Table 2.
Comparison of 37 Hoplolaimus species after Handoo and Golden (1992) and Ghaderi et al. (2020). The comparison includes eleven morphological and morphometric data and the presence or absence of male. Species included in this study are highlighted in bold and the measurements of body length (mm), stylet length and spicule length are in μm in range.
| Length | Lateral incisures | Gland nuclei | Stylet length | Labial annules | Longitudinal striae on basal lip annules | EP in relation to hemizonid | Intestinal post-rectal sac | Phasmids in relation to vulva | Tail annules | Males | Spicule length | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| H. abelmoschi | 1.5-1.8 | 2 | 3 | 42-47 | 5 | 25-28 | Anterior | Present | Both adjacent, one anterior & one posterior | 9-15 | Present | 44-47 |
| H. aegypti | 1.3-1.9 | 1 | 5-6 | 45-50 | 4 | 13-22 | Anterior | Present | One anterior & one posterior | 17-27 | Present | 54-65 |
| H. aorolaimoides | 0.8-0.9 | 4 | 3 | 31-35 | 4-5 | 6-13 | Posterior | Present | One anterior & one posterior | 6-17 | Present | 31-37 |
| H. bachlongviensis | 1.2-1.5 | 1 | 6 | 44-50 | 4.0 | 6 | Anterior | Absent | One anterior & one posterior | 9-13 | Unknown | - |
| H. californicus | 1.1-1.7 | 4 | 3 | 46-53 | 6 | 36 | Posterior | Present | Both posterior | 14 | Present | 45-55 |
| H. capensis | 1.6-2.1 | 2 | 3 | 46-58 | 5-6 | Unknown | Anterior | Present | One anterior & one posterior | 9-16 | Present | 51-70 |
| H. casparus | 1.2 | 0 | 3 | 39.7 | 3 | Unknown | Anterior | Absent | One anterior & one posterior | 12 | Present | 39-40 |
| H. caudifurcatus | 1.0-1.1 | - | >4 | 52-55 | 4-5 | Unknown | Anterior | Unknown | Unknown | 15-20 | Unknown | - |
| H. cephalus | 1.2 | 0 | 6 | 34 | Smooth | 0 | Anterior | Absent | One anterior & one posterior | 6 | Present | 33-38 |
| H. chambus | 1.2-1.6 | Breaks | 6 | 41-45 | 3 | 6 | Anterior | Present | One anterior & one posterior | 9-13 | Unknown | - |
| H. citri | 0.8-1.3 | 0 | 6 | 35-37 | 4 | 10-12 | Anterior | Absent | One anterior & one posterior | 12-15 | Present | 38-47 |
| H. clarissimus | 1.4-1.8 | 4 | 6 | 46-53 | 4 | 18-31 | Posterior | Present | One anterior & one posterior | 20-26 | Present | 55-61 |
| H. columbus | 1.3-1.8 | 1 | 6 | 40-48 | 3 | 10-15 | Anterior | Present | One anterior & one posterior | 16-22 | Rare | 37-53 |
| H. concaudajuvencus | 1.1-2.0 | 4 | 3 | 50-57 | 5-6 | 36 | Posterior | Absent | One anterior & one posterior | 7-14 | Present | 45-56 |
| H. diadematus | 1.1-1.8 | 2 | 3 | 47-52 | 3-4 | 20 | Anterior | Unknown | One anterior & one posterior | 9-13 | Present | 48-53 |
| H. dimorphicus | 1.1-1.6 | 0 | 6 | 34-36 | 2-3 | 18-21 | Anterior | Absent | One anterior & one posterior | 6-10 | Present | 36-41 |
| H. dubius | 1.1-1.3 | 1 | 6 | 31-42 | 3 | 14 | Anterior | Absent | One anterior & one posterior | 10-15 | Present | 37-44 |
| H. galeatus | 1.2-1.9 | 4 | 3 | 43-52 | 5 | 32-36 | Posterior | Present | One anterior & one posterior | 10-16 | Present | 40-52 |
| H. igualaensis | 1.1-1.5 | 4 | 3 | 40-50 | 5-6 | Unknown | Posterior | Unknown | Both posterior | 10-15 | Present | 36-43 |
| H. imphalensis | 1.0-1.4 | 1 | 3 | 34-37 | 3-4 | 29-30 | Anterior | Present | One anterior & one posterior | 12-14 | Present | 37-45 |
| H. indicus | 1.0-1.6 | 1 | 6 | 33-47 | 3-4 | 6-20 | Anterior | Present | One anterior & one posterior | 8-22 | Present | 34-42 |
| H. intermedius | 1.0-1.3 | Breaks | 3 | 37-40 | 4-5 | 6 | Anterior | Unknown | One anterior & one posterior | - | Present | 43-47 |
| H. jalalabadiensis | 1.2-1.7 | Breaks | 6 | 37-44 | 3-4 | 24-25 | Anterior | Present | One anterior & one posterior | 16-22 | Unknown | - |
| H. johani | 1.0-1.4 | Not visible | 3-6 | 21-30 | 3-5 | Unknown | Anterior | Unknown | Unknown | 14 | Present | 28-43 |
| H. maggentii | 1.0-1.4 | Absent | 3-5 | 43-48 | 3-5 | Unknown | Anterior | Unknown | Unknown | 8-17 | Unknown | - |
| H. magnistylus | 1.4-2.0 | 4 | 3 | 52-61 | 4-6 | 22-34 | Posterior | Absent | One anterior & one posterior | 12-17 | Present | 52-58 |
| H. pararobustus | 1.2-1.6 | Breaks | 3 | 41-44 | 4-5 | - | Anterior | Present | One anterior & one posterior | 9-15 | Present | 40-44 |
| H. pararobustus | 1.0-1.6 | 2-3 | 3 | 38-49 | 4-5 | 18-25 | Anterior | Present | One anterior & one posterior | 7-15 | Present | 40-57 |
| H. puertoricensis | 1.3-1.7 | 0 | 5 | 41-45 | 3 | 6-9 | Anterior | Absent | Both anterior | 19 | Unknown | - |
| H. puriensis | 1.1-1.3 | 4 | 4 | 33-37 | 4-5 | Unknown | Anterior | Absent | One anterior & one posterior | 12-13 | Present | 31 |
| H. smokyensis | 0.9-1.8 | 4 | 3 | 41-51 | 5-6 | 24 | Posterior | - | One anterior & one posterior | - | Present | 37-47 |
| H. sacchari | 1.1-1.2 | 4 | 6 | 34-35 | 3 | 8 | Anterior | Present | One anterior & one posterior | 9-10 | Present | 39-40 |
| H. seinhorsti | 1.3-1.7 | 1 | 5 | 38-44 | 4-5 | Unknown | Anterior | Absent | One anterior & one posterior | 13-16 | Unknown | - |
| H. seinhorsti | 1.1-1.6 | 1 | 6 | 40-49 | 4 | 8-12 | Anterior | Absent | One anterior & one posterior | 10-15 | Unknown | - |
| H. seshadrii | 1.5-1.8 | 0 | 6 | 42-43 | 3 | 20-22 | Anterior | Present | One anterior & one posterior | 14-18 | Unknown | - |
| H. singhi | 1.4-2.1 | 0 | 3 | 43-56 | 4 | Unknown | Anterior | Absent | One anterior & one posterior | 7 | Present | 52 |
| H. stephanus | 1.0-1.5 | 4 | 3 | 43-50 | 4 | 24-28 | Posterior | Present | One anterior & one posterior | 12 | Present | 30-38 |
| H. tabacum | 1.3-1.4 | 1 | 6 | 43-45 | 3-4 | Unknown | Anterior | Present | Both posterior | 12-15 | Unknown | - |
| H. tylenchiformis | 0.9-1.4 | 4 | 3 | 42-51 | 3-4 | 20-24 | Posterior | Present | One anterior & one posterior | 8-14 | Present | 31-38 |
Figure 1:
Light microscopy images of females of H. seinhorsti (A) whole bodies; (B-G) anterior body in lateral view showing SE pore opening and hemizonid (indicated by arrows), stylet and stylet knobs. DGO and median bulb; (H-K) esophageal region showing five gland nuclei (pointed by arrows); (L,M) scutella, lateral view; (N,O) vulva region in lateral view; (P) lateral incisures around mid-body; (Q-T) tail region showing anal opening, tail annuli number and lateral incisure.
Figure 2:
Scanning electron microscopy images of females of H. seinhorsti (A) whole body; (B-D) head region; (E,F) vulva region in ventral and lateral views; (G) scutellum anterior to vulva in lateral view; (H) scutellum posterior to vulva along with lateral incisures around the posterior region; (I-K) tails in lateral view showing anal openings.
Table 1.
Morphometric data of Hoplolaimus seinhorsti and Hoplolaimus pararobustus. All measurements are in μm (except for ratio) and in the form: mean ± sd (range). Species included in this study are presented in the first two columns of the table.
| Character | Hoplolaimus seinhorsti (Indonesian specimen) | Hoplolaimus pararobustus (Nigerian specimens) | Hoplolaimus pararobustus (Namibian specimens according to Marais et al., 2020) | Hoplolaimus pararobustus (Syntypes; according to Sher, 1963) | |||
|---|---|---|---|---|---|---|---|
| 15♀♀ | 16♀♀ | 10♂♂ | 21♀♀ | 21♂♂ | 3♀♀ | 5♂♂ | |
| L | 1521±143 (1280-1700) | 1322±107 (1161-1552) | 1233±50.7 (1171-1325) | 1100±76.1 (957-1245) | 925±65 (818-1018) | 1420-1520 | 1090-1250 |
| a | 26.2±1.3 (22.7-28.3) | 29.8±2.9 (24.7-36) | 33.8±1.6 (31.1-36.4) | 29±3.5 (23.7-36) | 29.2±4.2 (22-38.3) | 23-25 | 21-26 |
| b | 10.4±0.8 (7.0-11.4) | 9.5±0.9 (8-11.4) | 10.1±0.5 (9.2-10.7) | 7.4±0.7 (6.2-8.6) | 6.7±0.4 (6-7.5) | 8.8 | 7-7.4 |
| c | 53.7±5.7 (38.1-62.3) | 62.2±11.3 (42.6-88.2) | 39±1.6 (37-41.6) | 59.7±10.5 (46.9-80.6) | 34.7±4.3 (25-41) | 42-47 | 28-42 |
| c’ | 0.8±0.7 (0.7-0.9) | 0.7±0.1 (0.5-0.9) | 1.49±0.04 (1.4-1.5) | 0.8±0.1 (0.6-1.1) | 1.5±0.2 (1.3-2) | - | - |
| DGO | 5.3±0.5 (4.6-6.4) | 6.6±2.1 (3.7-9.6) | 6.2±1.2 (4.5-8.1) | 5±0.7 (3-6) | 4±0.9 (3-6) | - | - |
| V | 55.2±2.6 (50.2-59.3) | 59.4±3.5 (54-70.1) | - | 55±2.5 (49-67) | - | 57-62 | - |
| Stylet length | 42.1±1.7 (38.3-44.1) | 42.5±0.8 (41.3-43.8) | 42.4±1.1 (41.2-44.3) | 36±1.6 (34-40) | 34±1.3 (32-38) | 46-49 | 44-46 |
| Stylet cone length | 20.8±1.2 (18.2-23.2) | 21.7±1.1 (20.2-23.7) | 21.3±1.6 (19-23.3) | - | - | - | - |
| Stylet knob length | 7.7±0.4 (6.0-7.9) | 7.8±1.1 (5.7-9.1) | 5.3±0.7 (4.5-6.3) | 6±0.5 (5-7) | 5±0.6 (4-6) | - | - |
| Excretory pore from anterior | 151 ±7.91 (140-167) | 110±11.8 (85.4-128) | 89.2±7.1 (78.3-98.8) | 108±11.9 (82-123) | 93±13.6 (74-121) | - | - |
| Nerve ring from anterior | 118±4.9 (111-126) | 81.1±7.9 (70.8-94.8) | 79.8±7.1 (71.6-94.8) | - | - | - | - |
| Pharynx | - | 140±10.7 (114-156) | I23±3.3 (120-129) | - | - | - | - |
| Pharyngeal gland end from anterior | 200±15.7 (162-216) | 170±16.3 (143-201) | 139±8.1 (131-158) | - | - | - | - |
| Anterior phasmid of body length (%) | 36.1±2.2 (33-38.9) | - | - | - | - | 27-30 | 25-35 |
| Posterior phasmid of body length (%) | 81.1±0.9 (80-82) | - | - | - | - | 74-79 | 77-84 |
| Vulva from anterior end | 816±72.4 (715-912) | 784±61.3 (664-908) | - | - | - | - | - |
| Diameter at mid-body | 58.3±6.1 (44.3-66.6) | 44.5±2.6 (39.3-49.9) | 36.5±0.8 (35.3-37.6) | 39±4 (32-46) | 32±2.9 (27-38) | - | - |
| Anal body diameter | 33.8±2.9 (30.1-38.5) | 320±2.1 (28.2-37.1) | 21.3±0.7 (20.2-22.4) | 25±2.2 (21-29) | 17±1.3 (15-20) | - | - |
| Lip region height | 9.4±0.7 (8.1-10.4) | 7.8±1.2 (6.0-9.7) | 8.2±0.5 (7.5-9) | 7±0.5 (6-8) | 6±0.6 (5-7) | - | - |
| Lip region diameter | I6.3±0.9 (15.0-18.0) | 15.4±1.1 (13.4-17.6) | 11.9±1.0 (10-12.9) | 13±1 (12-15) | 12±0.9 (11-13) | - | - |
| Lip annulus | - | 4.2±0.4 (4-5) | 4.1±0.3 (4-5) | - | - | - | - |
| Tail annuli | 13-16 | - | - | - | - | - | - |
| Tail length | 28.4±3.1 (23.7-34.9) | 21.9±4 (15.7-30.4) | 31.6±1.0 (30-32.7) | 19±3.1 (15-24) | 27±3.5 (22-35) | - | - |
| Spicule length | - | - | 41.4±1.3 (44.7-48.5) | - | 38±2.3 (35-42) | - | 52-57 |
| Gubernaculum | - | - | 21.8±0.7 (19.2-21.8) | - | 17±2.3 (11-20) | - | 22-26 |
Figure 3:
Light microscopy images of females and males of H. pararobustus (A) whole body of female and male; (B-D) anterior body in lateral view showing SE-pore opening, stylet and stylet knobs. DGO and median bulb; (E) vulva region with no epiptygma in lateral view; (F) oocytes and ovary with sperm; (G) lateral incisures around mid-body; (H) scutella, lateral view; (I-M) female and male tail region showing anal opening, tail annuli number and lateral incisure.
Figure 4:
BI phylogenetic tree inferred from analysis of the D2-D3 expansion segment of 28S rDNA sequences from nine known and three unknown Hoplolaimus species using the GTR + I + G model. Bayesian posterior probabilities are given next to each node and H. seinhorsti and H. pararobustus is presented in bold. Intraspecific variation of a clade indicated by a bar is given to the right of the gray bars, nucleotide differences between sister clades is provided left to the gray bars. Red and blue bars represent species boundaries estimated by GMYC and bPTP methods, respectively. A star indicates data with too many non-nucleotide characters in the sequences.
Table 3.
Number of species according to the two species-delimitation methods (GMYC vs bPTP).
| Gene region | Number of species | ||
|---|---|---|---|
| Morphospecies (unidentified species included) | GMYC | bPTP | |
| D2-D3 of 28S rRNA | 10 | 27 | 9 |
| COI mtDNA | 8 | 13 | 13 |
| ITS rRNA | 7 | 16 | 7 |
| 18S rRNA | 4 | 6 | 5 |
Table 4.
List of unlabeled, mislabeled Hoplolaimus sequences on GenBank reassigned to corrected species in this study.
| Accession number | |||||||
|---|---|---|---|---|---|---|---|
| No | Species name on GenBank | 28S | COI | ITS | 18S | Remarks | Reassigned Species name/ Decision |
| 1 | Hoplolaimus sp. | KY639326 | KY639374 | Kolombia et al. (2017) provided these sequences as an outgroup, and thus lacking morphological data. The sequence form a maximally supported clade with H. pararobustus based on 28S D2-D3 rRNA, the bPTP output also confirmed that this sequence belongs to H. pararobustus. | H. pararobustus | ||
| 2 | Hoplolaimus sp. | EU586798 EU586797 EU626794 EU626795 | Bae et al. (2008) provided these sequences. These sequences forms a maximally supported clade with H. stephanus, the bPTP output also confirms that these sequences belong to a single species. | H. stephanus | |||
| 3 | Hoplolaimus sp. | OM218727 OM218726 OM218725 | Phylogenetic results and both species delimitation results (bPTP and GMYC) indicate that these sequences and H. columbus (KJ934150, KJ934149) sequences belong to a single species. | H. columbus | |||
| 4 | Hoplolaimus sp. | KP230658 KP230659 | KP303683 KP303684 | The sequences were originally provided by Holguin et al. (2015), and subsequently associated to the original description of H. smokyensis by Ma et al. (2019). | H. smokyensis | ||
| 5 | Hoplolaimus galeatus | KY849910 | This sequence is available on GenBank without morphological data. It forms a maximally supported clade with H. magnistylus (MF767410, Donald et al., 2013; EU626789, EU626790, Bae et al., 2008). Assuming that the sequences of H. galeatus EU626784, EU626786, HQ678710, HQ678711 are genuine. H. galeatus (KY849910) must be incorrectly assigned. | H. magnistylus | |||
| 6 | Hoplolaimus concaudajuvencus | KP303685 KP303686 | Holguin et al. (2015) provided these two sequences as unknown Hoplolaimus species in their paper although they were deposited on GenBank as H. concaudajuvencus. However, The sequences form a maximally supported clade with H. magnistylus (KP303623, KP303634, KP303681, KP303682, EU515325, EU515326, Bae et al., 2008; Holguin et al., 2015), also confirmed by bPTP and GMYC species delimitation. | H. magnistylus | |||
| 7 | Hoplolaimus stephanus | KP303639 | Holguin et al. (2015) provided this sequence as H. columbus in their paper, but deposited the sequence on GenBank as H. stephanus. This sequence forms a maximally supported clade with other H. columbus sequences. | H. columbus | |||
Figure 5:
BI phylogenetic tree inferred from analysis of COI mtDNA sequences from seven known and one unknown Hoplolaimus species using GTR + I + G model. Bayesian posterior probabilities are given next to each node, H. seinhorsti and H. pararobustus are given in bold. Intraspecific variation of a clade indicated by a bar is given to the right of the gray bars, nucleotide differences between sister clades is provided left to the gray bars. Red and blue bars represent species boundaries estimated by GMYC and bPTP methods, respectively.
Figure 6:
BI phylogenetic tree inferred from analysis of ITS rDNA sequences from seven known and four unknown Hoplolaimus species using the GTR + I + G model. Bayesian posterior probabilities are given next to each node and H. seinhorsti is provided in bold. Intraspecific variation of a clade indicated by a bar is given to the right of the gray bars, nucleotide differences between sister clades is provided left to the gray bars. Red and blue bars represent species boundaries estimated by GMYC and bPTP methods, respectively.
Figure 7:
BI phylogenetic tree inferred from analysis of 18S rDNA sequences from three known and two unknown Hoplolaimus species using GTR + I + G model. Bayesian posterior probabilities are given next to each node and H. pararobustus is given in bold. Intraspecific variation of a clade indicated by a bar is given to the right of the gray bars, nucleotide differences between sister clades is provided left to the gray bars. Red and blue bars represent species boundaries estimated by GMYC and bPTP methods, respectively.