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Characterization of Hoplolaimus seinhorsti and Hoplolaimus pararobustus (Tylenchina: Hoplolaimidae) from banana, with phylogeny and species delineation in the genus Hoplolaimus

Emmanuel Olajide
Emmanuel Olajide
, 
Phougeishangbam Rolish Singh
Phougeishangbam Rolish Singh
, 
Yao A. Kolombia
Yao A. Kolombia
, 
Merlin Kornelia Rumbarar
Merlin Kornelia Rumbarar
, 
Marjolein Couvreur
Marjolein Couvreur
 oraz 
Wim Bert
Wim Bert
   | 23 maj 2023
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Article Category: Research Paper
Data publikacji: 23 maj 2023
Zakres stron: -
Otrzymano: 19 lis 2022
DOI: https://doi.org/10.2478/jofnem-2023-0019
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© 2023 Emmanuel Olajide et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

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.
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.
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.

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.
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.
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.

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.
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.
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.
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.

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

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

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

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. EU586798EU586797EU626794EU626795 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. OM218727OM218726OM218725 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. KP230658KP230659 KP303683KP303684 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 KP303685KP303686 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
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