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

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 CO₂. 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).

DNA was extracted from single specimens as described by Zheng et al. (2003). Polymerase chain reaction (PCR) and sequencing were completed in two laboratories: LPN and IB, China. PCR conditions were as described by Ye et al. (2007). Several sets of primers were used for PCR: the forward D2A (5′-ACAAGTACCGTG AGGGAA AGTTG-3′) and the reverse D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers for amplifying the D2–D3 expansion segments of 28S rRNA gene (De Ley et al., 1999). Nearly full length 18S region was amplified with two sets of two sets of primers, the first set was 18s39F (5′-AAAGATTAAGCCATGCATG-3′) and 18s977R (5′-TTTACGGTTAGAACTAGGGCGG-3′). The second set was 18s900F (5′-AAGACGGACTACAGCGAAAG-3′) and 18s1713R (5′-TCACCTACAGCTACCTTGTTACG-3′) (Olson et al., 2017). PCR products were separated on 1% agarose gels and visualised by staining with ethidium bromide. PCR products of sufficiently high quality were purified for cloning and sequencing by Invitrogen, Shanghai, China or Quintara Biosciences CA, USA.

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; Archidona-Yuste et al., 2016). 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 × 10⁶ 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. 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 visualized using FigTree software V.1.42 (http://tree.bio.ed.ac.uk/software/figtree/).

Body elongated, slender, slightly tapering anteriorly and posteriorly, open C-shape when heat relaxed. Cuticle appearing smooth, 3.9 (3.5–4.0) μm thick near vulva, 8.6 (7.0–10.0) μm thick at tip tail, and marked by very fine, superficial, transverse striae mainly, in tail region. Lip region dome-shaped, continuous with the rest of the body. SEM observations showed a slit-like oral aperture surrounded by six inner and six outer labial papillae in en face view, labial lobe distinct, amphidial fovea stirrup-shaped, with conspicuous, crescent-shaped amphidal aperture having slightly rounded margins. Odontostyle straight or slightly arcuate, twice the length of odontophore, odontophore weakly developed, with slight basal swellings. Guiding ring located anteriorly ca more than three times the lip region width distance from anterior end. Nerve ring encircling pharynx, located slightly posterior to middle of pharynx. Oesophagus dorylaimoid, typical of genus. Anterior slender part of oesophagus usually with loop overlapping basal bulb. Basal bulb cylindrical, 108 (104–115) μm long and 24.5 (24.0–25.0) μm in diam. Dorsal oesophageal gland nucleus located 28 to 32 μm from the anterior end of oesophageal bulb, two ventro-sublateral nucleus located (SV1 = 61.2–63.5, SV2 = 59.1–61.3) μm from the anterior end of pharyngeal bulb. Cardia elongated, conoid shaped 9.8 (8.5–11.0) μm long. Reproductive system didelphic with reflexed ovaries, vulva a transverse slit, vagina perpendicular to body axis ca less than half of corresponding body diam. Inconspicuous, oocytes arranged in multiple rows, pre-rectum variable in length 403 (359–445) μm and rectum 29.5 (28.5–30.5) μm long, anus a small slit. Tail short, hemispherical (Figs. 1–4, Tables 1, 2).

Fig. 1

Fig. 2

Fig. 3

Not found.

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).

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′46″N; 120°07′′20″E”.

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.

Longidorus jonesi (Siddiqi, 1962)

Description

Body cylindrical, slightly tapering towards anterior end, open C- to spiral shape when heat relaxed. Cuticle appearing smooth, 3.7 (3.5–4.0) µm thick vulva, 7.9 (7.0–10.0) μm thick at tail tip. Lip region rounded, continuous with the rest of the body. SEM observations showing a slit-like oral aperture surrounded by six inner and six outer labial papillae en face view, amphidial fovea pouch like with pore-like amphidial apertures. Stylet guiding ring single, located 5.2–5.3 times lip region diameter from anterior end. Odontostyle long and narrow, 1.7 (1.6–1.9) times as long as odontophore, straight or slightly arcuate, odontophore moderately developed, with slightly swollen base, not set-off from esophageal contour. Oesophagus extending to a terminal oesophageal bulb with three nuclei, with one dorsal gland nucleus located at the beginning of bulb, i.e. (22%–23%) of the oesophageal bulb length, while the other two subventro-lateral nuclei located around the middle of bulb. Oesophageal basal bulb (91.4–112) long and (22–27) μm wide. Oesophageal intestinal valve conoid-oblong, 9.6 (9.0–10.5) μm long. Reproductive system with both genital branches equally developed, each 519 (439–577) μm long, with reflexed ovaries variable in length. Vulva in form of a transverse slit, located about mid-body, vagina perpendicular to body axis, less than half of corresponding body width, surrounded by well-developed muscles. Ovaries paired, roughly symmetrical, with oocytes arranged in a single file. Both sets of reproductive organs lying on left side of intestine. Pre-rectum distinct. Rectum about less than one anal body width long, anus a transverse slit. Tail conoid, obtusely rounded, with two pairs of caudal pores (Figs. 5–9; Tables 3, 4).

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

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.

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 characterized by the position of replacement odontostyle into odontophore, just posterior to base of functional odontostyle. Siddiqi (1962) mentioned three groups of juveniles where the first group have tails with elongated conoid and rounded terminus whereas the other groups have tails similar to the female. 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).

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′18″N; 120°06′60″E.”

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 first-stage 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.

Amplification of D2–D3 expansion segment of 28S rRNA and the partial 18S rRNA from P. sali and L. jonesi yielded a single fragment of ca 800 and 1,700 bp, respectively. Four new D2–D3 of 28S rRNA and four partial 18S rRNA gene sequences were obtained in the present study. D2–D3 expansion segments of 28S rRNA sequences of P. sali (MG729700–MG729701) showed 88% similarity with P. bikanerensis (Lal and Mathur, 1987; Siddiqi et al., 1993, JN032584) (differing in 94 nucleotides and 32 indels), 81% similarity with P. lusitanicus (Gutiérrez-Gutiérrez et al., 2017, KY750562) (differing in 156 bp and 71 indels); and 84-83% similarity with some Longidorus species such as L. macrosoma (Hooper, 1961, AY580055, AY601565) or L. helveticus (Lamberti et al., 2001, JN627415), differing in 130 to 132 bp and 52 indels. The partial 18S rRNA sequence for P. sali (MG729696–MG729697) showed high similarity (99–98% similar) with several Paralongidorus spp. such as P. bikanerensis (JN032586), P. maximus (Bütschli, 1874, AJ875152), and P. litoralis (Palomares-Rius et al., 2008, EU026158), differing in a range from 13 to 29 nucleotides and 7 to 8 indels. Intraspecific sequence diversity (uncorrected p-distance) of ribosomal markers among the studied specimens for P. sali was small (8–22 bp, 2–8 indels).

Finally, the Chinese population of L. jonesi (MG729702–MG729703) showed high similarity (99%) for D2–D3, 6 to 8 nucleotides and 1 to 4 indels with L. jonesi from Japan (KF552069). In addition, the two new partial 18S rRNA sequences of L. jonesi (MG729698–MG729699) showed high similarity (99%–98%, respectively) with L. litchi (Xu and Cheng, 1992, AY687996) and L. diadecturus (Eveleigh and Allen, 1982,AY283167, AY283166), differing in 25 to 33 nucleotides and 6 indels.

Phylogenetic trees reconstructed by the BI method for the two rRNA markers (D2–D3 expansion regions of 28S rRNA gene and the partial 18S rRNA) are presented in Figs. 10, 11. The D2–D3 segments of 28S rRNA gene tree based on a multiple edited alignment (100 sequences) of 749 total characters revealed a major clade for the majority of the Paralongidorus species, excluding P. bikanerensis and P. sali (Fig. 10). This tree is similar to the most recent phylogenetic analysis showed by Gutiérrez-Gutiérrez et al. (2017), Barsi and Luca (2017), and Palomares-Rius et al. (2013) showing P. bikanerensis in a position outside of the main clade for Paralongidorus. Longidorus jonesi is well-related phylogenetically to accession described from Japan (KF552069) (Palomares-Rius et al., 2013).

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.

Fig. 10

Fig. 11