Description of Prionchulus jonkershoekensis n. sp. (Nematoda: Mononchida), a new predatory species from South Africa

Abstract Prionchulus jonkershoekensis n. sp. is described from South Africa and illustrated using morphological, morphometric, and molecular techniques. This species is characterized by its body length (1.78–2.14 mm); the size of buccal cavity (38–44 × 24–31 µm), lower dorsal tooth position in relation to buccal cavity base, the position of amphidial aperture just above dorsal tooth apex, pars proximalis vaginae with almost straight walls, and tail 144–158 µm long with sickle shaped posterior third part. Phylogenetic analyses based on 18 S rDNA and 28 S rDNA of P. jonkershoekensis n. sp. revealed close relationships of the new species with Prionchulus punctatus and Prionchulus muscorum. This is also an additional geographical record for the genus from South Africa.

Species belonging to the genus Prionchulus Cobb, 1916 are predatory nematodes and have been described from terricolous and limnic habitats (Andrássy, 2006). Members of the genus are easily recognizable; however, species within the genus share high morphological similarities (Vu et al., 2018) making identification problematic. The genus has been revised several times by many taxonomists (Mulvey, 1967;Andrássy, 1985Andrássy, , 1993, with Susulovsky (2003, 2004) also redescribing a number of species from type material. Species lists of the Prionchulus have been revised and updated by different scientists in the last decade (Andrássy, 2006;Ahmad and Jairajpuri, 2010;Jana et al., 2010). Members of the family Mononchidae, to which the genus Prionchulus belongs, have been poorly studied in South Africa. Only one species, Prionchulus muscorum (Dujardin, 1845) Cobb, 1916 has previously been reported from the Kowynspas (Coetzee, 1968) in the Gauteng Province, South Africa.
The genus has a cosmopolitan distribution, with Europe being particularly rich in species (Andrássy, 2006). To date, 30 species have been described from this genus worldwide (Kim et al., 2018). Most of the species in the genus have been collected from moss, leaf litter, and forest soil (Susulovsky and Winiszewska, 2002;Winiszewska and Susulovsky, 2003;Orselli and Vinciguerra, 2007) associated with riverbanks and mountainous regions (Susulovsky and Winiszweska, 2006;Farahmand et al., 2009).
These predatory nematodes play an important role in bioregulation of litter and soil communities (Dipchikova et al., 2019). During a survey of the Jonkershoek Mountains in Stellenbosch, South Africa, samples were collected from seven localities to study nematodes associated with leaf litter. Among the various nematodes ex tracted, one population of Prionchulus was collected. A thorough investigation revealed that this was an undescribed species, and is described herein as Prionchulus jonkershoekensis n. sp. This species was characterized using both morphological and molecular techniques, its phylogenetic relationships are also discussed based on 18 S and 28 S rDNA genes.

Materials and methods
Nematode extraction and processing Prionchulus jonkershoekensis n. sp. specimens were extracted from leaf-litter samples collected from one locality in the Jonkershoek Mountains by using the Baermann tray method (Hooper and Evans, 1993). Nematodes were heat-killed and fixed using the glycerol-ethanol method of Seinhorst (1959) as modified by De Grisse (1969), and mounted on Cobb slides in anhydrous glycerin for identification purposes. Specimens for molecular analysis were stored in DESS solution (dimethyl sulphoxide, disodium EDTA, and saturated NaCl).
Measurements and drawings of mounted Prion chulus specimens were made with the aid of a Nikon 80i microscope equipped with a drawing tube. Micrographs were taken with an automatic camera system mounted (Axiocam ICc 5) on Zeiss Axiophot microscope. Using the drawings made with a drawing tube as a template, digital drawings were constructed in CorelDRAW X5.

Scanning electron microscopy
Mounted specimens were removed carefully from slides, hydrated in distilled water, dehydrated in a graded ethanol series, critical point dried, and coated with gold (Green, 1967), and observed under a JEOL JSM-7800F Field Emission Scanning Electron Microscope at 5 kV.

Phylogenetic analyses
The newly generated sequences were aligned using MUSCLE alignment tool (Edgar, 2004) in Geneious Prime 2020.2.3 (www.geneious.com) and consensus sequences were extracted and used for further analyses. The newly obtained consensus sequences of 18 S and 28 S rDNA genes were compared to those available for other species in Genbank using BLAST search. Two data sets were prepared for 18 S and 28 S genes using available mononchid sequences in Genbank; newly obtained sequences were also included. Both 18 S and 28 S rDNA datasets were aligned using MUSCLE (Edgar, 2004) in Geneious Prime 2020.2.3 (www.geneious. com). The General Time Reversible model with a Gamma distribution (GTR + G) was selected as the most appropriate nucleotide substitution model for both data sets according to jModelTest 2.1.10 (Darriba et al., 2012).
Bayesian inference (BI) analyses were conducted using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) implemented in Geneious Prime 2020.2.3. Two Markov chain Monte Carlo (MCMC) were run from a random starting tree for 2 million generations and trees were sampled every 100 generations. Burn-in sampled trees (25%) were discarded. The remaining trees were used to calculate the 50% majority rule consensus tree using the MCMC algorithm to estimate the Bayesian posterior probabilities (BPP) (Larget and Simon, 1999).
Cuticle moderately thick, with two layers, outer layer thinner than inner layer, with fine transverse striations. Cuticle appears more thickened at vulva and caudal region (2-4 µm). Lateral chord 27-38% of midbody diameter. Body pores obscure. Lip region rounded, offset by weak depression but of similar width with adjacent body, about 3-4 times as wide as high. Lips amalgamated, rounded to angular. Labial papillae slightly more conical and protruding than cephalic papillae.
Amphidial fovea cup-shaped, opening (oval to ellipsoid) at level of anterior denticle above dorsal tooth apex, 3-6 µm in diameter and occupying 1/5-1/6 of lip region width. Stoma consists of a vestibulum and a buccal cavity 1.4-1.7 times as long as wide, with 2-4 µm thick walls and tapering at its base, funnel shaped. Dorsal tooth of medium size situated in lower anterior half of buccal cavity, its apex sharply pointed, located 30-34 µm or 75-81% of the buccal cavity length from its base, its upper edge slightly curved, directed forward. Subventral denticles present, 11-14 in number, relatively large, orientated anteriorly, separated, arranged irregularly in some specimens. Two small foramina observed. Pharynx muscular, cylindrical, about 8-10 times as long as wide, surrounding basal part of stoma. Pharyngeal gland nuclei and outlets indistinct. Nerve ring located at 29-33% of the neck length.  Secretory-excretory pore visible, situated 30-38% of the neck length. Cardia oval to rounded, smooth, 0.3-0.5 times as long as wide, 0.2 times as long as body diameter at neck base, non-tuberculate. Intestine uniformly granulated; inner surface lined by short rod-like structures (bacillary layer).
Males: Not found.

Type locality and habitat
The type material was collected from leaf litter and moss near the First Waterfall hiking trail in the Jonkershoek mountains, Stellenbosch, South Africa (coordinates 33°59ʹ54.9ʺ S, 18˚58ʹ59.7ʺ E).

Type material
Holotype female (slide 51180), and one female and three juvenile paratypes (slide 51181) deposited in the National Collection of Nematodes (NCN), Agricultural Research Council-Plant Health and Protection (ARC-PHP), Pretoria, South Africa; 23 female paratypes deposited in the nematode collection of the University of the Free State (UFS), Bloemfontein, South Africa.

Etymology
The specific epithet refers to the geographical origin of the new species in the Jonkershoek Mountains in South Africa.

Molecular characterization
During the survey, two sequences for each gene were generated. However, since they were identical only one sequence of 18 S rDNA (

Phylogenetic relationships
The 18 S alignment was prepared using 47 sequences (739 nt long) in which 223 nt were variable, while the 28 S alignment contained 23 sequences (760 nt long), of which 494 nt were variable. Bayesian inference analyses using partial 18 S rDNA sequences clustered P. jonkershoekensis n. sp. closely with populations of P. punctatus and P. muscorum (Fig. 4). The 28 S rDNA Bayesian tree showed the new species forms a supported clade (BPP: 0.9) with three populations of P. punctatus (Fig. 5). Interestingly, both phylogenetic trees showed that Prionchulus and Clarkus Jairajpuri, 1970 are closely related genera.

Discussion
During this study, a population of Prionchulus representing a new species was found in South Africa and described herein as P. jonkershoekensis n. sp. using morphological, morphometrical, and molecular approaches. This also represents an additional geographical record for the genus Prionchulus. Two notable traits of the new species are its smaller buccal cavity dimensions and lower position of dorsal tooth. The phylogenetic position of P. jonkershoekensis n. sp. was resolved using sequences of partial 18 S and 28 S rDNA genes, however the monophyly of the genus Prionchulus was not established based on either of these genes. Although the new species shares some similarities with P. muscorum, phylogenetic analyses showed that this species and P. jonkershoekensis n. sp. are clearly distinct (Table 1).