Morphological and Molecular Characterization of Quinisulcius curvus from China

Abstract A stunt nematode species, Quinisulcius curvus, recovered from the rhizosphere of sea randa (Guettarda speciosa), is described and characterized herein based on integrative taxonomy. Morphometrics and distribution of all reported populations of Q. curvus are also discussed. The Chinese population of Q. curvus displayed slight variation in stylet length; however, the rest of the characters matches well with the original description. This is the first record of Q. curvus from Hainan, China, and the first molecular characterization for this species. Phylogenetic analysis based on partial 18S, 28S and ITS sequences placed Q. curvus with related stunt nematodes species, but clearly separated from them. The present study expanded the geographic record and provided molecular data on Q. curvus from China.

Unlike Tylenchorhynchus species, the biology and host association of Quinisulcius species are not well documented. So far, Q. capitatus and Q. acutus have been found damaging agronomic and horticultural crops including melon, maize, wheat, and sorghum (Claflin, 1984;Cuarezma-Teran and Trevathan, 1985;Khan et al., 1988;Khan and Khanzada, 1990;Singh et al., 2013;Todd et al., 2014). Therefore, it is imperative to correctly diagnose, and document the species identity in order to recognize the host association and geographic range of the species in question. In addition to that, information regarding the occurrence and distribution of plant-parasitic nematodes in agricultural or forestry areas is important to assess the damage potential of inhabiting species (Hafez et al., 2010). In majority of cases, nematode damage symptoms are frequently underestimated or misidentified to other stresses (Barker et al., 1994;Singh et al., 2013).
The nematode diversity is not well studied in Hainan Province, since the majority of nematological research was conducted on the more aggressive plant parasitic nematodes such as root-knot nematodes (Liu et al., 2005;Long et al., 2019;Zhuo et al., 2008), cyst forming nematodes (Zhuo et al., 2013) and virus vector nematodes (Li et al., 2020;Luo et al., 2001).
Therefore, detailed samplings were conducted with a focus to determine the identity of ectoparasitic nematodes in Hainan Province. In this study, a population of stunt nematodes was recovered from the rhizosphere of a shrub, sea randa in 2019. No above ground symptoms were observed on the host. The population was detected in high density (200-300/100 g of soil) as compared to other soil nematodes. Preliminary examination showed that the species has five lateral lines which is a salient characteristic of genus Quinisulcius. Therefore morphological and molecular characterization were performed, and the results were compared with the nominal species of Quinisulcius. The morphological characters of the population confirm the close resemblance to Quinisulcius curvus (Williams, 1960) Siddiqi, 1971. Literature studies indicated that the species was described decades ago in the rhizosphere of Sugarcane from Mauritius with scarce morphological details. It also elaborated that Q. curvus was reported from Dominican Republic, Pacific Islands, Martinique, Thailand, India but without morphometrical or morphological characterization (Bohra and Baqri, 2005;Bridge, 1988;Cadet et al., 1994;Román, 1968;Toida et al., 1996), moreover some reports are in languages other than English. Besides, we also noted that of 17 nominal species, sequencebased information of Quinisulcius is only available for Q. capitatus. Therefore, the objective of the present study was i) to provide detailed morphological, morphometrical and molecular characterization of Q. curvus ii) study the phylogenetic relationships of Q. curvus with other stunt nematode species.

Isolation and morphological observation of nematodes
Rhizosphere soil samples were collected from sea randa. Nematodes were extracted from soil and root samples of sea randa using the modified Cobb sieving and flotation-centrifugation method (Jenkins, 1964). Nematode suspension contained mixture of herbivores (Dorylaimids), fungivores (Aphelenchoides, Filenchus, Aphelenchus spp.) bacterivores (Rhabditis sp.) and populations of spiral and Quinisulcius sp. Since Quinisulcius was the most abundant species in the soil suspension, the females were collected individually from the mixture of soil nematodes and studied under light micrscope. For preliminary examinations, fresh Quinisulcius females were transferred to a drop of distilled water, heat relaxed and observed under a Zeiss microscope. For additional morphological and morphometric studies, nematodes were killed and fixed in hot formalin (4% formaldehyde) and processed to ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969) and mounted on permanent slides. Measurements were made on mounted specimens, light micrographs and illustrations were produced using a Zeiss microscope equipped with a Zeiss AxioCam MRm CCD camera.

DNA extraction, PCR and sequencing
DNA samples were prepared according to Li et al. (2008). Four sets of primers (synthesis by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify sequences of the near full-length 18S region, D2-D3 expansion segments of 28S, and ITS of ribosomal RNA genes (rDNA). The 18S region was amplified with primers 988F/1912R and 1813F/2646R (Holterman et al., 2006). The 28S D2-D3 region was amplified with primers D2A/D3B (De Ley et al., 1999), and the ITS was amplified using primers TW81/AB28 (Tanha Maafi et al., 2003). PCR conditions were as described by Ye et al. (2007) and Li et al. (2008). PCR products were separated on 1% agarose gels and visualized by staining with ethidium bromide. PCR products with high quality were purified for cloning and sequencing by Invitrogen, Shanghai, China.

Phylogenetic analyses
Sequenced DNA fragments from the present study (after discarding primer sequences and ambiguously aligned regions) and other stunt nematode sequences obtained from GenBank were used in the phylogenetic reconstruction. Outgroup taxa for each dataset were selected based on previously published studies (Handoo et al., 2014;Munawar et al., 2021;Nguyen et al., 2019). Multiple sequence alignments of the newly obtained and published sequences were made using the FFT-NS-2 algorithm of MAFFT V.7.450 (Katoh et al., 2019). Sequence alignments were visualized with BioEdit (Hall, 1999) and manually 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 conducted based on Bayesian inference (BI) using MRBAYES 3.2.7a (Ronquist and Huelsenbeck, 2003). The best-fit model of DNA evolution was calculated with the Akaike information (AIC) of JMODELTEST V.2.1.7 (Darriba et al., 2012). The best-fit model, the base frequency, the proportion of invariable sites, substitution rates and the gamma distribution shape parameters in the AIC were used for phylogenetic analyses. BI analyses were performed under a transitional model, with a rate of variation across sites (TIM3 + G) for the partial 28S region; a transversional model with a proportion of invariable sites and a rate of variation across sites (TVM + I + G) for ITS; and a transitional model with a proportion of invariable sites and a rate of variation across sites (TIM1ef + I + G) for 18S region. These BI analyses were run separately per dataset with four chains for 2 × 10 6 generations. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis. 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. Posterior probabilities (PP) are given on appropriate clades. Trees from all analyses were edited by FigTree software V. 1.4.4 (Rambaut, 2014).

Male
Not found.

Taxonomic notes
Quinisulcius curvus was originally described from Mauritius in the rhizosphere of sugarcane (Williams, 1960). Since then, the species has been reported from Dominican Republic, Pacific Islands, Martinique, Thailand, India, Pakistan in the rhizosphere of sugarcane, grapes, maize, tuber and vegetable crops (Bohra and Baqri, 2005;Bridge, 1988;Cadet et al., 1994;Hussain et al., 2016;Mizukubo et al., 1993;Román, 1968;Toida et al., 1996). Though it has been reported from several countries but morphometrical and morphological data was only provided in few  reports (Table 1). Morphometrically, it is observed that the Hainan population of Q. curvus has a shorter stylet than the type population (11.5-13.5 µm vs 17.0 µm) and other reported descriptions (Table 1), but close to a Thailand population (11.5-13.5 µm vs 13.5-15 µm) (Mizukubo et al., 1993), suggesting a high intraspecific variability on this character. However, the rest of morphometrics and morphological characters e.g. body habitus, lip and tail morphology, and lateral field characters posterior to the phasmid correspond well with the original description. Vulva and spermatheca morphology was not described in the original or subsequent descriptions. The vulva of the Hainan population has fine lips and apparently covered with a vulval flap. The spermatheca is nonfunctional, weakly developed and irregularly shaped.
No sperm was observed in the spermatheca. Male was not described in the original description or in the subsequent descriptions (Li et al., 1986;Mizukubo et al., 1993;Williams, 1960), same as in the Hainan population. Out of 17 nominal species of Quinisulcius, males were not reported for 10 species (Geraert, 2011). It can be speculated that Q. curvus is a parthenogenetic species as evidenced by the empty spermatheca.

Molecular characterisation and phylogeny
The sequences of nearly full-length 18S (1649 bp, NW628178-NW628179), ITS region of rDNA (806 bp, Figure 3: Phylogenetic relationships within selected genera of subfamily Telotylenchinae and subfamily Merliniinae as inferred from Bayesian analysis using the 28S of the rRNA gene sequence dataset with the TIM3 + G model. Posterior probability of more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold. NW628172-NW628173) and 28S D2-D3 region (742 bp, NW628174-NW628175) of Q. curvus were obtained in the present study. Phylogenetic relationships among the subfamily Merliniinae and subfamily Telotylenchinae nematodes were determined separately for each dataset using BI (Figs. 2-4). These subfamilies were selected because of the genus Quinisulcius is morphologically close to both of them.
The phylogenetic analyses of Q. curvus are presented in Figures 2-4. The trees inferred from 18S, and 28S analyses separated into two distinct clades (PP = 1.00, 1.00, respectively) which comprises species of subfamilies Telotylenchinae and Merliniinae; however, in ITS tree Telotylenchinae appears separate in three different groups (Fig. 4). In all the trees, both Quinisulcius species (Q. capitatus and Q. curvus) are clustered together and grouped with members of Telotylenchinae, except for 18S tree (Fig. 2), in which both species appeared on separate branches among species of Tylenchorhynchus. However, in 28S and ITS trees (Figs. 3 and 4), Q. curvus and Q. capitatus grouped together as a subclade within species of Tylenchorhynchus. We also anticipate that with the inclusion of more sequences from Quinisulcius and species from other genera within these subfamilies will certainly clarify the phylogenetic positioning of Q. curvus and Q. capitatus, as well as the monophyletic or paraphyletic status of the genus Quinisulcius and the subfamilies Merliniinae and Telotylenchinae.
Our results suggest that Merliniinae and Telotylenchinae appears clearly separated with 18S and 28S ribosomal markers (Figs. 2 and 3), confirming morphological separations, and appearing as monophyletic separated groups. Nevertheless, in ITS Telotylenchinae appears as a polyphyletic group (Fig. 3). Consequently, additional species characterization under integrative taxonomic approaches are needed to confirm this hypothesis by studying several species of other genera within these subfamilies.

Remarks
The host associations or preference of Q. curvus has not been well documented. It was initially reported from the sugarcane rhizosphere in Mauritius (Williams,Figure 4: Phylogenetic relationships within selected genera of subfamily Telotylenchinae and subfamily Merliniinae as inferred from Bayesian analysis using the ITS of the rRNA gene sequence dataset with the TVM3 + I + G model. Posterior probability of more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold.  (Williams, 1960) Siddiqi, 1971 This study Williams, 1960Mizukubo et al., 1993Li et al., 1986 Origin 1960), subsequently it has been detected in sugarcane growing areas of Martinique and Australia (Nobbs, 2013;Román, 1968). From these reports, it can be speculated that Q. curvus has a host preference for sugarcane. In addition to that, Q. curvus has also been reported from other countries in the rhizosphere of different horticultural crops, although no considerable plant damage was reported (Table 1). Quinisulcius curvus was also reported in Henan Province of China from the rhizosphere of Brassica caulorapa, but the diagnosis of this population was based on a single specimen (Li et al., 1986, Table 1). In our opinion, a morphological identification based on single nematode is doubtful. Therefore, the status of the Henan population of Q. curvus needs further sampling and confirmation. It has been noted that Q. curvus has always been reported in association with principle agricultural or horticultural crops. In this study, we found Q. curvus in the rhizosphere of shrub sea randa i.e. G. speciosa, which is a new host record for this species. The discovery of Q. curvus from China highlights the need to update the list of distribution of these nematodes. Moreover, comprehensive surveys will likely uncover other Quinisulcius species from China.