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

Male

Not found.

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′46″N; 120°07′′20″E”.

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.

Longidorus jonesi (Siddiqi, 1962)

Description

Female

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).

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 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).

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′18″N; 120°06′60″E.”

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 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.

Phylogenetic relationships of Paralongidorus sali and Longidorus jonesi

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

Discussion

Longidorus species are widely distributed in China. Until now, fifteen Longidorus species (viz. L. camelliae (Zheng et al., 2003) L. fangi (Xu and Cheng, 1991), L. fursti (Heyns et al., 1987), L. hangzhouensis (Zheng et al., 2003), L. henanus (Xu and Cheng, 1992), L. jiangsuensis (Xu and Hooper, 1990), L. jonesi, L. juglans (Xu et al., 2017), L. litchii (Xu and Cheng, 1992), L. macromucronatus (Siddiqi, 1962), L. martini (Merny, 1966), L. moniloides (Heyns, 1966), L. pawneensis (Luc and Coomans, 1988), L. pisi (Edward et al., 1964) and L. intermedius (Kozlowska and Seinhorst, 1979)) have been reported from 13 provinces, i.e., Anhui, Beijing, Fujian, Gansu, Hebei, Henan, Hunan, Jiangsu, Liaoning, Shandong, Shanxi, Yunnan and Zhejiang (Guo et al., 2011; Xu et al., 2017).

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). These phylogenetic results are congruent with recently published data by Pedram et al. (2012), Palomares-Rius et al. (2013), Gutiérrez-Gutiérrez et al. (2017), or 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, 2005), 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.