Morphological and molecular characterization of Hoplolaimus pararobustus (Schuurmans Stekhoven and Teunissen, 1938) Sher 1963 with its first report on Zea mays roots in Namibia

Abstract In the summer of 2018, specimens of a Hoplolaimus population were extracted from a maize root sample collected near Stampriet, Namibia. This population was identified as Hoplolaimus pararobustus and is described and illustrated based on its morphological, morphometric, and molecular characteristics. To our knowledge, this is the first report H. pararobustus from maize roots. Females of the population had a mean body and stylet length of 1,100 µm and 36 µm, respectively. Esophagus with three nuclei in three pharyngeal glands. Lateral field reduced, ranging from a very faint line to just breaks in striae. The males were shorter than the females with a mean body length of 925 µm and the stylet slightly shorter, with a mean length of 34 µm. Phylogenetic analyses using partial sequences of 18 S and the expansion fragment D2–D3 of 28 S rDNA genes showed the close relation of this species and H. columbus. This Namibian population of H. pararobustus is the first Hoplolaimus species from Africa to be molecularly characterized.

Hoplolaimus pararobustus has been reported from inside roots of banana (Whitehead, 1959) giving rise to dark-brown pustules that eventually result in necrotic cortical tissue situated around the heads of the feeding sites of the nematodes and eventually elongated ulcerated lesions on the roots (Siddiqi, 1974). It has also been reported, at a population density of 200 individuals per gram of tissue, in corms and roots of banana where they were likely to cause damage to the crop (Sikora et al., 2018). Phylogenetic analysis of Hoplolaimus spp. using D2-D3 expansion of 28 S and internal transcribed spacer (ITS1) ribosomal DNA sequences resolved the phylogeny of the genus and were useful in molecular identification of Hoplolaimus spp. (Bae et al., 2008). In addition, the PCR-RFLP method was applied by different researchers to evaluate the genetic diversity of Hoplolaimus spp. (Robbins et al., 2009;Bae et al., 2009). Later, a species-specific primer was developed to distinguish Hoplolaimus stephanus Sher, 1963 from another similar species viz. Hoplolaimus galeatus (Cobb, 1913) Thorne, 1935(Ma et al., 2011. Moreover, sequences of the actin gene were successfully used for phy logenetic studies of Hoplolaimus spp. (Ma et al., 2011). High genetic variability among the Hoplolaimus populations in soybean-growing areas in the USA was reported when their genetic diversity was evaluated based on sequences of ITS1 ribosomal DNA and COI mitochondrial DNA genes (Holguin et al., 2015). Although the evolutionary relationships of Hoplolaimus spp. have been studied, phylogenetic analyses of this genus within the subfamily Hoplolaiminae are still lacking. Therefore, this study aimed to characterize a population of a Hoplolaimus isolated in Namibia using both morphological and molecular approaches, which is presented herein as H. pararobustus. To our knowledge, this is the first report of H. pararobustus from maize (Z. mays) roots.

Materials and methods
Nematode extraction and morphological studies Nematodes were extracted from roots using an adapted sugar centrifugal flotation method (Marais et al., 2017). Females and males were fixed in a heated 4% formaldehyde plus 1% propionic acid (FPG) solution, dehydrated in a glycerine solution, and mounted in glycerine on glass slides using a wax ring method (Marais et al., 2017). Measurements and drawings of the mounted specimens were done with a Nikon LABOPHOT-2 microscope equipped with a Nikon 1.25× drawing tube. All measurements were done at ×1,000 magnification. Curved structures were measured along the median line. Morphometrics were used in the descriptions with standard morphometric calculations and terms used throughout the paper (Siddiqi, 2000). Specimens were deposited in the National Collection of Nematodes (NCN), Biosystematics, Agricultural Research Council (ARC) -Plant Health and Protection (PHP), Pretoria.

Taxonomy and phylogenetic analyses
The newly generated 18 S and 28 S rDNA sequences of the Namibian nematode population were compared to those available in GenBank using a BLAST search. For phylogenetic tree construction, available sequences of the subfamily Hoplolaiminae were retrieved from GenBank for 18 S and 28 S data sets. Both data sets were aligned using MUSCLE (Edgar, 2004) in Geneious Prime 2020.0.4 (https//:www. geneious.com). The jModelTest 2.1.10 program (Darriba et al., 2012) was used to identify the best nucleotide substitution model. The General Time Reversible with an invariable site and a gamma distribution (GTR + I + G) and General Time Reversible with a Gamma distribution (GTR + G) were the most suitable models for 18 S and 28 S data sets, respectively. Bayesian analysis was accomplished using MrBayes 3.2.2 (Huelsenbeck and Ronquist, 2001) in Geneious Prime 2020.0.4 (https//:www. geneious.com); the chain was run for 3×10 6 generations for each locus. After discarding burnin samples (25%), posterior probability (PB) of the Bayesian trees was estimated using the Markov chain Monte Carlo (MCMC) algorithm (Larget and Simon, 1999) based on the 50% majority rule. Heterodera schachtii Schmidt, 1871 and Globodera rostochiensis (Wollenweber, 1923) Skarbilovich, 1959 were, respectively, used as outgroups for the 18 S and 28 S phylogenetic trees.

Description Female
Habitus slightly curved ventrad, C-shape, S-shape or curved into a complete spiral with head and tail end overlapping. Body length of (957-1,245 μ m). Cuticular annules distinct about 2 µm wide. Lip region broadly rounded and well set off from body usually with four distinct annules but sometimes with three on the one side and four on the other side. Basal annulus sometimes larger than others. Longitudinal striae on basal annulus faint. Labial framework well sclerotized. Stylet well-developed (34-40 μ m) long with metenchium and telenchium almost equal in length. Stylet knobs tulip-shaped with two or more projections anteriorly. Median bulb round, muscular (15-20 μ m long), 13-17 μ m wide with a prominent centrally located valve, 4-6 μ m long and 3-5 μ m wide. Nerve ring encircling the isthmus. Excretory pore situated from opposite anterior part of median bulb to opposite the middle of the esophagus, 82-123 μ m from anterior end, at 61-85% of esophagus length. Esophagus with three nuclei in each of the three esophageal glands extending dorsally over the intestine. Hemizonid two annules long and situated from opposite excretory pore, nine annules posterior to it. Hemizonion not seen. Esophagus glands overlap 15-44 μ m long. Vulva a transverse slit with epiptygma folded into the vagina. Spermatheca small, oval or round, empty or filled with rounded sperm. Lateral field reduced; very faint line to just breaks in striae. Two or three very faint incomplete incisures can sometimes be seen in the lateral field area. Caudalid not seen. Intestine does not overlap the rectum. Phasmids: two enlarged scutella situated anterior and

Diagnosis and relationships
Hoplolaimus pararobustus belongs to the group in which the lateral field is degenerate, not showing the regular compliment of four incisures and three and not six esophageal gland nuclei (Van den Berg and Quénéhervé, 2012). The current Namibian specimens are considered to be H. pararobustus because of the presence of a well set off, broadly round lip region with four annuli, lateral field represented by a single incisura, and two or three very faint incomplete incisures that can sometimes be seen in the lateral field area. Esophagus with three nuclei in three pharyngeal glands, no overlap of the rectum, and presence of males. The Namibian specimens correspond to the descriptions of Fortuner (1991), Larizza et al. (1998), and Van den Berg and Quénéhervé (2012), but for the lower range of stylet length of females (34-40 µm vs 37.5-49 µm) and males (32-42 µm vs 35-46 µm), expansion of the reported range for the position of the vulva (V = 49-67% vs V = 51-62.1%), and a shorter spicule (35-42 µm vs 40-57 µm) than that reported (Van den Berg and Buckley, 1987;Fortuner, 1991;Larizza et al., 1998). Molecular analyses indicated that H. columbus Sher, 1963 is the closest to the Namibian population of H. pararobustus. Hoplolaimus columbus was described from soybean in the USA and is currently reported from India, Pakistan, Vietnam, Egypt, and the USA (Shafiee and Osman, 1971;Fortuner, 1991). This lance nematode belongs to the group in which the lateral field is represented by one indistinct incisure and six esophageal gland nuclei, one or two sometimes indistinct (Fortuner, 1991), this is in contrast with the three nuclei observed in the Namibian specimens.  Rotylenchus Filipjev, 1936 andPeltamigratus Sher, 1963 were the closest genera to the genus Hoplolaimus, respectively ( Figure 3).

JOURNAL OF NEMATOLOGY
belongs to the group in which the lateral field is degenerate, not showing the regular complement of four incisures and with three and not six esophageal gland nuclei (Van den Berg and Quénéhervé, 2012). The Namibian specimens correspond to the redescription of Fortuner (1991), but the lower range of the body and stylet length of females and males, the position of the vulva more anterior, and spicule length is shorter than that previously reported (Fortuner, 1991;Larizza et al., 1998;Van den Berg and Quénéhervé). Hoplolaimus pararobustus is commonly found in Namibia, reported from both cultivated and noncultivated areas, but this is according to our knowledge the first report from maize roots. This study reported the first molecular characterization of an African population of Hoplolaimus, in this case H. pararobustus. We resolved the evolutionary relationship of H. pararobustus based on partial 18 S and 28 S rDNA sequences. The constructed Bayesian tree using 18 S data set showed that H. pararobustus is in a well-supported clade with two populations of H. galeatus and one population of H. columbus, with H. columbus being the closest to the Namibian population of H. pararobustus. Ultimately, the monophyletic nature of the genus was confirmed using both phylogenetic trees.