Morphological and molecular characterization of Butlerius butleri Goodey, 1929 (Nematoda: Diplogastridae) from South Africa: First report

Abstract Two populations of a Butlerius species were recovered from compost in two gardens in Potchefstroom, North-West Province, South Africa. Although the genus has previously been reported from South Africa, no species of the genus has ever been identified in the country. Based on morphological, morphometric, and molecular studies, the specimens were identified as Butlerius butleri and are herein reported for the first time from South Africa. The South African specimens are 1,082 to 1,423 µm long, a = 40.8 to 47.6; b = 4.7 to 5.8; c = 4.0 to 6.0; c′ = 117 to 16.3; V = 44 to 47%. Cuticle with evenly spaced punctations. Reproductive system didelphic, amphidelphic, both branches equal in length. Four large glands opening into proximal part of uterus. Males with prominent sphincter present in mid-region of vas deferens. Spicules 36 to 43 μm long, gubernaculum 23 to 31 µm long, nine pairs of genital papillae, three pre-cloacal and six post-cloacal, formula: v1, v2, v3d/v4, ad, ph, v5, 6, 7, pd. The v5, 6, 7 clusters greatly separated, left subventral group at level of phasmid, right subventral group at level of posterior dorsal papilla. Although there were some differences, the South African populations of the species compare well to all know descriptions of the species. Phylogenetic analysis based on partial small subunit (SSU) rDNA sequences showed that both South African populations of B. butleri are in a maximally supported sister relation with an Iranian population of this species. Based on large subunit (LSU) rDNA sequences, the two populations of B. butleri clustered together in a well-supported clade.

from specimens found in rotting plant material in South Korea. More recently Shokoohi et al. (2015) described a population from vermicompost in Iran and presented the first molecular characterization of the species.
In South Africa the genus Butlerius was first reported in 1961 (Heyns, 1961) and are commonly found in soil samples (Dr Marais, ARC-PHP, personal communication). However, populations of the genus are very rarely formally reported in the country. Species of the genus are yet to be reported or described from South Africa. During sampling of compost matter from two different localities in Potchefstroom, North-West Province, South Africa, two populations of the genus Butlerius were found and identified as B. butleri and is herein reported for the first time from South Africa using morphological and molecular techniques.

Materials and methods
Sampling, nematode extraction, and fixation Soil samples were collected from two home garden compost heaps (garden 1/population 1: 26°42′25.3″S 27°06′25.9″E; garden 2/population 2: 26°41′17.4″S 27°06′05.6″E) in Potchefstroom, North-West Province South Africa. Following, the samples were transported in cooler boxes to the North-West University and stored at 6 to 8°C until further processing. Nema todes were extracted from soil samples using the adapted decanting and sieving followed by the sugar centrifugal-flotation method (Marais et al., 2017). The extracted nematodes were fixed in a heated 4% formaldehyde plus 1% propionic acid (FPG) solution, dehydrated in a glycerin solution following De Grisse (1969) and mounted in glycerin on glass microscope slides.

Morphological characterization
Measurements and drawings of the mounted specimens were done with a Zeiss Axio Imager A2 equipped with an Axiocam ERc5s camera and iPad with the Labscope imaging application as a drawing tube. All measurements and identifications were done at 1,000 × magnification. Curved structures were measured along the median line. All measurements in the descriptions are given in micrometers (µm) and in the form: mean ± standard deviation (range). All specimens were deposited in the National Collection of Nematodes (NCN), Biosystematics, Agricultural Research Council -Plant Health and Protection (ARC-PHP), Pretoria.

DNA extraction and PCR
One specimen from each population was transferred into an Eppendorf tube containing 15 µl of ddH 2 O and DNA was extracted from each population using the chelex method (Musapa et al. 2013) modified by Rashidifard et al. (2019) as follow: 20 μ l chelex-100 (5% w/v) and 5 μ l proteinase K (20 mg ml −1 ) were added to each tube containing the nematodes, the tubes were vortexed (15 sec) and centrifuged at 8,000 rpm for 10 sec. Finally, the tubes were incubated at 56°C for 2 hr followed by incubation at 95°C for 10 min before they were stored at −20°C. The following DNA markers were used to amplify the small and large sub units (SSU and LSU) genes, respectively: SSU F04 (5′-GCTTGTCTCAAAGATTAAGCC-3′), SSU R26 (5′-CATTCTTGGCAAATGCTTTCG-3′) (Blaxter et al., 1998), and D2A (5′-ACAAGTACCGTGAGGG AAAGTTG-3′), D3B (5′-TCGGAAGGAACCAGCTAC TA-3′) (Subbotin et al., 2006). Following DNA extraction, the polymerase chain reaction (PCR) was carried out using an Eppendorf Mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany); more details are provided in Table 1 contained 12.5 μ l master mix (Promega Corporation, USA), 1 μ l of each of the primers (i.e. forward and reverse), 5 μ l DNA, and 5.5 μ l ddH 2 O. Four microliters of PCR product were loaded on a 1% agarose gel to check the DNA quality. The DNA was stained using GelRed and then visualized under a UV transilluminator. The PCR product was stored at −20°C before sequencing by inqaba biotec™, South Africa (www.inqaba-southafrica.co.za).

Phylogenetic analyses
Selection of the appropriate taxa for SSU phylogenetic analysis was done according to Shokoohi et al. (2015). For the LSU analysis the available sequences for Diplogastridae, as well as the outgroups, were obtained from NCBI GenBank. The sequences of selected taxa for each gene were aligned using the MUSCLE tool (Edgar, 2004) implemented in Geneious Prime® 2019.2.1 (https:// www.geneious.com). Post-editing for each alignment was done using Gblock program (version 0.91b) (http://phylogeny.lirmm.fr/phylo_cgi/one_task. cgi?task_type=gblocks) with all three less stringent parameters. The jModelTest program 2.1.10 (Darriba et al., 2012) was used to identify the best nucleotide substitution model. General time reversible model with a Gamma distribution (GTR + G) was selected as the most appropriate model for SSU and D2-D3 LSU analyses. Bayesian inference (BI) was performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) Geneious Prime® 2019.2.1 and the chains were running for 2 × 10 6 generations for both the SSU and LSU datasets. After discarding a 25% burnin sample to estimate the posterior probabilities of the phylogenetic trees, the Markov Chain Monte Carlo (MCMC) algorithm was used (Larget and Simon, 1999) to estimate the posterior probabilities (PI) with the 50% majority rule. One population of Odontopharynx longicaudata De Man, 1912 was selected as outgroup for each dataset.

Diagnosis
The South African specimens of B. butleri are characterized by a 1,082 to 1,423 µm long body, cuticle with evenly spaced punctations, reproductive system didelphic, amphidelphic (V = 44-47%), both branches equal in length, four large glands opening into proximal part of uterus, males with prominent sphincter present in mid-region of vas deferens, spicules 36 to 43 μm long, gubernaculum 23 to 31 µm long , nine pairs of genital papillae, three pre-cloacal and six post-cloacal, formula: v1, v2, v3d/v4, ad, ph, v5, 6, 7, pd. The v5, 6, 7 clusters greatly separated, left subventral group at level of phasmid, right subventral group at level of posterior dorsal papilla. The two South African specimens were compared to the original description of B. butleri by Goodey (1929) as well as the redescription of the species by Ahmad et al. (2009) and a population reported by Shokoohi et al. (2015).

Discussion
Two populations of Butlerius were collected in 2019 from garden compost in Potchefstroom, South Africa and characterized as B. butleri. Although differences were observed between the South African populations and the other populations of B. butleri reported previously, the South African specimens were conspecific to the species and represent the first report of the species B. butleri in South Africa. The populations of B. butleri described herein broaden the morphometric range of the species as the South African populations measured smaller than both the South Korean and Iranian populations of the species. The constructed phylogenetic tree based on SSU sequences confirmed the phylogeny published by Shokoohi et al. (2015) where Butlerius populations represent a monophyletic group and were closely related to the genera Diplogatrellus Paramonov, 1952 andPseudodiplogasteroides Körner, 1954. However, Butlerius can be distinguished from Diplogastrellus based on the shape of the stoma (barrel shaped in Butlerius vs tube shaped in Diplogastrellus) and length of the cephalic setae (longer in Butlerius as compared to Diplogastrellus). Furthermore, Butlerius could be separated from Pseudodiplogasteroides based on the shape of the stoma (barrel shaped in Butlerius vs tube shaped in Pseudodiplogasteroides) and the presence of remnants of haustrulum in the terminal pharyngeal bulb of Pseudodiplogasteroides that is not present in Butlerius. The evolutionary relationship of B. butleri based on partial LSU sequences revealed a close relation of this genus and Diplogasteroides De Man, 1912, however, these genera are differentiated by the shape of the stoma (barrel shaped in Butlerius vs tube shaped in Diplogasteroides) and the shape of the dorsal tooth in the stegostom (thorn-like in Butlerius vs dorsal tooth formed by three rods, lateral ones distally diverging in Diplogasteroides). Despite of novel phylogenetic relationships of B. butleri based on partial LSU rDNA sequence, monophyly of the genus could not be solely confirmed due to lack of sequences for the other members of the genus in the GenBank. Ultimately, adding further molecular data to the current phylogeny in the future would improve our knowledge and provide better resolution on taxonomy of the genus.