In South Africa the genus
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. Nematodes 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.
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.
One specimen from each population was transferred into an Eppendorf tube containing 15 µl of ddH2O and DNA was extracted from each population using the chelex method (Musapa et al. 2013) modified by Rashidifard et al. (2019) as follow: 20 μl chelex-100 (5% w/v) and 5 μl 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′-ACAAGTACCGTGAGGGAAAGTTG-3′), D3B (5′-TCGGAAGGAACCAGCTACTA-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. The amplification tube contained 12.5 μl master mix (Promega Corporation, USA), 1 μl of each of the primers (i.e. forward and reverse), 5 μl DNA, and 5.5 μl ddH2O.
Polymerase chain reaction steps used for amplification of the SSU and LSU rDNA genes.
35 cycles | |||||
---|---|---|---|---|---|
Primers | Initial denaturation | Denaturation | Annealing | Extension | Final extension |
SSU F04/SSU R26 | 94°C 3 min | 94°C 45 sec | 54°C 45 sec | 72°C 45 sec | 72°C 6 min |
D2A/D3B | 94°C 3 min | 94°C 45 sec | 56°C 45 sec | 72°C 45 sec | 72°C 6 min |
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 (
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 (
Light microscope pictures of females of
Light microscope pictures of males of
Measurements: Table 2.
Morphometrics of two populations of
Population 1 | Population 2 | |||
---|---|---|---|---|
Characteristics | Female ( |
Male ( |
Female ( |
Male ( |
L | 1,315 ± 90.2 (1,166-1,397) | 1,193 ± 110.1 (1,082-1,365) | 1,423.0* | –, 1,399 |
|
979 ± 75.2 (880-1,086) | 887 ± 68.2 (783-1,009) | 1,140 ± 69.7 (1,093-1,220) | 1,148, 1,136 |
|
44.5 ± 2.7 (40.8-47.6) | 47.6 ± 6.0 (37.5-54.2) | 39.9* | –, 43.4 |
|
32.5 ± 2.2 (30.2-35.3) | 35.3 ± 3.3 (29.3-39.6) | 32.8 ± 1.9 (31.0-34.8) | 36.8, 35.3 |
|
5.3 ± 0.4 (4.7-5.8) | 5.2 ± 0.2 (4.9-5.4) | 5.5* | –, 5.4 |
|
3.9 ± 0.3 (3.5-4.1) | 3.9 ± 0.2 (3.5-4.3) | 4.3 ± 0.2 (4.1-4.5) | 4.8, 4.4 |
|
4.6 ± 0.9 (4.0-6.0) | 3.9 ± 0.4 (3.4-4.6) | 4.5* | –, 5.3 |
|
14.5 ± 2.0 (11.7-16.3) | 13.5 ± 3.1 (8.7-16.9) | 13.3* | –, 9.2 |
|
45.7 ± 1.3 (43.5-46.6) | 39.2 ± 3.2 (36.3-44.4) | 45.3* | –, 50.2 |
G1 | 12.6 ± 1.4 (11.4-14.8) | – | 13.0* | –, – |
G2 | 12.4 ± 0.9 (11.5-13.5) | – | 13.8* | –, – |
Body width at midbody | 31 ± 3.1 (26-35) | 25 ± 2.9 (20-30) | 35 ± 1.1 (34-36) | 31, 32 |
Labial region diameter | 19 ± 1.9 (18-23) | 17 ± 1.7 (14-20) | 19 ± 0.7 (18-19) | 18, 19 |
Cephalic setae length | 8 ± 0.7 (8-9) | 8 ± 0.9 (7-10) | 7 ± 0.5 (7-8) | 7, 7 |
Length of stoma | 26 ± 1.5 (23-28) | 24 ± 1.7 (21-27) | 27 ± 0.6 (27-28) | 26, 26 |
Stoma width | 11 ± 1.1 (9-13) | 10 ± 1.3 (8-12) | 12 ± 0.9 (12-13) | 12, 12 |
Cheilostom length | 12 ± 0.8 (11-13) | 10 ± 2.2 (5-13) | 14 ± 1.6 (12-15) | 13, 12 |
Gymnostom length | 7 ± 1.0 (6-9) | 7 ± 2.0 (5-10) | 6 ± 0.4 (6-7) | 6, 6 |
Stegostom length | 7 ± 1.1 (6-9) | 5 ± 1.1 (4-7) | 7 ± 0.8 (6-8) | 7, 8 |
Dorsal tooth length | 9 ± 1.2 (7-10) | 7 ± 0.9 (6-8) | 7 ± 0.7 (6-7) | 9, 7 |
Subventral tooth length | 5 ± 0.9 (4-6) | 4 ± 0.4 (4-5) | 5 ± 0.7 (4-5) | 5, 4 |
Corpus (procorpus and metacorpus) | 137 ± 3.2 (133-141) | 125 ± 7.2 (113-140) | 144 ± 2.5 (142-147) | 138, 134 |
Postcorpus (isthmus and basal bulb) | 115 ± 5.3 (107-120) | 102 ± 8.6 (86-118) | 121 ± 5.2 (116-126) | 102, 125 |
Pharynx (anterior end to base of basal bulb) | 252 ± 8.1 (241-262) | 227 ± 15.3 (199-258) | 265 ± 6.4 (258-269) | 240, 259 |
Excretory pore from anterior | 191 ± 45.4 (163-243) | 156 ± 9.4 (146-171) | 157 ± 2.5 (155-158) | –, 173 |
Nerve ring from anterior | 140 ± 6.3 (133-149) | 123 ± 10.6 (100-140) | 146 ± 3.5 (143-150) | 138, 134 |
Metacorpus width | 22 ± 2.5 (19-26) | 20 ± 1.2 (18-22) | 28 ± 3.7 (25-32) | 20, 23 |
Basal bulb width | 22 ± 2.6 (18-25) | 18 ± 1.5 (16-21) | 27 ± 0.6 (27-28) | 18, 22 |
Cardia length | 9 ± 1.3 (8-12) | 9 ± 3.1 (6-14) | – | 7, – |
Anterior genital tract length | 166 ± 27.8 (129-205) | – | 196 ± 11.8 (185-209) | – |
Posterior genital tract length | 157 ± 15.2 (133-184) | – | 188 ± 7.1 (182-196) | – |
Body width at vulva | 31 ± 3.2 (26-36) | – | 35 ± 1.5 (34-36) | – |
Vulva from anterior end | 608 ± 42.5 (542-671) | – | 683 ± 49.5 (645-739) | – |
Vulva-anus distance | 376 ± 32.1 (338-415) | – | 457 ± 27.2 (427-481) | – |
Vagina length | 12 ± 2.5 (10-16) | – | 14 ± 2.1 (12-16) | – |
Rectum length | 32 ± 2.6 (29-36) | – | 31 ± 5.3 (28-37) | – |
Body width at anus/cloaca | 20 ± 1.6 (18-22) | 24 ± 3.2 (19-28) | 24 ± 0.4 (24-25) | 26, 29 |
Testis length | – | 496 ± 72.0 (392-589) | – | 607, 703 |
Spicules length | – | 40 ± 2.7 (36-43) | – | 39, 43 |
Gubernaculum length | – | 27 ± 2.6 (23-31) | – | 30, 29 |
Tail length | 290 ± 46.4 (225-325) | 310 ± 43.2 (242-356) | 317.0 | –, 263 |
Phasmid posterior to anus | 29 ± 1.5 (28-31) | 36 ± 4.3 (30-44) | – | –, 37 |
Female (
Male (
Soil samples were collected from two home garden compost heaps (1: 26°42′25.3′′S 27°06′25.9′′E, 2: 26°41′17.4′S 27°06′05.6′E) in PotchefstroomNorth-West Province, South Africa. Slides containing 10 females and 12 males were deposited in the NCN (ARC-PHP, Biosystematics, Pretoria).
The South African specimens of
The South African specimens of
In comparison to the other species of the genus the South African populations of
The Nucleotide BLAST (Blastn) search based on the partial SSU sequences (MN710517 and MN710518) showed > 99% similarity to a
Bayesian topology based on partial SSU sequences confirmed the well-supported sister relationship (PB: 0.99) of South African populations (MN710517 and MN710518) with another population of
Bayesian phylogenetic tree with 50% majority rule of
Bayesian phylogenetic tree with 50% majority rule of
Two populations of