A new rare nematode Nothocriconemoides hangzhouensis n. sp. (Nematoda: Criconematidae) from Hangzhou, China

Nematodes were extracted from soil and root samples using the modified Cobb sieving and flotation-centrifugation method (Jenkins, 1964). For morphometric studies, nematodes were killed and fixed in hot formalin (4% with 1% glycerol) and processed in glycerin (Seinhorst, 1959). The measurements and light micrographs of nematodes were made with a Nikon Eclipse Ni-U 931845 compound microscope. For the SEM examination, the nematodes were fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde, washed three times in 0.1 M cacodylate buffer, post-fixation 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 with 6 to 10-nanometer thickness and the micrographs were made with 3 to 5 kV operating system (Maria et al., 2018a).

DNA was extracted by transferring individual nematodes into the Eppendorf tube containing 16 μL ddH₂O. Nematodes were crushed using a sterilized pipette tip, the tubes were centrifuged at 12,000 rpm for 1 min and frozen at −68°C for at least 30 min. Tubes were heated to 85°C for 2 min, and then, 2 μL proteinase K and PCR buffer solution were added. The tubes were incubated at 56°C for 1 to 2 hr and, then, at 95°C for 10 min. After incubation, these tubes were cooled to 4°C and used for conducting PCR (Zheng et al., 2003). Several sets of primers (synthesized by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify the partial 18 S, ITS region, D2–D3 of 28 S of rDNA and partial coxI fragments. Primers for amplification of partial 18 S were 18s900–18s1713 (Olson et al., 2017). Primers for amplification of ITS were TW81-AB28 (Joyce et al., 1994). The primers for amplification of D2–D3 of 28 S were D2A and D3B (De Ley et al., 1999). And, finally, the primers used for coxI amplification were COI-F5 (5’-AATWTWGGTGTTGGAACTTCTTGAAC-3’) and COI-R9 (5’-CTTAAAACATAATGRAAATGWGCWAC WACATAATAAGTATC-3’) (Powers et al. 2014). PCR conditions were as described by Ye et al. (2007) and Powers et al. (2010). PCR products were evaluated on 1% agarose gels stained with ethidium bromide. PCR products of sufficiently high quality were sent for sequencing by Invitrogen (Shanghai, China).

Newly obtained sequences of Nothocriconemoides hangzhouensis n. sp. (D2-D3 expansion segments of 28 S, ITS, partial 18 S rRNA, and partial coxI) and the available sequences of other criconematid nematodes obtained from NCBI were used for phylogenetic analyses. Outgroup taxa for the dataset were chosen according to previous published data (Afshar et al., 2019; Maria et al. 2019). Multiple alignments of the different sequences were made using the FFT-NS-2 algorithm of MAFFT v. 7.205 (Katoh and Standley 2013). Sequence alignments were manually visualized using BioEdit (Hall 1999) and edited by Gblocks ver. 0.91b (Castresana 2000) in the Castresana Laboratory server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) 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 3.1.2 (Ronquist et al., 2012). The best-fit model of DNA evolution was obtained using JModelTest V.2.1.7 (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 given to MrBayes for the phylogenetic analyses. An unlinked general time-reversible model with invariable sites and a gamma-shaped distribution (GTR fo li + G) was used for the D2-D3 expansion segments of 28 S rRNA, ITS, partial 18 S, and partial coxI. These BI analyses were run separately per dataset using four chains for 2 × 10⁶ generations for all of the molecular markers. A combined analysis of the three genes was not undertaken due to some sequences not being available for all species. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority-rule consensus tree. Bayesian posterior probabilities (BPP) are given on appropriate clades. Trees from all analyses were visualized using FigTree software V.1.42 (http://tree.bio.ed.ac.uk/software/figtree/).

Nothocriconemoides hangzhouensis n. sp.

(Figs 1–4; Table 1).

Figure 1:

Figure 2:

Figure 3:

This population was found in the rhizosphere of Ulmus sp. from Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang Province, P.R. China, on February 2019. The geographical position of the sampling site is E: 120 °4´54˝ N: 30 °17´5.

Holotye female and 13 female paratypes (slide numbers ZJU-30-01-ZJU-30-03) were deposited in the nematode collection of Zhejiang University, Hangzhou, China. Four females and two juveniles paratypes on two slides (Slide numbers T-7353, T-7356) and ten additional females on two slides were (T-7354-55) deposited at USDA nematode collection, Beltsville, Maryland, USA. The Zoobank code is as follows: LSID urn:lsid:zoobank.org:pub:E977B880-9EF3-4E2C-BB29-4BD970D63F0A

The species epithet refers to the City name where the species was detected.

The new species can be characterized by wider body annuli that have fine longitudinal striations that look like annulus bearing rough cuticular margins, and 2 to 3 anastomoses at the posterior half of the body. The first cephalic annulus is rounded and expanded enclosing the lip region, and the second annulus is narrow, offset, collar like. Four prominent submedian lobes are present. Excretory pore is 3 to 4 annuli posterior to esophageal bulb. Vagina is straight and vulva is closed. The ventral side of postvulval annuli is inverted, in majority of individuals. Anus is indistinct and located on the next annuli posterior to vulva. Tail is short, with forked or branched terminus.

The genus only contains two species; It can be differentiated from N. crenulatus by having shorter stylet 64.4 to 75.5 vs 87 to 96 µm long, less number of body annuli R = 35.0 to 39.0 vs 60 to 68, less number of annuli between vulva and tail terminus RV = 2.0 to 3.0 vs 6 to 8, location of anus (next annuli to vagina vs 3 to 4 annuli posterior to vagina), and tail terminus morphology (terminal annulus forked or branched vs button shaped).

The new species differs from N. lineolatus by less number of body annuli R = 35.0 to 39.0 vs 57 to 64, less number of annuli between vulva and tail terminus RV = 2.0 to 3.0 vs 7 to 9, location of anus (next annuli to vagina vs 3 to 5 annuli posterior to vagina), anastomosis (2-3 vs 1), lip annuli (2 vs 4), submedian lobes (separate as four vs connected as 2 subdorsal and sublateral lobes), position of excretory pore (3 to 4 annuli posterior to esophageal bulb vs at the same level of esophageal bulb), vulval lip ornamentation (absent vs present), and tail terminus morphology (terminal annulus forked or branched vs bifid or irregular).

The new species was molecularly characterized using partial 18 S, D2-D3 of 28 S, ITS and coxI sequences and obtained sequences were deposited in the GenBank. As this is the sole species of genus Nothocriconemoides with molecular characterization, in order to predict the closely related species all the named criconematid species were included in the phylogenetic analysis.

In 18 S tree (Fig. 5), Nothocriconemoides hangzhouensis n. sp. (MN879878-MN879881) is a sister species of Criconema demani (Micoletzky, 1925) (MH828134), Discocriconemella sinensis (Maria et al., 2019) (MK253543) and Neolobocriconema serratum (Khan and Siddiqi, 1963; Mehta and Raski, 1971) (MH668972). The pairwise sequence identities of the new species with its sister species are 97.53 to 98.9% (5-20 bp difference). This clade is further grouped with Bakernema inequale (Taylor, 1936; Mehta and Raski, 1971) (MF094923) and species of Mesocriconema (Andrássy, 1965), Lobocriconema (De Grisse and Loof, 1965) and D. limitanea (Luc, 1959; De Grisse and Loof, 1965) (MF095032).

Figure 5:

In 28 S tree (Fig. 6), Nothocriconemoides hangzhouensis n. sp. (MN879889-MN879890) clustered with species of Mesocriconema, Criconemoides (Taylor 1936), Lobocriconema and Neobakernema (Ebsary 1981) but it is sister species of Discocriconemella sinensis (MK253537) and Criconemoides informis (Micoletzky, 1922; Taylor, 1936) (AY780970). The pairwise sequence identities of the new species with its sister species are 84.93-89.01% (70-82 bp difference).

Figure 6:

The majority of criconematid species were not characterized for ITS sequences, and based on the available sequences, the ITS tree (Fig. 7), was constructed. It indicated that Nothocriconemoides hangzhouensis n. sp. (MN876029-MN876030) is a sister species of Discocriconemella sinensis (MK253546). The sequence similarity between the new species and the sister species is 81.80% (130 bp difference). This clade further grouped with species of Criconemoides, Lobocriconema, Mesocriconema, Neobakernema and D. limitanea.

Figure 7:

In the coxI tree (Fig. 8), Nothocriconemoides hangzhouensis n. sp. (MN867795-MN867799, MN867800) clustered with species of the genera Bakernema, Criconemoides, Lobocriconema, Mesocriconema, Neobakernema, Neolobocriconema, but it is a sister species of Discocriconemella sinensis (MK249990) and Criconemoides informis (MF770692). The pairwise sequence identities of new species with its sister species are 82.7 to 89.0%.

Figure 8: