Morphological and Molecular Characterization of Paralongidorus sali Siddiqi, Hooper, and Khan, 1963 with a Description of the First-Stage Juvenile and Male of Longidorus jonesi Siddiqi, 1962 from China

Longidorids are economically important plant-parasitic nematodes because several species are virus vectors. Populations of Paralongidorus sali and Longidorus jonesi, isolated from woody perennials of Hangzhou, Zhejiang, China, were characterized molecularly and morphologically. The morphometric data of the Chinese populations of both species were compared with other populations. The present study provided a first record of the occurrence of Paralongidorus in China coupled with description of the first-stage juvenile and male of L. jonesi. Phylogenetic analysis based on 18S and D2–D3 expansion segments of 28S gene indicated that L. jonesi clustered with L. jonesi reported from Japan and P. sali grouped with P. bikanerensis from Iran. Considering the pathological and economic importance of this group of nematodes, the study emphasized the need of updated descriptions from accurately identified specimens, isolation of sufficient material for examination, and molecular and phylogenetic analysis for a better understanding and diagnostics of Longidorid nematodes.

The family Longidoridae Thorne, 1935 comprises a group of migratory plant-parasitic species that damage a wide range of wild and cultivated plants through direct feeding on root cells and the transmission of several plant-pathogenic viruses (Decraemer and Robbins, 2007;Decraemer and Chaves, 2012). Members of these genera are known to transmit nepoviruses and are regulated by quarantine inspections in many countries (Gutiérrez-Gutiérrez et al., 2016). Longidorus and Paralongidorus belong to family Longidoridae, both are globally distributed and have 160 and 90 known species, respectively Esmaeili et al., 2016).
Some species of Paralongidorus have controversial status due to synonymization of Longidoroides (Khan et al., 1978) and Siddiqia (Khan et al., 1978), and some Paralongidorus species have been wrongly included and belong to genus Longidorus (Decraemer and Coomans, 2007). The major difference used to separate Longidorus, Longidoroides, and Paralongidorus is the shape of amphids (pouch like in Longidorus and Longidoroides vs. funnel/stirrup shaped in Paralongidorus) and the opening of amphidial aperture (pore-like in Longidorus vs. slit-like in Longidoroides and Paralongidorus) (Oliveira and Neilson, 2006). Several new species of Paralongidorus have published with complete molecular characterization and scanning electron microscopy (SEM) observations (Palomares-Rius et al., 2008Pedram et al., 2012;Gutiérrez-Gutiérrez et al., 2017;Barsi and Luca, 2017) which enables the discrimination between Longidorus and Paralongidorus species. However, there is no molecular evidence to distinguish Longidoroides species which leaves the status of this genus as junior synonym of Paralongidorus as suggested by Decraemer and Coomans (2007).
During a routine nematological survey of Hangzhou, Zhejiang Province, eastern China, two populations of longidorid nematodes were isolated from the rhizosphere of woody perennials. The population isolated from Cyclobalanopsis glauca (Thumb.) Oerst, 4 juvenile stages were recovered and identified as Longidorus jonesi, the population from Castanopsis sclerophylla (Lindl.) Schottky, 3 juvenile stages were recovered and was identified as Paralongidorus sali. Robbins et al. (1995) reported 3 juveniles stages of L. jonesi and no first stage juvenile or male were observed additionally this is the first report of genus Paralongidorus found in China. Therefore, the objectives of the present study were to: (i) provide updated morphological descriptions of first-stage juvenile P. sali, and male of L. jonesi, (ii) characterize the molecular data of both species using the D2-D3 expansion segments of 28S rRNA and partial 18S rRNA gene sequences, and (iii) demonstrate the phylogenetic relationships of both species with related species.

Materials and methods
Nematode sampling, extraction and morphological study Nematodes were extracted from soil samples using modified Baermann funnel method for 24 hr. For morphometric studies, nematodes were killed and fixed with hot formalin (4% with 1% glycerol), and processed in glycerine (Seinhorst, 1959) as modified by De Grisse (1969). The measurements and light micrographs of nematodes were performed using 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, (the mixture contained = 25 ml of 8% paraformaldehyde, 10 ml of 25% glutaraldehyde, 50 ml of 0.2 M phosphate buffer, and 15 ml distilled water) washed three times in 0.1 M 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 to 10 nanometer thickness and the micrographs were made at 3 to 5 kv operating system (Maria et al., 2018).

Phylogenetic analysis
D2-D3 28S segments, and partial 18S rRNA sequences of different Longidorus and Paralongidorus species from GenBank were used for phylogenetic reconstruction. Rotylenchus paravitis (JX015422) for D2-D3 of 28S and Tylencholaimus mirabilis (EF207253), Xiphinema rivesi (HM921344) for 18S tree were selected as outgroup taxa for each dataset following previous published studies (He et al., 2005;Holterman et al., 2006;Gutiérrez-Gutiérrez et al., 2013;. Multiple sequence alignments of the different genes were made using the Q-INS-i 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 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). Percentage similarity between sequences was calculated using the sequence identity matrix in BioEdit. For that, the score for each pair of sequences was compared directly and all gap or place-holding characters were treated as a gap. When the same position for both sequences had a gap it was not treated as a difference. Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). 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. General time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for the D2-D3 segments and a transitional of invariable sites model with invariable sites and a gamma-shaped distribution (TIM2 + I + G) for the 18S rRNA gene.
These BI analyses were run separately per dataset using four chains for 2 × 10 6 generations for all of molecular markers. A combined analysis of the two 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.

Juveniles
Three juvenile stages (J1, J2, and J4) were found and they were basically similar to adults, except for their smaller size, shorter tails, and sexual characteristics ( Fig. 4). Tail becomes progressively wider after each moult. Juvenile stages are distinguishable by relative body lengths, functional and replacement odontostyle (Robbins et al., 1995).

Locality and habitat
The population was found in the rhizosphere of Castanopsis sclerophylla from botanical garden, Hangzhou, Zhejiang Province, China on July 1, 2017. The geographical position of the sampling site was "30°15¢46²N; 120°07²20²E."

Remarks
Paralongidorus sali was originally described from India by Siddiqi et al. (1963), later on it was reported from Korea by Choi and Duan (1998) and this is the first report from China. Males were not described in original or any other report; similarly males   were also not found in this population as well. The Chinese population is slightly longer than the original description and subsequently has slightly longer odontostyle (112-124.5 vs. 98-107) µm and odontophore (66-74 vs. 52-62) µm but all the morphometric variation is in the range of Korean population. The morphology fits well with the original description except, for slight morphometrical values. We consider these small intraspecific differences are due to the geographical variability.

Male
Very rare, morphologically similar to female except for genital system. Male genital tract diorchic with testes opposed, containing multiple rows of spermatogonia. Tail conoid, rounded, with 7 to 8 adcloacal supplements.

Juveniles
Four juvenile stages (J1, J2, J3, and J4) were found and they were basically similar to adults, except for their smaller size, shorter tails, and sexual characteristics (Fig. 9). The first-stage juvenile of L. jonesi was The first-stage and second-stage juveniles of the Hangzhou population fits well with this description as they both have a long conoid, rounded peg, except the J1 has a slimmer and shorter tail as compared with the J2. The tails of other stages becomes progressively wider after each moult. All of the stages are distinguishable by relative body lengths, functional, and replacement odontostyle (Robbins et al., 1995).

Locality and habitat
The population was found in the rhizosphere of Cyclobalanopsis glauca from botanical garden, Hangzhou, Zhejiang Province, China on July 1st, 2017. The geographical position of the sampling site was "30°15¢18²N; 120°06¢60²E."

Remarks
Longidorus jonesi was originally described from India by Siddiqi (1962), later on it was reported from Jiangsu Province of China by Xu and Hooper (1990). They have found L. jonesi from two localities, i.e., Nanjing and Suzhou. Palomares-Rius et al. (2014) reported another population from Japan. The original description and none of the reported population described the firststage juvenile or male. In the population found in Hangzhou, Zhejiang Province, the first-stage juvenile and male were detected and described for the first time.
The females of Hangzhou population showed slightly smaller V value (44-51.8 vs. 50-52.4) as compared with original description, odontostyle (138-143 vs. 107-120) μm and odontophore (71-87 vs. 66-73) μm were slightly longer, but these morphometrics correspond well with the Japanese population. The morphology fits well with the original description, except for slight morphometrical values. We consider, these small intraspecific differences are due to the geographical variability.
Similarly, the 50% majority rule consensus BI tree of a multiple alignment including 96 18S rRNA sequences and 1,679 bp alignment length (Fig. 11) showed a clear phylogenetic relationship of P. sali with P. bikanerensis in both datasets and also outside of the main clade for Paralongidorus.
To date, there are no reports of Paralongidorus species from China, this genus is known to have global distribution with maximum diversity found from Asia and Africa (Palomares-Rius et al., 2008). Similarly, P. sali was originally described from India and later on it was reported from Korea and now it has also been found in China. In the same way, the other longidorid species under investigation, i.e., L. jonesi, was originally described from India and later on it was only reported from China and Japan, suggesting the possible prevalence of both species is localized in Asian countries.
Our phylogenetic analysis based on 18S and D2-D3 expansion segments of 28S sequences L. jonesi clustered well with L. jonesi from Japan and other Longidorus species while in both trees P. sali clustered with P. bikanerensis. In 18S tree, P. sali and P. bikanerensis form a separate clade with species of Longidorus and Paralongidorus (Fig. 11) while in 28S tree both species forms a separate clade with Longidorus species (Fig. 10) Barsi and Luca (2017). Based on morphological and SEM observations, both species belongs to the genus Paralongidorus and interestingly both species originally were described from India. Considering the separate position of P. sali and P. bikanerensis in phylogenetic trees we assume these two species are molecularly intermediate between Longidorus and Paralongidorus. However, we strongly emphasized the need of further study to support this assumption. In the past, longidorid species identification was mainly based on morphological characters and hierarchical cluster analysis (Ye and Robbins, 2003, 2004, however, with the advent of molecular sequencing and phylogenetic studies, the species identification is more reliable and equitable. In conclusion, this study provided a first record of the occurrence of Paralongidorus species from China coupled with detail morphological and molecular characterisation of P. sali, description of the first-stage juvenile of L. jonesi, additionally provided the SEM observations of both species in order to  elucidate the lip morphology and amphidial aperture of both species in detail. The systematics and diagnostics of Longidorid nematodes are important because of regulatory and management issues attributed to this group of nematodes as virus vectors. Thus, we suggest updated descriptions from accurately identified specimens, collection of sufficient materials for examination and close observations based on our present knowledge and molecular analysis are necessary for better understanding of the current distribution and host association of longidorid nematodes.