shape of vagina (straight vs. sigmoid), and shape of postvulval body (elongated conoid with bilobed terminus vs. convex conoid, terminus subacute, and directed dorsal).

From D. sinensis by having more number of annuli on the body (80–86 vs. 64–69), absence of rudimentary submedian lobes (vs. present), excretory pore (conspicuous vs. inconspicuous), the position of excretory pore (1–2 annuli posterior to esophageal bulb vs. at the base of it), and tail terminus (bilobed vs. lobed).

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.

Figure 4

Figure 5

Figure 6

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 (PP = 0.96–1.00), the new species grouped in the first clade with C. geraerti (MN738725-MN738727), C. informis (KU722386, AY780970), D. sinensis (MZ011403-MZ011404), and D. limitanea (MZ254775) whereas D. hengsungica grouped with X. macrodora and occupied the basal clade.

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.