Integrative Taxonomy and Molecular Phylogeny of the Plant-Parasitic Nematode Genus Paratylenchus (Nematoda: Paratylenchinae): Linking Species with Molecular Barcodes

Pin nematodes of the genus Paratylenchus are obligate ectoparasites of a wide variety of plants that are distributed worldwide. In this study, individual morphologically vouchered nematode specimens of fourteen Paratylenchus species, including P. aculentus, P. elachistus, P. goodeyi, P. holdemani, P. idalimus, P. microdorus, P. nanus, P. neoamblycephalus, P. straeleni and P. veruculatus, are unequivocally linked to the D2-D3 of 28S, ITS, 18S rRNA and COI gene sequences. Combined with scanning electron microscopy and a molecular analysis of an additional nine known and thirteen unknown species originating from diverse geographic regions, a total of 92 D2-D3 of 28S, 41 ITS, 57 18S rRNA and 111 COI new gene sequences are presented. Paratylenchus elachistus, P. holdemani and P. neoamblycephalus are recorded for the first time in Belgium and P. idalimus for the first time in Europe. Paratylenchus is an excellent example of an incredibly diverse yet morphologically minimalistic plant-parasitic genus, and this study provides an integrated analysis of all available data, including coalescence-based molecular species delimitation, resulting in an updated Paratylenchus phylogeny and the corrective reassignment of 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 COI gene sequences that were previously unidentified or incorrectly classified.


Introduction
The plant-parasitic nematode (PPN) genus Paratylenchus Micoletzky, 1922, commonly known as pin nematodes, are obligate ectoparasites of a wide variety of plants, including herbs, shrubs and trees, that are distributed worldwide and cause various symptoms in their host plants [1][2][3][4][5]. This genus was reviewed by Tarjan [6], who provided the first key to the species. In subsequent years, several attempts were made to split the genus and group its representatives into new genera. The genus Gracilacus Raski, 1962, was proposed for members of the Paratylenchus species with stylet lengths longer than 48 µm [7]. The validity of Gracilacus was supported by Thorne and Malek [8], Raski and Luc [9], Maggenti et al. [10], Raski [11], Esser [12], Andrássy [13] and Yu et al. [14], while Siddiqi [15] treated it as a subgenus of Paratylenchus. Gracilacus was synonymised with Paratylenchus by Brzeski [16], and it was recognized in further works of Siddiqi and Goodey [17], Geraert [18], Brzeski [19], Nguyen et al. [20], Decraemer and Hunt [21], Van den Berg et al. [22], Ghaderi et al. [23], Table 1. List of Paratylenchus populations included in this study. Accession numbers of three ribosomal RNA genes (D2-D3 of 28S, ITS and 18S) and a mitochondrial gene (COI) fragments are provided for 18 identified and 14 unidentified Paratylenchus species. Accession numbers in italics are ones generated in this study.  Figure 1, Table 2): Heat relaxed specimens open C-to J-shape. Lateral field with three lateral lines. Deirids not observed (not necessarily an indication that they are absent). Cephalic region rounded, low, sometimes appearing slightly truncated, submedian lobes not protruded. Stylet 52-61 µm long, cone 80-91% of stylet length, knobs 2-4 µm across. Pharynx well developed, about one-third of body length. Secretoryexcretory pore between median bulb and isthmus level. Spermatheca rounded to slightly oval and filled with sperm cells. Prevulval swelling not prominent. Vulval flaps very small and can be visible under LM. Vulval located at 71-76% of body length from anterior end. Vagina straight to slightly oblique, reaching to almost half of body width. Anus obscure. Tail 18-25 µm long, tapers gradually to a finely or bluntly rounded terminus.
Remarks: Males were not found. Female morphology and morphometrics matched very well with P. aculentus. This species has been reported earlier in Belgium [53]. Although the D2-D3 and 18S sequences pointed towards P. colinus, the current population had no cuticular ornamentations present in the anterior part of the body and female bodies were not swollen and submedian lobe protrusions were not seen, which are important characteristics for P. colinus. According to Ghaderi et al. [23], P. aculentus is part of Group 9 of the Paratylenchus species with stylet lengths longer than 40 µm, three lateral lines and absence of vulval flaps. Here, we confirm the presence of small vulval flaps in P. aculentus, clearly supported by SEM. This was also an observation originally made by Brzeski [19]. Paratylenchus aculentus should, therefore, be placed in Group 8 with P. colinus and P. idalimus; furthermore, the close affinity of our P. aculentus population with P. colinus is also molecularly supported by the very conserved 18S rRNA gene fragment.   Figure 2, Table 3): Heat relaxed specimens open C-to J-shape. Lateral field with four lateral lines. Deirids visible under SEM. Cephalic region conicalrounded to sometimes truncated. En face square-shaped, showing poorly developed submedian lobes, two pronounced lateral ridges and small indistinct dorso-ventral ridges around oral opening, two slit-like amphidial openings laterally. Stylet 20-22 µm long, cone 61-68% of stylet length and knobs 3-4 µm across. Pharynx well developed, about one-fourth of body length. Hemizonid commonly above secretory-excretory pore about two body annuli long. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca rounded to oval and filled with sperm cells. Vulval flaps rounded, prominent. Vulva located at 80-83% of body length from anterior end. Vagina oblique, reaching to half of body width. Tail 21-29 µm long, conical, thin and terminus from spicate to pointed or minutely rounded.
Molecular characterisation: Two D2-D3 of 28S, four ITS, four 18S rRNA and four COI gene sequences were generated for the first time from this species without intraspecific sequence variations.
Remarks: Males were not found. This species is reported for the first time in Belgium and has only been recorded in Poland and Slovakia before in Europe [19,54,55]. Female morphology and morphometrics agree well with the original description [56] and also with descriptions of other populations [19,31]. Paratylenchus elachistus can be separated from its closest species, Paratylenchus minutus, Lindford in Lindford, Oliveira & Ishii, 1949, by a longer body length (0.23-0.34 mm vs. 0.19-0.31 mm), a more robust and longer stylet (19-25 µm vs. 15-21 µm) and a more slender tail, commonly with spicate to finely rounded tail termini.   Figure 3, Table 2): Heat relaxed specimens C-to J-shape. Lateral field with four lateral lines. Deirids observed under LM. Cephalic region conical-rounded, submedian lobes not protruding except in two freshly killed specimens where small protrusions were seen under LM. Stylet 47-59 µm long, cone 78-90% of stylet length, stylet guide faintly seen, knobs 3-5 µm across. Pharynx well developed, about one-third of body length. Secretory-excretory pore around median bulb level. Spermatheca oval to elongate, filled with sperm cells. Vulval flaps present. Vulva located at 78-82% of body length from anterior end, in one female a short post-vulva sac observed. Vagina oblique and reaching to two-third of body width. Tail 26-32 µm long, conoid with variable terminus from finely rounded to bluntly rounded and rarely pointed.
Molecular characterisation: Three D2-D3 of 28S, one ITS, two 18S rRNA and three COI gene sequences were generated for the first time for this species without intraspecific sequence variations.
Remarks: Males were not found. Females morphology and morphometrics agree well with former P. goodeyi descriptions [18,19,57]. This species was originally described from the Netherlands and has been reported from many European countries, including Belgium. Paratylenchus goodeyi is one of the 22 species of the Group 10 of Paratylenchus after Ghaderi et al. [23] with stylet length more than 40 µm, four lateral lines and presence of vulval flaps. This species is comparable to other members of the group namely Paratylenchus ivorensis Luc & de Guiran, 1962, Paratylenchus pandatus (Raski, 1976) Siddiqi, 1989 and P. straeleni with females having more or less conical-rounded heads, stylet lengths in the range 40-61 µm (except for P. pandatus for which, a stylet length up to 68 µm was rarely reported). However, the vulvae of P. goodeyi and P. straeleni are located more posteriorly (77-88%) than that of the other two species (70-78%); P. ivorensis and P. goodeyi have been reported with variable tail termini, while P. pandatus and P. straeleni have been found usually with finely rounded to sub-acute female tail termini.

Paratylenchus holdemani
Females (Sample AR3; Figure 4, Table 3): Heat relaxed specimens C-to J-shape. Lateral field with four lateral lines. Deirids not observed. Cephalic region slightly truncated, submedian lobes not protruded. En face showing four small submedian lobes, four irregular ridges around oral opening, slit-like lateral amphidial openings. Stylet 19-26 µm long, cone 61-77% of stylet length, knobs 3-4 µm across. Pharynx well developed, about one-fourth of body length. Secretory-excretory pore commonly between mid-isthmus and end bulb level. Spermatheca rounded, filled with sperm cells. Vulval flaps prominent. Vulva located at 81-90% of body length from anterior end. Vagina oblique and reaching to two-third of body width. Tail 20-30 µm long, conoid with regularly finely rounded to sometimes bluntly rounded or digitate terminus. Males: Two males were obtained from Sample AR3 and one from Sample BE20. Their conspecificity with the females was confirmed by identical D2-D3 of 28S rRNA and COI gene sequences identified from the AR3 and BE20 males, respectively. The males had an average stylet length of approximately 12 µm and spicule length of 21 µm.
Molecular characterisation: Three D2-D3 of 28S, one 18S rRNA and three COI gene sequences were generated from the AR3 females, whereas two D2-D3 of 28S, one ITS, one 18S rRNA and four COI gene sequences were generated from the BE20 females. No sequence from either population showed any intraspecific variations. The D2-D3 sequences were found to be identical to P. bukowinensis sequences that originated from Italy [37] and Belgium [47]; however, morphological data for these populations are not available for comparison and both are considered here as representatives of P. holdemani.
Remarks: This species has been reported for the first time in Belgium and has only been reported in the Czech Republic in Europe [19]. The morphology and morphometrics of the AR3 population agree well with the original description [58] and with the population from the Czech Republic [19]. Although our D2-D3 sequences were identical to a P. bukowinensis sequence (AY780943), the female morphology of this Belgian population is different from P. bukowinensis descriptions.
Most importantly, the average stylet length (22.5 µm) of our population is shorter than for many previously reported P. bukowinensis populations. In addition, the cephalic region of P. bukowinensis is more rounded than that of P. holdemani. Paratylenchus holdemani is comparable to P. hamatus and Paratylenchus baldaccii Raski, 1975, but is distinguishable from both species by a shorter stylet length of 22.5 ± 2.0 (19-26) µm vs. always above 26 µm. In this study, greater variation in the tail termini was observed in our P. holdemani population compared to the other two species.

Paratylenchus idalimus
Females (Samples BE19 and BE20, two nearby localities; Figures 5 and 6, Table 2): Heat relaxed specimens J-or open C-shape. Lateral field with three lateral lines. Deirids not observed. Cephalic region conical-truncate, submedian lobes well developed and protruding. Stylet 84-93 µm long of which 83-89% is cone, prominent stylet guide, knobs about 4 µm across. Pharynx well developed, occasionally reaching up to half of body length. Secretory-excretory pore around level of stylet knobs which is above median bulb level. Spermatheca small, rounded, usually filled with sperm cells. Vulval lips slightly protruding. Vulval flaps reduced and small, sometimes easily overlooked. Vulva located at 77-79% of body length from anterior end. Vagina oblique, often reaching to two-third of body width. Anus obscure. Tail 22-28 µm long, conoid with subacute to finely rounded terminus.
Molecular characterisation: Two identical D2-D3 of 28S and two identical 18S rRNA gene sequences were generated from the BE20 population, whereas one D2-D3 sequence, identical to that of the BE20 sequence, was generated from the BE19 population. These sequences were generated for the first time for this species.
Remarks: Males and swollen females were not found. Only one juvenile was recorded from the BE20 population with a stylet length of 42 µm. This is the first time the species has been reported in Europe. Female morphology and morphometrics based on seven females from both populations (three from BE19 and four from BE20) agree well with the description of the slender female by Raski [7] in the USA. This species and P. colinus are the only two members of Group 8 of Paratylenchus [23]. It differs from P. colinus in having a longer stylet (84-93 µm vs. 56-72 µm), more pronounced protrusion of submedian lobes, slightly posterior position of vulva (77-79% vs. 69-78%) and absence vs. presence of cuticular ornamentation in anterior body.   Table 3): Body small, heat relaxed specimens open C-to 6-shape. Lateral field with four lateral lines. Deirids not observed. Cephalic region conical-truncate, submedian lobes sometimes slightly protruding. En face showing four submedian lobes and slit-like lateral amphidial openings. Stylet 11-15 µm long, cone 45-60% of stylet length. Pharynx about one-fifth of body length. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca rounded, empty or filled with sperm cells. Vulval flaps prominent. Vulva located at 79-83% of body length from anterior end. Vagina oblique, reaching to half of the body width. Post-vulval uterine sac not seen. Tail 28-36 µm long, conoid and terminus pointed to subacute to sometimes finely rounded. Molecular characterisation: Three D2-D3 of 28S, three ITS, four 18S rRNA and three COI gene sequences were generated without intraspecific variability; the ITS and the COI sequences are new for this species. Only 300 bp of the D2-D3 sequences were found to be homologous with four P. microdorus sequences from Germany (MF325254-MF325257; 98% similarity; 5 bp difference). The 18S rRNA sequences are 98-99% similar with P. microdorus from the Netherlands (AY284632 and AY284633; 8-15 out of 880 bp difference).
Remarks: Males were not found. Female morphologies and morphometrics agree well with the original description [59] and other populations [16,31], except for a slightly shorter stylet length (11-15 µm vs. 13-18 µm). Wide variations in the tail termini have been reported for this species [31]. However, for the BE9 population, finely rounded to subacute female tail termini were commonly observed. This species is comparable to Paratylenchus recisus Siddiqi, 1996, Paratylenchus variabilis Raski, 1975 and P. veruculatus, with a female stylet length within 11-17 µm, four lateral lines, presence of vulval flaps, secretoryexcretory pore at the posterior part of pharynx and vulva located at 78-87% of body length. However, they differ from each another in having conical-truncate heads with sometimes slightly protruded submedian lobes in P. microdorus, broadly rounded to truncated head with central swallow depression in P. recisus, rounded to almost hemispherical head in P. variabilis and low and broadly rounded head in P. veruculatus. Only P. microdorus and P. veruculatus males have been reported to have weak stylets, while stylets in the males of the other two species are degenerated.
2.1.7. Paratylenchus nanus Females (Sample BE11; Figure 8, Table 4): Heat relaxed specimens open C-to J-shape. Lateral field with four lateral lines. Deirids not observed. Cephalic region conical-rounded, in some specimens with sloping sides to rounded end, submedian lobes not protruding under LM. En face square shaped, revealing four submedian lobes, four distinct ridges around oral opening, lateral ridges slightly larger than dorso-ventral ridges, and two slit-like lateral amphidial openings. Stylet 27-31 µm long, cone 67-78% of stylet length and knobs 3-5 µm across. Pharynx well developed, about one-fourth of body length. Hemizonid just above secretory-excretory pore, about two body annuli long. Secretoryexcretory pore between isthmus and end bulb level. Spermatheca rounded and filled with sperm cells. Vulval flaps present. Vulva located at 82-86% of body length from anterior end. Vagina oblique, reaching up to half of body width. Tail 19-26 µm long, conoid, often more pronounced curvature on dorsal side ending with sub-acute to finely rounded terminus.
Molecular characterisation: Seven D2-D3 of 28S, four ITS, four 18S rRNA and seven COI gene sequences were generated without any intraspecific sequence variations among four P. nanus populations-AR3, BE1, BE11 and BE18. The D2-D3 and the ITS sequences were, respectively, identical to KF242194, KF242197 and KF242267, KF242268 of P. nanus from Van den Berg et al. [22] Remarks: Only in the BE11 population was a sufficient number of females recovered to allow morphological and morphometrical data comparisons, which agreed well with the original description [60] and subsequent descriptions of P. nanus [19,22,58]. Van den Berg et al. [22] reported two sibling species of P. nanus with different genotypestype A and type B (the latter of which was recently transferred to P. projectus) [61]. This correction suggests that the available 28S (MN720102-MN720103) and COI (MN734387 and MN734388) sequences of P. nanus from South Korea [48] were misidentified as they were found to be identical to the P. projectus sequences. Paratylenchus nanus is very similar to P. projectus and P. neoamblycephalus. It differs from P. projectus in having a conical-rounded vs. more trapezoid head shape and sperm-filled vs. empty spermathecae. It is differentiated from P. neoamblycephalus by more rounded vs. oval spermathecae and a conoid tailwith pronounced curvature on the dorsal side ending with a subacute or finely rounded terminus vs. a conoid tail with subacute terminus or almost acute tip. Furthermore, in our study we also observed that the ridges around the oral opening of the freshly killed specimens protruded more in P. neoamblycephalus compared to P. nanus when observed under LM.    Males: Two males were obtained with very thin stylets in freshly killed specimens, which were not visible after fixation, and spicules of 24 µm long. Their conspecificity with the females was confirmed by identical COI and D2-D3 sequences.
Molecular characterisation: Four D2-D3 of 28S, five ITS, six 18S rRNA and eight COI gene sequences were generated without intraspecific sequence variations. The 18S and the COI sequences are new for this species. The D2-D3 sequences were found to be identical to KF242189 and KF242190 of an unidentified Paratylenchus sp.6 from the USA [22], which is considered here as P. neoamblycephalus. However, the D2-D3 sequences were only 89% similar (79 out of 710 bp difference) with MG925221 and 92% similar (43 out of 546 bp difference) with MK506807 of P. neoamblycephalus from the USA and Iran, and named here as type A and type B, respectively. Interestingly, we observed 17 ambiguous nucleotide sites in the American P. neoamblycephalus type B sequence, which was found to be similar to P. projectus (previously P. nanus type B; KF242198-KF242201; 98% similarity; 16-20 out of 690 bp difference) after Van den Berg et al. [22,61]. On the other hand, the Iranian P. neoamblycephalus sequence [24] was similar to P. nanus (KF242194 and KF242197; 95% similarity; 27 out of 575 bp difference) [22]. Furthermore, our ITS sequences were only 74% similar (222 out of 865 bp difference) to MK506794 of P. neoamblycephalus type A generated from the same Iranian population.
Remarks: This species is reported for the first time in Belgium. Female morphology and morphometrics agree well with the original description from Germany [18] and to subsequent descriptions from Poland [19]. Paratylenchus neoamblycephalus is very similar to P. nanus and a comparison is provided above.
2.1.9. Paratylenchus straeleni Females (Sample BE15; Figure 10, Table 2): Heat relaxed specimens J-to C-shape. Lateral field with four lateral lines. Deirids clearly visible under SEM. Cephalic region conical-rounded to sometimes slightly truncated, submedian lobes not protruded. Stylet straight to slightly curved, 54-59 µm long, cone 76-83% of stylet length, knobs 3-5 µm across. Pharynx roughly one-fourth of body length. Secretory-excretory pore between isthmus and end bulb level. Spermatheca rounded to sometimes slightly ovoid and filled with sperm cells. Vulval flaps distinct. Vulva located at 80-84% of body length from anterior end. Vagina oblique, occasionally reaching to two-third of body width. Tail conical, 31-41 µm long, and terminus sharply pointed to minutely rounded.
Males: Two males were recovered without stylets and with spicule lengths of 20 and 22 µm, respectively. Their conspecificity with the females was confirmed by identical COI sequences.
Molecular characterisation: One D2-D3 of 28S, two ITS (99% similarity; 4 out of 830 bp difference), three identical 18S rRNA and five identical COI gene sequences were generated from the BE15 population. From another population (BE11), single D2-D3, ITS and 18S sequences and three identical COI sequences were also generated. All the sequences from both populations showed no intraspecific variation, except for the ITS sequences. The 18S sequences were 99% similar (3-5 out of 930 bp difference) with P. straeleni from the Netherlands (AY284630 and AY284631). The D2-D3 sequences were 97-99% similar (11-18 out of 700 bp difference) with four P. straeleni sequences-i.e., MK506804 from Iran [24], KM875547 from Turkey [42], and KF242235 and KF242236 from the USA [22]. The COI sequences were generated for the first time for this species. Remarkably, the Belgian ITS sequences were only 62% similar (295 bp difference) to the Iranian P. straeleni sequence (MK506791) of Hesar et al. [24].
Remarks: Female morphology and morphometrics agree well with the original description, also from Belgium [62], and subsequent descriptions of globally distributed P. straeleni populations [31]. This species is comparable to P. goodeyi as described above.

Paratylenchus veruculatus
Females (Sample BE20; Figure 11, Table 3): Heat relaxed specimens open C-shape to slightly ventrally curved. Lateral field with four lateral lines. Deirids not observed. Cephalic region broadly rounded, submedian lobes not protruding. En face rectangular with indistinct submedian lobes, four irregular ridges around oral opening and lateral amphidial openings. Stylet 13-15 µm long, cone 60-65% of stylet length, knobs about 3 µm across. Pharynx roughly one-fourth of total body length. Hemizonid two body annuli long, usually visible just above secretory-excretory pore. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca rounded and filled with sperm cells, young females with empty spermatheca also seen. Vulval flaps prominent. Vulva located at 84-90% of body length from anterior end. Vagina oblique and long, reaching up to three-fourth of body width. Tail 14-19 µm long, conoid with often broadly rounded to sometimes finely rounded terminus. Molecular characterisation: Five D2-D3 of 28S (99% similarity; 1-2 out of 720 bp difference) and two identical 18S rRNA and seven COI gene (97-100% similarity; 12-13 out of 410 bp difference) sequences were generated for the first time for this species.
Remarks: Female morphology and morphometrics agree well with the original description [63] and with other populations [19,31]. This species is comparable to other species with a short stylet such as P. microdorus, P. recisus and P. variabilis (see also above).

Paratylenchus sp.2
Females (Sample BE15; Figure 12, Table 4): Heat relaxed specimens open C-shape. Lateral field with four lateral lines. Deirids observed under SEM. Cephalic region conicalrounded, sometimes slightly trapezoid, submedian lobes not protruding under LM. En face square-shaped, showing four rounded, poorly separated submedian lobes, four ridges around oral opening, lateral ridges more prominent and larger than dorso-ventral ridges. Stylet 27-31 µm long, cone 64-71% of stylet length, knobs about 4 µm across. Pharynx well developed, about one-fourth of body length. Hemizonid just above secretory-excretory pore, about two body annuli long. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca rounded to occasionally slightly ovoid, filled with sperm cells. Vulval flaps prominent. Vulva located at 81-84% of body length from anterior end. Vagina oblique, reaching up to two-third of body width. Tail 23-29 µm long, conoid, slender and terminating with finely rounded tip.
Molecular characterisation: Two identical sequences each of D2-D3 of 28S, ITS, 18S rRNA as well as the COI gene were generated. The D2-D3 and ITS sequences were found to be, respectively, identical to KF242220 and KF242221 and 99% similar (five out of 750 bp difference) to KF242243 of Paratylenchus sp.2, which was identified as a member of the P. hamatus species complex [22]. The 18S and COI sequences were generated for the first time.
Remarks: Males were not found. The female morphology and morphometrics are in agreement with the description of P. hamatus [64]. Based on morphology and D2-D3 and ITS sequences, Van den Berg et al. [22] considered P. hamatus as a species complex containing several species, including P. hamatus sensu stricto collected from the type locality, and Paratylenchus sp.1 and Paratylenchus sp.2, collected from other places in California. Paratylenchus sp.1 is identified as representative of P. tenuicaudatus. Paratylenchus sp.2 is morphologically similar with P. hamatus sensu stricto but differs based on D2-D3 and ITS sequences [22], and this species appears to be not only present in the USA (California) but also in Belgium and Kyrgyzstan.  Molecular characterisation: Three identical D2-D3 of 28S, one ITS rRNA and two identical COI gene sequences were generated.
Remarks: No males were found. Female description is based on only three freshly killed specimens, while sufficient specimens are needed for a comprehensive species characterisation. The female morphology is close to P. variabilis, P. veruculatus and Paratylenchus vexans Thorne and Malek, 1986. These four species have more or less broadly rounded heads with non-protruding submedian lobes, stylet lengths in the range of 12-18 µm, four lateral lines, sperm-filled spermathecae, vulval flaps and conoid tails with more or less rounded termini. However, our population appears to have a stronger cephalic sclerotisation and slightly more anteriorly located vulvae (80-82% vs. 80-87%) compared to the other three species. This species is a sister to P. microdorus in the D2-D3 tree (96% similarity; 27 out of 740 bp difference), ITS tree (93% similarity; 37 out of 530 bp difference) as well as the COI tree (91% similarity; 36 out of 420 bp difference). It can, however, be readily morphologically distinguished from P. microdorus (see above). Pharynx about one-fourth of body length. Hemizonid just above secretory-excretory pore, about two body annuli long. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca empty. Vulval flaps prominent, commonly rounded. Vulva located at 82-85% of body length from anterior end. Vagina oblique reaching up to half of body width. Tail 18-26 µm long, conoid with finely rounded to bluntly rounded terminus, sometimes dorsally sinuate.
Molecular characterisation: Seven D2-D3 of 28S (99% similarity; one out of 730 bp difference), four ITS, five 18S rRNA and eleven COI gene sequences were generated without intraspecific sequence variation.
Remarks: Males were not found. The female morphology and morphometrics is close to P. projectus and Paratylenchus neoprojectus Wu and Hawn, 1975. The cephalic region of females were seen with both rounded to trapezoid shape, secretory-excretory pore located between mid-isthmus to end bulb level, empty spermatheca and tail termini which fit both the above two species. However, the molecular data appears to be different from any available sequences including that of P. projectus. A comparative study of this species with type specimens of P. neoprojectus and its molecular information should further confirm whether or not this species is P. neoprojectus. Pharynx about one-fourth of body length. Hemizonid just above secretory-excretory pore, about two body annuli long. Secretory-excretory pore between mid-isthmus and end bulb level. Spermatheca oval to elongated and filled with sperm cells. Vulval flaps rounded to oval and very prominent. Vulva located at 81-84% of body length from anterior end. Vagina oblique, reaching up to two-third of body width. Tail 20-26 µm long, conoid with regularly bluntly rounded terminus. Males: Heat relaxed specimen curved slightly ventrally, about the same body length as females but slightly slender. Cephalic region conoid and rounded. Stylet and pharynx degenerated. Secretory-excretory pore at about one-fifth of body length from anterior end. Spicule arcuate ventrally, about 21.5 µm in length. Gubernaculum 3-5 µm long. Tail conical with finely rounded tip. Conspecificity of males with females was confirmed by identical D2-D3, 18S and ITS sequences.
Molecular characterisation: Five D2-D3 of 28S, three ITS, four 18S rRNA and three COI gene sequences were generated without intraspecific sequence variations. The D2-D3, 18S and COI sequences were found to be identical, respectively, to MN783707, MN783708, MN783668-MN783670 and MN782407-MN782413 of Paratylenchus sp.F [47], while the ITS sequences were generated for the first time.
Remarks: Specimens belong to the same population as Paratylenchus sp.F in Etongwe et al.'s work [47]. Detailed morphological reanalysis revealed very close similarity to P. nanus. Nevertheless, the submedian lobes of this species appear to be somewhat more rounded than that of P. nanus based on SEM images and the vulval flaps also appear to be more pronounced and rounded compared to that of the latter. However, these characteristics need careful additional observations based on more specimens from both species. All four gene sequences of Paratylenchus sp.F were closest to the sequences of P. elachistus and phylogenetic analysis revealed their highly supported (PP > 90%) sister relationship. However, this species is morphologically different from P. elachistus en face, with rounded vs. poorly differentiated submedian lobes, stylet lengths of 25-30 µm vs. 20-22 µm and bluntly rounded vs. spicate to pointed tail termini.

Phylogenetic and Species Delimitation Analysis
The D2-D3 domains of the 28S rRNA gene alignment (744 bp long) included 128 sequences of 31 Paratylenchus species and three outgroup species. Forty-nine new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the analysis of the D2-D3 alignment contained three highly supported major clades and a weakly supported one ( Figure 16, PP < 70%). The molecular species delimitation based on the generalized mixed-yule coalescent (GMYC) and Poisson tree process (bPTP) methods revealed 66 and 63 putative species, respectively, a result that is largely congruent with former species delineations. However, P. projectus, P. straeleni, P. minor and P. shenzhenensis were further divided into 6, 5 (four according to bPTP), 2 and 2 separate lineages, respectively.
The ITS rRNA gene alignment (995 bp long) included 99 sequences of 37 Paratylenchus species and three outgroup species. Thirty-six new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the analysis of the ITS alignment contained four highly supported major clades ( Figure 17). Results of molecular species delimitation showed a high discrepancy between the models used-i.e., 48 putative species based on GMYC vs. 56 species based on bPTP. Additionally, molecular species delimitation based on the GMYC and bPTP methods did not correspond to species demarcation based on morphology and clade support; for example, virtually all individual sequences of P. chongqingensis and P. shenzhenensis were delineated as separate species. Figure 16. Phylogenetic relationships within populations and species of Paratylenchus, as inferred from Bayesian analysis using the D2-D3 of 28S rRNA gene sequence dataset with the GTR + I + G model. Posterior probability of more than 70% is given for the appropriate clades. Newly obtained sequences are indicated in bold. 1 = originally identified as P. nanus, 2 = originally identified as P. bukowinensis, 3 = originally identified as Paratylenchus sp., 4 = originally identified as Paratylenchus sp.8, 5 = originally identified as Paratylenchus sp.E, 6 = originally identified as Gracilacus sp. 7 = originally identified as Paratylenchus sp.5 and 8 = originally identified as Paratylenchus sp.6. Black and grey bars represent species boundaries estimated by generalized mixed-yule coalescent (GMYC) and Poisson tree process (bPTP) methods, respectively (only differences with GMYC provided).

Figure 17.
Phylogenetic relationships within populations and species of Paratylenchus as inferred from Bayesian analysis using the ITS rRNA gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold. 1 = originally identified as P. nanus and 2 = originally identified as Paratylenchus sp. Black and grey bars represent species boundaries estimated by GMYC and bPTP methods, respectively.
The 18S rRNA gene alignment (899 bp long) included 88 sequences of 31 Paratylenchus species and two outgroup species. Fifty-four new sequences were obtained for this study. The Bayesian 50% majority rule consensus tree inferred from the analysis of the partial 18S sequence alignment contained four highly supported major clades ( Figure 18). Molecular species delimitation failed to delimit well established species-for example P. goodeyi, P. veruculatus, P. nanus and P. neoamblycephalus were identified as belonging to the same species. Furthermore, both models provided highly varied results (14 putative species according to GMYC vs. 26 according to bPTP), reducing the confidence in said results. Figure 18. Phylogenetic relationships within populations and species of Paratylenchus, as inferred from Bayesian analysis using the 18S rRNA gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold. 1 = originally identified as P. dianthus and 2 = originally identified as P. nanus. Black and grey bars represent species boundaries estimated by GMYC and bPTP methods, respectively.
The COI gene alignment (745 bp long) included 130 sequences of 31 Paratylenchus species and three outgroup species. Seventy-one new sequences were included in this analysis. The Bayesian 50% majority rule consensus tree inferred from the analysis of the COI sequence alignment contained four moderate (Figure 19, PP = 70-90%) or highly supported major clades. Both employed species delineation methods, GMYC and bPTP, provided exactly the same 54 putative species delineations. These results were largely consistent with those obtained using other methods. However, P. enigmaticus, P. microdorus and P. veruculatus were subdivided into different species despite these sequences originating from the same population and their corresponding D2-D3 sequences being similar.
Paratylenchus straeleni was appointed as a species complex with nine putative species. Statistical parsimony networks showing the phylogenetic relationships between different isolates of P. straeleni and P. enigmaticus based on COI sequences are given in Figure 19B,C. The maximum variation of sequences for P. straeleni was found to be 9.1%.
Taking both morphological and molecular evidence together, we have been able to reassign a total of 49 Paratylenchus sequences, including 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 COI gene sequences, to their appropriate species (Table 5). However, we cannot exclude that in future, the identification of Paratylenchus species made in this study may be improved in light of new datasets.

Discussion
The genus Paratylenchus sensu lato, with 124 valid species, is an important plantparasitic group consisting of several commonly occurring and economically important species such as P. bukowinensis, P. dianthus, P. hamatus, P. nanus, P. neoamblycephalus and P. projectus, which are difficult to separate solely based on morphology [14,23,25,[31][32][33][34][35][36]. Female morphological traits are the most commonly used features for the identification of Paratylenchus populations, with the relative lengths of stylet cones and the positions of the secretory-excretory pores and vulvae as the most informative traits [31,67], while several ratios such as a, c and c' show high intraspecific variation. Given the limited species-specific female traits, some characteristics of males and juveniles-such as the presence or absence of stylet and male spicule length-may also be used to supplement the available data. However, care must be taken-for example, the occasional observance of a thin stylet in freshly killed juveniles or males that was invisible once the specimens were fixed highlights the importance of reporting this characteristic from both freshly killed and fixed specimens. Further complicating Paratylenchus taxonomy is the presence of mixture of species within one locality and sample [31]-an observation which calls for precaution concerning the conspecificity of several life stages. Indeed, the presence of multiple species in a soil sample was amply illustrated in our study. Seventy five percent of our investigated soil samples contained multiple species, with up to five different Paratylenchus species present in the same sample. This is, to the best of our knowledge, one of the highest numbers of species of one plant-parasitic nematode genus present in a single soil sample. More suitable morphological characters such as ridges around the oral opening or distinct to fused submedian lobes in face view also appear to be usefully informative but were only clearly revealed in our study with supporting evidence from SEM; additionally, the small vulval flaps in P. aculentus confirmed in this study have often been overlooked in previous studies under LM. Scanning electron microscopy is known to be important in nematode taxonomy [68][69][70], and this is especially true for the genus Paratylenchus as demonstrated in this study.
Nevertheless, even if all existing morphological tools are carefully employed, it remains impossible for all Paratylenchus species to be morphologically delineated, owing to the existence of cryptic species such as P. aquaticus [22]. The extensive use of new molecular data in the current study has demonstrated a remarkable molecular diversity in Paratylenchus, with several additional cryptic species being potentially present. The most obvious example is P. straeleni, which comprises 9, 5 and 4 putative species according to COI-(both GMYC and bPTP), D2-D3 (GMYC) and D2-D3 (bPTP)-based molecular species delimitation methods, respectively. It is noteworthy that the P. straeleni COI sequences have clearly clustered according to geographical location, as revealed by the COI haplotype network. The problems of morphologically delineating the Paratylenchus species have been further demonstrated in our study by the difficulties experienced in distinguishing between Paratylenchus sp.2, Paratylenchus sp.D and Paratylenchus sp.F, which were found to be very similar to P. hamatus, P. projectus/P. neoprojectus and P. nanus, respectively. A formal description with an appropriate diagnosis can only be developed for these putative new species following detailed observations of additional specimens and a thorough comparison with type materials of the known species.
Taken together, it is abundantly clear that molecular data are essential in advancing Paratylenchus taxonomy. Unfortunately, the several sequences published for Paratylenchus have serious limitations. One such issue is that the majority of the available D2-D3, 18S and ITS rRNA sequences have either not been linked to morphological data or have been associated with poor morphological data, thereby rendering them unreliable for use in identification purposes. For example, sequences of P. aculentus, P. leptos, P. microdorus, P. neoamblycephalus, etc., are not currently linked to reliable and clear morphological data and any subsequent identification based on these sequences may, therefore, lead to the deposition of further sequences under incorrect names [51]. An additional problem identified with the currently available 18S sequences, which are often relatively short (700-800 bp), is that several Paratylenchus species were detected with almost identical sequences. It is clear that to render these conserved sequences useful, complete or nearly complete lengths of the 18S rRNA gene (1600-1800 bp) will be required to allow species delimitation [71][72][73][74].
In the present study, we have also applied DNA-based species delimitation approaches to infer putative species boundaries on a given phylogenetic input, based on two different models [75,76] and four gene fragments (D2-D3 of 28S, ITS, 18S rRNA and COI). These coalescence-based species delimitation methods are rapidly gaining popularity in studies on closely related species that are difficult to distinguish based on phenotypic features, and have been applied to various eukaryotic groups [77,78]. However, despite plant-parasitic nematodes being a morphologically minimalistic group par excellence, such methods have been rarely applied to this group; nevertheless, they appear to be largely congruent with traditional methods [79][80][81]. Conversely, we have observed a remarkable discrepancy among the genes used, showing a poor link between DNA species delimitation and other methods, including a discrepancy between the employed models. The ITS and 18S rRNA genes gave, respectively, a likely overestimation and underestimation of the number of putative species, while for COI and D2-D3 of 28S rRNA genes, we observed, to a certain extent, an agreement with traditional methods, albeit with a likely overestimation of the number of species in several cases. This was not unexpected, as it has been exemplified by several studies that methods of species delimitation based on the coalescent model tend to overestimate phylogenetic lineages [52,77,82]. Both approaches (bPTP and GMYC) are similar in the fact that they identify significant changes in the pace of branching events on the tree. However, GMYC uses time to identify branching rate transition points, whereas, in contrast, bPTP directly uses the number of substitutions. Based on real and simulated data, both methods yield, in general, similar results [76,83]. This is the case for our COI-based output (identical results) and the D2-D3-based output (two differences, bPTP being more conserved). If differences have been observed, bPTP usually yields a more conservative delimitation than GMYC [76,80,83]. This is contrary to our unexpected ITS and 18S results and reduces the trust in the latter. Counterintuitively, the mutation rate of a chosen marker does not have a direct influence on its effectiveness to detect species. Mitochondrial markers reveal clearer discontinuities between interspecific divergence and intraspecific variation because of their faster coalescence within species lineages compared with nuclear loci, not necessarily because of their higher mutation rates [84,85]. The discrepancy between ITS and other delimitation methods in this study agrees with previous observations pointing to an unclear transition between species-level and population-level genetic distance for ITS [78]. Furthermore, it has been indicated that species delimitation based on single gene trees has serious limitations due to gene tree-species tree incongruence-confusions caused by processes including incomplete lineage sorting, trans-species polymorphism, hybridisation and introgression [78]. Multilocus approaches provide a posteriori double-check for contamination, sequencing errors or mitochondria-specific pitfalls [86]-for example, the high COI gene sequence variations within P. enigmaticus, P. microdorus and P. veruculatus observed in this study, despite these sequences originating from the same population. Although both nuclear and mitochondrial sequences were provided consistently from the same morphologically vouchered individuals, this study was restricted to the use of only single-locus data since only a limited number of other Paratylenchus individuals (and plant-parasitic nematodes in general) are linked to the same two genes. A further rigorous acquisition of both D2-D3 of 28S and COI gene sequences, which appear most promising for species delimitation in plant-parasitic nematodes (see [74]), will allow for more substantiated coalescence-based, multilocus species delimitation in plant-parasitic nematodes. Nevertheless, based on all obtained evidence, our findings support the proposition of Puillandre et al. [87], Padial et al. [88] and Qing et al. [80], that DNA-based species delimitation methods are important tools for the exploration of species delineation in diverse groups, but that identification of any new putative species will require further corroboration by an integrative taxonomic approach.

Nematode Populations
Nematode samples used in this study were collected from various localities (Table 1). Bulk soil samples of about 500 mL from 15-20 cm depths were collected from twelve locations in Belgium using a shovel. They were subsequently stored at 4 • C until nematode extraction. Nematodes were extracted from soil using a modified Baermann's method [89] or a rapid centrifugal flotation method [90]. Nematode extracts were observed under a stereo microscope. Paratylenchus populations were picked out in an embryo glass dish and stored in tap water at 4 • C for further analysis.

Morphological Study
Morphological study of nematodes was carried out using both heat relaxed and fixed specimens mounted on temporary and permanent slides, respectively. For preparation of a temporary mount of a nematode, a Cryo-Pro label (VWR International) was cut into two halves and stuck at the centre of a glass slide creating a small parallel gap between them. A single nematode was then transferred in a drop of distilled water to the glass slide in the centre of the gap. The nematode was then heat relaxed by passing over a flame a few times and covered with a glass coverslip. The specimen was then examined, photographed and measured using an Olympus BX51 DIC Microscope (Olympus Optical, Tokyo, Japan), equipped with an Olympus C5060Wz camera [91]. After recording morphological data, the specimen was recovered from the slide by adding a few drops of water from one end of the gap and collecting the nematode that was flushed out on the other end of the gap. The recovered specimens were subsequently used to extract genomic DNA as described in the next section.
A small nematode suspension of the remaining nematodes was heated in an embryo glass dish with a few drops of Trump's fixative ((2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M Sorenson buffer (sodium phosphate buffer at pH = 7.5)) in a microwave (700 Watts) for 3-4 sec and leaving it at room temperature for 1 h and at 4 • C for 24 h and followed by gradually transferring to anhydrous glycerine, as described in Singh et al. [92]. The fixed specimens were then mounted in glycerine on glass slides and were studied as above using the camera-equipped microscope. Species identification was carried out both at Nematology Research Unit of Ghent University and National Plant Protection Organization, Wageningen, the Netherlands.
For scanning electron microscopy, specimens fixed in Trump's fixative were washed in 0.1 M phosphate buffer (pH = 7.5) and dehydrated in a graded series of ethanol solutions, critical point-dried with liquid CO 2 , mounted on stubs with carbon tabs (double conductive tapes), coated with gold of 25 nm, and photographed with a JSM-840 EM (JEOL) at 12 kV [92].

Phylogenetic and Species Delimitation Analysis
The new sequences for each gene (D2-D3 of 28S, ITS, 18S rRNA and COI) were aligned using Clustal X 1.83 [104] with their corresponding published gene sequences [22,24,[32][33][34][35][36][37][38][39][42][43][44][45][46][47][48][49][50]. Outgroup taxa for each dataset were chosen based on previously published data [105]. Sequence datasets were analysed with Bayesian inference (BI) using MrBayes 3.1.2 [106] under the GTR + I + G model. BI analysis was initiated with a random starting tree and was run with four chains for 1.0 × 10 6 generations for 18S and ITS rRNA gene alignments, 5.0 × 10 6 generations for D2-D3 of 28S rRNA gene alignment and 9.0 × 10 6 generations for COI gene alignment. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. The log-likelihood values of the sample points stabilised after approximately 1,000 generations. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analysis. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PPs) are given on appropriate clades. Sequence analyses of alignments were performed with PAUP * 4b10 [107]. Pairwise divergences between taxa were computed as absolute distance values and as percentage mean distance values based on whole alignment with adjustments for missing data.
The COI gene alignments for P. straeleni and P. enigmaticus were used to construct phylogenetic network estimation using statistical parsimony, as implemented in POPART software [108].
Species delimitation of Paratylenchus in this study was undertaken using an integrated approach that considered morphological and morphometric evaluations combined with molecular-based phylogenetic inference (tree-based methods) and coalescent-based molecular species-delimitation methods. Putative species boundaries on a given phylogenetic input tree were inferred using a Bayesian implementation of the Poisson tree processes (bPTP) method [76] and using the generalized mixed-yule coalescent (GMYC) method [75]; see Qing et al. [80] for more details. Ultrametric trees were constructed using BEAST v1.10.4 [109] based on D2-D3, ITS, 18S and COI sequences, respectively. Default prior distributions were used and analyses were run for 1 × 10 7 generations, saving trees every 1 × 10 3 generations. The final trees were produced after removing 2,000 samples (20%) as burn-ins, and the maximum clade credibility tree was calculated using TreeAnnotator 1.10.4 [109]. Finally, for the bPTP method, an unrooted Bayesian 50% majority-rule consensus tree, containing only ingroups and unique haplotypes, was uploaded on the online server [110] and 1 × 10 5 Markov chain Monte Carlo (MCMC) generations were performed. The same tree was also uploaded on the GMYC web server [111] using the single threshold method.

Conclusions
An integrative approach by linking DNA sequences and morphological characters represents the best way to move nematode taxonomy forward. Creating this link involves the rigorous generation of multiple DNA sequences from individual morphologically vouchered nematode specimens, which, in the current study, resulted in the first molecular characterisations for five species, the first COI sequences for eight species and, most importantly, the reassignments of 18 D2-D3 of 28S, 3 ITS, 3 18S rRNA and 25 COI gene sequences, which had been unidentified or misidentified.
This study showed that Paratylenchus is a case in point, representing an incredibly diverse yet morphologically minimalistic plant-parasitic genus. Our recommendations for future protocol in Paratylenchus taxonomy, which are also valid for integrative nematode taxonomy, are: (1) to include SEM in new descriptions or re-descriptions; (2) to use juvenile and male traits after their conspecificity is irrefutably proven using molecular data; (3) to unequivocally link elaborate morphological data with both nuclear D2-D3 of 28S rRNA and mitochondrial COI gene sequences; (4) to employ caution when performing molecular identification using partial 18S rRNA gene fragments only; (5) to make use of the promising molecular species delineation methods to establish species boundaries, but base this on multilocus data and merely use it as one of the elements of integrative taxonomy.  Data Availability Statement: The datasets generated during and/or analyzed during the currentstudy are available from the corresponding author on reasonable request.