This work is licensed under the Creative Commons Attribution 4.0 International License.
The type species Rotylenchus robustus (De man, 1876) was originally described as Tylenchus robustus by De man (1876). In a publication concerning free-living nematodes and their relation to the parasitic nematodes, Filipjev (1934) proposed the new genus Rotylenchus based on the type species T. robustus (De man, 1876).
Herein, the new species Rotylenchus rhomboides n. sp. is characterized and a population of R. buxophilus is reported for the first time on yam (Dioscorea tokoro Makino). Both species were described based on morphology and morphometrics along with molecular characteristics and phylogeny of the D2-D3 expansion segment of 28S rDNA, ITS rDNA, and COI mtDNA sequences.
Materials and methods
Sampling and nematode extraction
The soil and root samples were collected around the rhizosphere of banana (Musa basjoo Siebold & Zucc. ex Iinuma) (GPS coordinates N: 51°2′6.8″, E: 3°43′22.7″) and Yam (Dioscorea tokoro) (GPS coordinates: N: 51°2′6.9″, E: 3°43′22.6″) at the Botanical garden of Ghent University. The nematodes were extracted from soil and roots by the modified Baermann tray method (Whitehead and Hemming, 1965).
Morphological characterization
Nematodes were fixed in Trump’s fixative (2% paraformaldehyde + 2.5% glutaraldehyde in a 0.1 M Sorenson buffer (Sodium phosphate buffer at pH 7.3)), then dehydrated for mounting in glycerin on permanent slides following the method described by Singh et al. (2018).
Microphotographs and drawings were made from permanent slides by using an Olympus BX51 DIC Microscope equipped with a drawing tube and digital camera. Measurements were obtained based on light microscopic pictures using the software ImageJ 1.51. Illustrations were made by Illustrator ® CS 3 based on pencil drawings and SEM pictures. For scanning electron microscopy, specimens were processed and viewed following the procedure of Steel et al. (2011).
Molecular characterization
Digital light microscope pictures were taken from temporary slides as morphological vouchers. Then, the nematodes were cut into pieces and put in the Eppendorf tubes with 20 µl of WLB (50 mM KCl; 10 mM Tris pH 8.3; 2.5 mM MgCl2; 0.45% NP 40 (Tergitol Sigma); 0.45% Tween 20) and were frozen for at least 10 min at −20°C. One μl proteinase K (1.2 mg ml−1) was added before the incubation in a PCR machine for 1 hr at 65°C and 10 min at 95°C and centrifugation for 1 min at 14,000 rpm. Finally, the samples were stored at −20°C before running PCR (Singh et al., 2018).
The 5′-end of the 28S rDNA region was amplified using the primers DP391/501 (Nadler et al., 2006) with the PCR reaction started at 94°C for 4 min, followed by 5 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 2 min. This step was followed by 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min and finished at 12°C for 10 min. For ITS rDNA region, the primers Vrain2F/Vrain2R (Vrain et al., 1992) were used with the PCR reaction started at 94°C for 4 min, followed by 50 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 2 min. The cytochrome c oxidase subunit 1 (COI mtDNA) gene was amplified using the primers JB3/JB4 according to the protocol of Derycke et al. (2010). The successful PCR reactions were purified and sequenced commercially by Macrogen Europe (Amsterdam, Nederland).
The forward and backward sequences were assembled in Geneious R11 (www.geneious.com) to get the consensus sequences. All the contigs were used for the BLAST search on GenBank to check for the closely related species (Altschul et al., 1997). Multiple alignments of the different sequences of each gene were made using MUSCLE in MEGA 7 (Kumar et al., 2016). The phylogenetic trees were created by using MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001) Add-in in Geneious R11 (www.geneious.com) under the nucleotide substitution models that were selected by using MEGA 7 (Sharma et al., 2012) based on the BIC criterion. The selected models were HKY + G for the D2-D3 of 28S rDNA and ITS rDNA sequences, and GTR + G + I for COI mtDNA sequences. The Markov chains were set with 1 × 106 generations, 4 runs, 20% burn-in, and subsampling frequency was 500 generations (Huelsenbeck and Ronquist, 2001). For D2-D3 of 28S rDNA data set, Helicotylenchus dihystera (accession number: AB933471) and Helicotylenchus multicinctus (accession number: MF401446) were chosen as outgroups. The outgroups for ITS rDNA data set were Hoplolaimus columbus (accession number: FJ485623) and Hoplolaimus seinhorsti (accession number: KX446971), and Scutellonema brachyurus (accession number: JX472089) and Scutellonema truncatum (accession number: KX959308) were selected for COI mtDNA data set.
Cluster analysis and web-based key
The new species was compared with 103 described species, based on the tabular key of Castillo and Vovlas (2005). This was done using Hierarchical Cluster analysis implemented in the software Primer 6 (Clarke and Gorley, 2006) using Bray–Curtis similarity measure with the percent similarity between species defined by the average of the multiple characters. The 11 characters of 103 described species and Rotylenchus rhomboides n. sp. in the analysis are: (A) Lip annulation: 1: absent or smooth labial region, 2: labial region with 2 to 3 annuli, 3: labial region with four annuli, 4: labial region with five annuli, 5: labial region with six annuli, 6: labial region with 7 to 8 annuli, 7: labial region with 9 to 10 annuli; (B) Labial region shape: 1: labial region hemispherical, 2: labial region rounded, 3: labial region conoid, 4: labial region truncate; (C) Lateral field areolation: 1: areolated only in esophageal region, 2: areolated in esophageal region and irregularly areolated at mid-body, 3: areolated in esophageal region and incompletely areolated at mid-body, 4: areolated in esophageal region and near phasmids, 5: areolated along whole length of body except at tail region, 6: areolated along whole length of body including the tail region, 7: incompletely areolated along whole body; (D) Body longitudinal striations: 1: punctated along body annuli, 2: longitudinally striated in esophageal region, 3: longitudinally striated over whole body, 4: without body striations; (E) Stylet length: 1: stylet shorter than 30 µm, 2: stylet between 30 and 35.9 µm, 3: stylet between 36 and 40.9 µm, 4: stylet longer than 41 µm; (F) Dorsal esophageal gland outlet (DGO) to stylet base: 1: DGO less than 2 µm, 2: DGO between 2 and 6.9 µm, 3: DGO between 7 and 12 µm, 4: DGO > 12 µm; (G) Dorsal esophageal gland overlapping: 1: esophageal gland overlapping less than 5 µm, 2: esophageal gland overlapping by 6 to 20.9 µm, 3: esophageal gland overlapping by 21 to 30.9 µm, 4: esophageal gland overlapping by 31 to 40.9 µm, 5: esophageal gland overlapping by >41 µm; (H) Tail shape: 1: hemispherical, 2: rounded, 3: conoid, 4: pointed, 5: with ventral projection; (I) Ratio V: 1: ratio V < 50%, 2: ratio V of 50 to 70%, 3: ratio V > 70%; (J) Presence of males: 1: males present, 2: males absent; (K) Phasmid position: 1: well anterior to level of anus (>five annuli anterior to anus), 2: at level of anus (from five annuli anterior to five annuli posterior to anus), 3: well posterior to level of anus (>five annuli posterior to anus).
In order to facilitate the identification of Rotylenchus spp., based on a cluster analyses a web-based key was developed. The domain for this web-based key was registered from the website: www.awardspace.com. The interface of this web-based key was written using Notepad++ v7.5.6 and the algorithm was based on Bray–Curtis similarity measure. The web-based key can be freely accessed at http://nematodeidentification.mypressonline.com/category/identification-tool/.
Rotylenchus rhomboids n. sp., (drawings ♀). (A) entire body; (B) en face view; (C) head region; (D) esophageal region; (E) lateral field at vulva region; (F) vulva region; (G) tail region; (H) lateral field at tail region. (B, C based on SEM illustrations) (A, E, F, G, H: Holotype) (Scale bar: A: 25 µm; B: 5 µm; C: 20 µm; D–H: 25 µm).
Figure 2
Rotylenchus rhomboides n. sp. (LM ♀). (A) esophageal region; (D) vulva region; (E) lateral field at vulva region with a rhomboid widening (see arrow); (G) entire body; (H) tail region; (I) lateral field with phasmid at tail region (SEM ♀); (B) anterior body region, lateral view; (C) en face view (arrow indicates amphid aperture); (F) vulva region, lateral view; (J) vulva region, ventral view; (K) tail region of female, lateral view; (L) tail region of female, oblique ventral view; (M) tail region of juveniles (D, E, G, H, I: Holotype; others paratypes) (Scale bar: B, C: 1 µm; A, D–M: 25 µm).
Figure 3
BI phylogenetic tree generated from the D2-D3 of 28S rDNA region data set with the HKY + G model (94 parameters). Bayesian posterior probabilities are given next to each node. Sequences of Rotylenchus rhomboides n. sp. and R. buxophilus (Belgium) are in bold.
Figure 4
BI phylogenetic tree generated from ITS sequences with HKY + G model (70 parameters). Bayesian posterior probabilities are given next to each node. Sequences of Rotylenchus rhomboides n. sp. and R. buxophilus (Belgium) are in bold.
Figure 5
BI phylogenetic tree generated from COI sequences with the GTR +G + I model (77 parameters). Bayesian posterior probabilities are given next to each node. Sequences of Rotylenchus rhomboides n. sp. and R. buxophilus (Belgium) are in bold.
Figure 6
The cluster analysis of 104 species (included Rotylenchus n. sp.) based on Bray–Curtis similarity measure of 11 ranked features. Rotylenchus rhomboides n. sp. is in bold. The dotted line indicates 85% similarity.
Figure 7
Belgian population of Rotylenchus buxophilus (LM ♀). (A) entire body; (B) esophageal region; (C) lateral field at vulva region; (D) vulva region; (E) lateral field with phasmid at tail region; (F) tail region (Scale bar: A–F: 25 µm).
Morphometric data of Rotylenchus rhomboides n. sp. All measurements are in μm (except for ratio) and in the form: mean ± sd (range).
Character
Holotype female
Paratype females
n
1
12
Body length (L)
827
861 ± 101 (634–1049)
a = L/MBD
28.5
31.8 ± 2.8 (27.8–35.1)
b’ = L/Anterior end to the end of esophageal gland
7.6
6.9 ± 0.5 (5.6–7.6)
c = L/Tail length
55
63.3 ± 10.7 (50.3–85.5)
c’ = Tail length/ABD
68
0.7 ± 0.1 (0.5–0.8)
V = Anterior end to vulva/L × 100
59
56 ± 2.6 (53–61)
Lip height
6
6 ± 0.7 (5–7)
Lip width
11
10 ± 0.8 (8–11)
Stylet length
35
33 ± 1.5 (31–37)
Conus length
17
17 ± 0.8 (16–18)
Shaft length
14
13 ± 0.9 (12–14)
Knob height
3
3 ± 0.4 (3–4)
Dorsal gland opening from stylet base
4
3 ± 0.9 (2–5)
Anterior end to secretory-excretory pore
100
107 ± 10.6 (77–123)
Anterior end to nerve ring
78
86 ± 9.9 (60–96)
Anterior end to esophageal gland end
108
125 ± 10.5 (108–146)
Esophagus overlapping
12
14 ± 2.3 (9–19)
Max body diameter (MBD)
29
27 ± 2.3 (23–31)
Anal body diameter (ABD)
22
20 ± 1.8 (16–23)
Tail length
15
14 ± 2.0 (9–16)
Tail annuli number
11
10 ± 1.0 (8–11)
Comparison of the matrix code of Rotylenchus rhomboides n. sp. with other closely related species.
Species
A
B
C
D
E
F
G
H
I
J
K
Rotylenchus rhomboides n. sp.
3–4
2
1
2
2
2
2
1–2
2
2
2
R. buxophilus from Belgium
4
1
1
4
2
2
3
3
2
2
1
R. buxophilus
4
1
1
4
2
2
3
3
2
2
1
R. corsicus
4
1
1
2
2
2
2
3
2
2
1
R. goodeyi
3
1
1
4
2
2
4
2
2
1
2
R. gracilidens
3
1
1
2
2
2
3
1
2
1
1
R. incultus
4
1
1
4
1
2
3
1
2
1
1
R. laurentinus
4
1
1
3
2
2
3
1
2
1
2
R. pumilus
4
1
1
4
1
2
2
1
2
1
2
R. rugatocuticulatus
3
2
1
2
3
2
4
1
2
1
1
Rotylenchus species and populations used and sequenced in the present study.
Accession number
Species
28S
ITS
COI
Host
Locality
H. dihystera
AB933471
Sugarcane
Japan
H. multicinctus
MF401446
Banana
Greece
H. columbus
FJ485623
Unknown
USA
H. seinhorsti
KX446971
Unknown
China
R. arasbaranensis
KJ765335
KJ765345
Grasses
Iran
KJ765336
KJ765346
Grasses
Iran
R. brevicaudatus
JX015419
Grasses
Australia
JX015420
Grasses
Australia
JX015431
Grasses
Australia
R. buxophilus
JX015421
–
USA
KJ765337
Euonymus sp.
Iran
KJ765338
Euonymus sp.
Iran
KJ765340
Euonymus sp.
Iran
KU722390
–
Iran
JX015432
Unknown
USA
JX015433
Unknown
USA
KJ765347
Euonymus sp.
Iran
R. buxophilus
MK114140
MK114142
MK114155
Dioscorea tokoro
Belgium
MK114141
MK114143
MK114156
Dioscorea tokoro
Belgium
R. castilloi
KM251415
Fagus orientalis Lipsky (oriental beech tree)
Iran
R. cazorlaensis
JX015399
Quercus faginea (Portuguese oak)
Spain
JX015400
Quercus faginea (Portuguese oak)
Spain
R. conicaudatus
HQ700698
Grasses
Iran
R. cretensis
KT288060
Olive
Greece
R. cypriensis
KP843116
Prunus sp.
Iran
KT288062
Olive
Greece
R. dalikhaniensis
KM042108
Ruscus hyrcanus Woronow
Iran
R. eximius
KX398058
Tamarix smyrnensis
Greece
KX669231
KX669244
Olive
Tunisia
KX669232
Olive
Tunisia
KX669233
KX669245
Olive
Tunisia
JX015401
Pistacia lentiscus (lentisc)
Italy
JX015402
Pistacia lentiscus (lentisc)
Italy
R. goodeyi
DQ328756
Olea europaea subsp. silvestris (wild olive)
Spain
R. incultus
KX669234
Olive
Tunisia
KX669235
Olive
Tunisia
KX669236
Olive
Tunisia
JX015403
Vitis vinifera (grapevine)
Spain
KX669246
Olive
Tunisia
KX669247
Olive
Tunisia
KX669248
Olive
Tunisia
KX669249
Olive
Tunisia
R. iranicus
HQ700697
Unidentified forest tree
Iran
R. jaeni
JX015404
Quercus suber (cork tree)
Spain
R. laurentinus
DQ328757
JX015405
Pistacia lentiscus (lentisc)
Italy
JX015406
Pistacia lentiscus (lentisc)
Italy
JX015407
Pistacia lentiscus (lentisc)
Italy
EU280798
Pistacia lentiscus (lentisc)
Spain
R. magnus
JX015409
Ilex aquifolium (holly)
Spain
JX015410
Ilex aquifolium (holly)
Spain
R. paravitis
JX015422
JX015415
Helianthus annuus (sunflower)
Spain
JX015416
Helianthus annuus (sunflower)
Spain
R. pumilus
JX015423
JX015435
Urtica sp.
USA
JX015436
Urtica sp.
USA
R. rhomboidesn. sp.
MK114123
MK114127
MK114144
Musa basjoo
Belgium
MK114124
MK114128
MK114145
Musa basjoo
Belgium
MK114125
MK114129
MK114146
Musa basjoo
Belgium
MK114126
MK114130
Musa basjoo
Belgium
MK114131
Musa basjoo
Belgium
MK296738
Musa basjoo
Belgium
R. robustus
JX015424
JX015413
Pinus pinea (stone pine)
Spain
JX015425
JX015438
Grasses
USA
JX015426
JX015439
JX015411
Grasses
USA
JX015440
Grasses
USA
JX015414
Pinus pinea (stone pine)
Spain
R. sardashtensis
KP843115
KP843113
Unidentified grasses
Iran
R. striaticeps
KJ765343
Unidentified forest tree
Iran
KJ765344
Unidentified forest tree
Iran
KU722389
Unknown
Iran
KP718973
Unknown
Iran
R. unisexus
EU373674
Citrus aurantium (citrus)
Spain
EU373675
Citrus aurantium (citrus)
Spain
R. urmiaensis
KP718966
KP718971
Corylus spp.
Iran
KP718967
KP718972
Corylus spp.
Iran
R. vitis
JN032581
JX015417
Vitis vinifera (grapevine)
Spain
JX015418
Vitis vinifera (grapevine)
Spain
S. brachyurus
JX472089
Sansevieria laurentii
USA
S. truncatum
KX959308
Unknown
USA
Note: Newly added sequences are indicated by bold font.
Description
Females
Body relatively small, habitus spiral or C-shaped (Figs 1A, 2G). Cuticle clearly annulated with irregular longitudinal striations in anterior region; annuli 1.6 to 2 μm wide at mid-body. Lateral fields with four lines at mid-body, beginning anteriorly at 7 to 8th annulus as two lines forming one band, third line appearing at level 10 to 11th annulus; mid-ridge at vulva level with a characteristic rhomboid widening. Regular areolation of lateral fields observed only in esophageal region (Figs 1C, E, 2B, E, F). Labial region rounded, offset from rest of body, bearing 4 to 5 annuli, divided longitudinally; labial disc rounded to hexagonal, marked from rest of labial region, but not elevated (Figs 1B, C, 2B, C). Stylet robust; basal knobs rounded, 3 to 4 μm high. Dorsal esophageal gland opening 2 to 5 μm posterior to stylet base. Procorpus cylindrical, with slight depression just anterior to median bulb; median bulb well developed, rounded, or broadly oval; isthmus slender, encircled by nerve ring; esophageal glands sacciform, overlapping intestine dorsally. Secretory-excretory pore usually located just posterior to the hemizonid. Hemizonid distinct ca 1.5 to 2 body annuli long, located around esophago-intestinal junction level (Figs 1D, 2A). Reproductive system didelphic-amphidelphic, genital branches equally developed; vulva slightly posterior to mid-body, without distinct epiptygma; outstretched ovaries with a single row of oocytes; spermatheca mostly oval, without sperm (Figs 1A, F, 2D, F, G). Tail short, varying in shape from hemispherical to rounded, with 8 to 11 annuli ventrally; terminus striated. Phasmid opening relatively large 1.6 (1.2–2) μm, pore-like, located 3 to 5 annuli anterior to level of anus (Figs 1G, H, 2H, I, K, L).
Male
Not found.
Molecular characterization
28S rDNA
Four sequences of 5′-end region of 28S rDNA were obtained, 916 to 1057 bp long, with an intraspecific variation of 1 to 4 positions (0.1–0.5%). The muscle alignment comprised 46 sequences, 1,080 positions. The sequences of D2-D3 of 28S rDNA Rotylenchus rhomboides n. sp. differ by 24 to 74 positions (90–96% similar) compared to the sequences of all other Rotylenchus species in this study. These sequences were most similar to the sequences of R. buxophilus with 24 to 28 different positions (95–96% similar). The Bayesian interference phylogenetic tree based on the D2-D3 of 28S rDNA sequences showed that the sequences of Rotylenchus rhomboides n. sp. have a maximally supported sister relation with R. buxophilus, R. pumilus (Perry et al., 1959; Sher, 1961), R. incultus (Sher, 1965), R. goodeyi, and R. laurentinus (Scognamiglio and Talamé, 1972) (Fig. 3).
ITS rDNA
Six ITS rDNA sequences were obtained, 1,026 to 1,339 bp long, with differences on 0 to 4 nucleotides (99–100% similar). The muscle alignment included 34 nucleotide sequences and 1,444 positions. The sequences of Rotylenchus rhomboides n. sp. differed by 158 to 357 positions (60–83% similar) compared to all other Rotylenchus species in this study. The sequences of Rotylenchus rhomboides n. sp. are most similar to the sequence of R. buxophilus with 138 to 210 different positions (80–83% similar). The ITS tree topology showed small changes compared to D2-D3 tree topology. The sequences of Rotylenchus rhomboides n. sp. have a sister relationship to R. incultus and R. laurentinus (0.97 PP) (Fig. 4).
COI mtDNA
Three COI mtDNA sequences were obtained (442 bp long). The sequences of Rotylenchus rhomboides n. sp. varied 5 to 8 positions (2–3% different) compared to each other. The muscle alignment consisted of 35 nucleotide sequences, 445 positions long. The sequences of Rotylenchus rhomboides n. sp. differed by 47 to 101 positions (72–86% similar) compared to other Rotylenchus species in this study. The sequences of Rotylenchus rhomboides n. sp. are most similar to the sequence of R. buxophilus with 47 to 48 different positions (85–86% similar). The phylogenetic tree based on the COI mtDNA showed few changes compared to the D2-D3 of 28S and ITS rDNA trees. In that, the sequences of Rotylenchus rhomboides n. sp. also have a maximal sister relation to R. laurentinus, R. incultus, and R. buxophilus, but there were few changes in the position of each species (Fig. 5).
Diagnosis and relationships
Rotylenchus rhomboides n. sp. is characterized by a combination of the following traits: a rounded labial region with four annuli; an annulated body cuticle with irregular longitudinal striations in the anterior region; lateral field with four lines forming three ridges at mid-body, areolated only at esophageal region; a rhomboid widening at the mid-ridge at level of the vulva; a robust stylet of average length (31–37 μm); esophageal glands shortly overlapping the intestine dorsally; female reproductive system with oval spermatheca without sperm and a vulva located slightly posterior to mid-body; hemispherical or rounded tail tip with relatively large phasmids (1.2–2 µm) located 3 to 5 annuli anterior to the level of the anus; male not found. According to Castillo and Vovlas (2005), the matrix code for this new species is: A3–4, B2, C1, D2, E2, F2, G2, H1-2, I2, J2, and K2. Rotylenchus rhomboides n. sp. differs from all Rotylenchus species by the presence of the rhomboid widening of the mid-ridge of the lateral field at vulva level.
Rotylenchus rhomboides n. sp. is also different from all other species according to the dichotomous key of Castillo and Vovlas (2005) as well as the comparison with other species from the studies of Vovlas et al. (2008), Atighi et al. (2011), Cantalapiedra-Navarrete et al. (2012, 2013), Talezari et al. (2015), and Golhasan et al. (2016). By using the hierarchical cluster analysis of all the characters that were used in the tabular key of Castillo and Vovlas (2005), all 103 valid species and Rotylenchus rhomboides n. sp. were separated into groups that have the highest similarities. This cluster analyses can also be done by using web-based key at: http://nematodeidentification.mypressonline.com/category/identification-tool/. The new species was grouped together with the most similar species, namely R. corsicus, R. gracilidens (Sauer, 1958), and R. rugatocuticulatus (Sher, 1965) (Fig. 6). They shared more than 85% similarity to each other and less than 80% similarity to all other species. The shared characteristics of this group are the labial region having 4 to 5 annuli, body longitudinal striations being restricted to the esophageal region, DGO at 2 to 6.9 μm from the base of the stylet and ratio V between 50 and 70%.
However, Rotylenchus rhomboides n. sp. can be distinguished from R. corsicus by 3 out of the 11 main characters used in the tabular key of Castillo and Vovlas (2005), including B: labial region shape (rounded vs hemispherical); H: tail shape (rounded to hemispherical without mucron vs conoid to rounded with mucron in some specimens); K: phasmid position (located 3–5 annuli anterior to anus level vs 5–16 annuli anterior to anus level) (Table 2). Morphometrically, Rotylenchus rhomboides n. sp. differs from R. corsicus by having a smaller maximum body diameter (23–31 μm vs 48–57 μm) and smaller c’ value (0.5–0.8 vs 1.0–1.3).
Rotylenchus rhomboides n. sp. differs from R. gracilidens by 4 out of 11 compared characters in tabular key, including B: Labial region shape (rounded labial region vs hemispherical labial region); G: Dorsal esophageal gland overlapping (between 6 and 20.9 μm vs between 21 and 30.9 μm); J: Presence of males (absent vs present); K: phasmid position (phasmids 3–5 annuli anterior to the anus level vs 15–20 annuli anterior to the anus level).
Rotylenchus rhomboides n. sp. differs from R. rugatocuticulatus by 4 out of 11 compared characters in tabular key, including E: stylet length (between 30 and 35.9 µm vs between 36 and 40.9 µm); G: dorsal esophageal gland overlapping (between 6 and 20.9 μm vs between 31 and 40.9 μm); J: Presence of males (absent vs present); K: phasmid position (phasmids 3–5 annuli anterior to the anus level vs 8–10 annuli anterior to the anus level). Moreover, Rotylenchus rhomboides n. sp. has different vulva structure (without epiptygma vs double epiptygma), a smaller body length (0.6–1 mm vs 1–1.3 mm).
Rotylenchus rhomboides n. sp. is phylogenetically close to R. buxophilus, R. goodeyi, R. laurentinus, R. pumilus, and R. incultus. Rotylenchus rhomboides n. sp. can also be differentiated from the phylogenetically closely related species by the morphological features. Rotylenchus rhomboides n. sp. differs from R. buxophilus by 5 out of 11 compared characters in the tabular key including B: Labial region shape (rounded labial region vs hemispherical labial region), D: Body longitudinal striations (longitudinally striated in esophageal region vs without body striation), G: Dorsal esophageal gland overlapping (between 6 and 20.9 μm vs between 21 and 30.9 μm), H: tail shape (rounded to hemispherical vs dorsally convex-conoid), K: phasmid position (phasmids 3–5 annuli anterior to the anus level vs 6–13 annuli anterior to anus level). Rotylenchus rhomboides n. sp. also has a smaller body length (861 μm vs 1090 μm), and larger c value (63.3 vs 43).
Rotylenchus rhomboides n. sp. differs from R. goodeyi by 4 out of 11 compared characters in tabular key of Castillo and Vovlas (2005), including B: Labial region shape (rounded labial region vs hemispherical labial region); D: Body longitudinal striations (longitudinally striated in esophageal region vs without body striation); G: Dorsal esophageal gland overlapping (between 6 and 20.9 μm vs between 31 and 40.9 μm); J: Presence of males (absent vs present). Rotylenchus rhomboides n. sp. can also be differentiated from R. goodeyi by having different number of lip annuli (4–5 annuli vs 3–4 annuli), smaller body length (0.86 mm vs 0.97 mm), different phasmid position (phasmids 3–5 annuli anterior to the anus level vs 1–11 annuli anterior to anus level).
Rotylenchus rhomboides n. sp. differs from R. laurentinus by 4 out of 11 compared characters in tabular key of Castillo and Vovlas (2005), including B: Labial region shape (rounded labial region vs hemispherical labial region); D: Body longitudinal striations (longitudinally striated in esophageal region vs longitudinally striated over whole body); G: Dorsal esophageal gland overlapping (between 6 and 20.9 μm vs between 21 and 30.9 μm); J: Presence of males (absent vs present). Rotylenchus rhomboides n. sp. also differs from R. laurentinus by having a vulva without distinct epiptygma vs vulva with posterior epiptygma present overlap less conspicuous anterior one.
Rotylenchus rhomboides n. sp. differs from R. pumilus by 4 out of the 11 main compared characters in tabular key including B: labial region shape (rounded vs hemispherical); D: Body longitudinal striations (longitudinally striated in esophageal region vs without body striation), E: stylet length (31–37 μm vs 23–28 μm); J: Presence of males (absent vs present).
Rotylenchus rhomboides n. sp. differs from R. incultus by 6 out of the 11 main compared characters in the tabular key including B: labial region shape (rounded vs hemispherical); D: Body longitudinal striations (longitudinally striated in esophageal region vs without body striation); E: stylet length (31–37 μm vs 24–28 μm); J: Presence of males (absent vs present); K: phasmid position (located 3–5 annuli anterior to anus level vs 13–14 annuli anterior to anus level).
Etymology
Name of the new species regarded from rhomboideus (modern Latin), which refers to typical rhomboid enlargement in the lateral field at vulva level.
Type host and locality
Rotylenchus rhomboides n. sp. was recovered from soil and root samples from the rhizosphere of banana (Musa basjoo) in the botanical garden of Ghent University, Belgium (GPS coordinates N: 51°2′6.8″, E: 3°43′22.7″).
Type material
Holotype female and four paratype females, all in one slide, are deposited at the Ghent University Museum, Zoology Collections, collection number UGMD 104367. Four paratype females in one slide were deposited at National Plant Protection Organization, Wageningen Nematode Collection (WaNeCo). Additional paratypes (five females and three juveniles in two slides) are available in the UGent Nematode Collection (slide UGnem-189–190) of the Nematology Research Unit, Department of Biology, Ghent University, Ghent, Belgium. ZooBank ID: urn:lsid:zoobank.org:act:EF3FEED6-E804-4EFE-BCC2-12CBF072ADA3.
Morphometric data of Rotylenchus buxophilus from Belgium. All measurements are in μm (except for ratio) and in the form: mean ± sd (range).
Character
Females
n
15
Body length (L)
972 ± 87 (818–1065)
a = L/MBD
30.1 ± 1.9 (27.2–32.5)
b″ = L/Anterior end to the end of esophageal gland
7.0 ± 0.8 (5.3–8.2)
c = L/Tail length
40.1 ± 4.1 (34.6–49.8)
c″ = Tail length/ABD
1.2 ± 0.1 (1.0–1.4)
V = Anterior end to vulva/L × 100
53 ± 3 (48–58)
Lip height
7 ± 0.3 (6–8)
Lip width
11 ± 0.6 (10–12)
Stylet length
36 ± 1.6 (32–38)
Conus length
18 ± 1.1 (16–20)
Shaft length
14 ± 0.7 (13–14)
Knob height
4 ± 0.6 (3–5)
Dorsal gland opening from stylet base
4 ± 1.2 (3–7)
Anterior end to secretory-excretory pore
123 ± 10 (105–139)
Anterior end to nerve ring
99 ± 7 (87–109)
Anterior end to esophageal gland end
140 ± 11 (124–157)
Esophagus overlapping
22 ± 5 (18–28)
Max body diameter (MBD)
32 ± 2 (29–37)
Anal body diameter (ABD)
21 ± 1.6 (19–24)
Tail length
24 ± 3.1 (18–28)
Tail annuli number
14 ± 1.3 (12–16)
The measurements of known and new populations of Rotylenchus buxophilus.
Female
R. buxophilus (Belgian population)
R. buxophilus Golden (1956)
R. buxophilus Sher (1965)
R. buxophilus = R. sheri Jairajpuri (1963)
R. buxophilus Geraert and Barooti (1996)
R. buxophilus Wouts and Sturhan (1999)
Host, location
Yam (Dioscorea tokoro), Belgium
English boxwood (Buxus sempervirens var. suffruticosa L.), USA
camellia (Camellia japonica L.), France
Cedrus libani var. deodara, India
Magnolia sp. L., Iran
Cabbage tree (Cordyline sp. Comm, ex R.Br.), New Zealand
n
15
20
20
10
4
6
L
972 (818–1065)
1,090
920–1,310
900–1,200
850–1,070
1,020–1,250
V
53 (48–58)
55
52–58
52–56
54–58
55–58
STL
36 (32–38)
33.5
34–38
35–39
34
38–40
a
30.1 (27.2–32.5)
31
28–38
27–35
25–32
32–43
b
7.0 (5.3–8.2)
7
6.4–8.9
5.5–7.0
6–8
7.3–8.7
c
40.1 (34.6–49.8)
43
36–48
38–47
33–45
41–58
c′
1.2 (1.0–1.4)
–
–
–
1.1–1.4
0.8–1.2
Morphological characterizations of Rotylenchus buxophilus in Belgium
Females
Body relatively large, habitus mostly spiral (Fig. 7A). Cuticle clearly annulated. Lateral field with four lines at mid-body, areolated at esophageal region (Fig. 7B, C). Labial region hemispherical, bearing five annuli. Stylet robust; basal knobs rounded. Median bulb rounded to oval; isthmus slender, encircled by nerve ring; esophageal glands sacciform, overlapping intestine dorsally 18–28 μm. Secretory-excretory pore anterior to esophago-intestinal junction level; hemizonid distinct ca 1.5 to 2 body annuli long, just anterior to secretory-excretory pore (Fig. 7B). Reproductive system didelphic-amphidelphic, branches equally developed; outstretched ovaries with a single row of oocytes; vulva at mid-body level, with double distinct epiptygma; spermatheca inconspicuous, without sperms (Fig. 7A, D). Tail dorsally convex-conoid shape; pore-like phasmids located 9 to 16 annuli anterior to anus level (Fig. 7E, F).
Male
Not found.
Molecular characterization
All the molecular analyses of the Belgian population of Rotylenchus buxophilus were executed together with the sequences of Rotylenchus rhomboides n. sp. as above.
28S rDNA
Two sequences of 5′-end region of 28S rDNA were obtained (844 and 1,047 bp long). These sequences differ from other sequences of R. buxophilus (accession numbers: KJ765338, KJ765340, KJ765337, and KU722390) by 1 to 4 nucleotides (99–100% similar) and they differ from other Rotylenchus species by 9 to 71 nucleotides (85–97% similar). In the resulting phylogenetic tree, R. buxophilus from Belgium is in a well-supported clade (0.99 PP) together with all other sequences of R. buxophilus and R. pumilus (Fig. 3).
ITS rDNA
Two sequences of ITS rDNA sequences were obtained, 1,175 and 1,249 bp long. The ITS sequences of the Belgian population of R. buxophilus differ from other sequences of R. buxophilus (accession numbers: KJ765347, JX015435, JX015432, and JX015433) by 6 to 7 nucleotides (97–98% similar). The interspecific differences to other Rotylenchus sequences are 158 to 339 nucleotides (66–81% similar). Rotylenchus buxophilus from Belgium has a maximal relationship to other sequences of R. buxophilus and R. pumilus (Fig. 4).
COI mtDNA
Two sequences of COI mtDNA sequences were obtained, 444 and 445 bp long. The COI mtDNA sequences of the Belgian population of R. buxophilus are identical to the sequence of R. buxophilus on GenBank (accession number: JX015398) and these sequences differ from other Rotylenchus species in this study by 50 to 103 nucleotides (72–88% similar). Rotylenchus buxophilus from Belgium was placed in a maximal supported clade together with other sequences of R. buxophilus (Fig. 5).
Remarks
The morphological characteristics of the Belgian population of R. buxophilus resemble the original description of R. buxophilus. The few minor variations were observed, including the number of lip annuli (5 annuli vs 4–5 annuli), V value (48–58 vs 55), stylet length (32–38 μm vs 33.5 μm), a value (27.2–32.5 vs 31), and c value (34.6–49.8 vs 43). However, these differences are insignificant and the measurements of other population also show some variations (Table 5).
According to Castillo and Vovlas (2005), the matrix code for this population is: A4, B1, C1, D4, E2, F2, G3, H3, I2, J2, and K1. This matrix code is identical to that of the type population of R. buxophilus.
The BI phylogenetic trees on Figures 3–5 showed a very close relationship of the sequences from our population compared to the sequences of R. buxophilus and R. pumilus from GenBank. However, our population can be differentiated from R. pumilus by 3 out of 11 compared characters in tabular key of Castillo and Vovlas (2005), including E: stylet length (32–38 μm vs 23–28 μm); H: tail shape (conoid vs hemispherical); J: presence of males (absent vs present); K: phasmid position (located 9–16 annuli anterior to anus level vs located immediately posterior to latitude of anal opening). Furthermore, the females of our population have a larger body length (818–1065 µm vs 600–700 µm), smaller V value (48–58 vs 58–64), and larger a value (27.2–32.5 vs 20.0–23.7).
Host and locality
The Belgian population of Rotylenchus buxophilus was recovered from soil and root samples from the rhizosphere of Yam (Dioscorea tokoro) in the botanical garden of Ghent University, Belgium (GPS coordinates: N: 51°2′6.9″, E: 3°43′22.6″).
Voucher specimens
All the permanent slides (19 females in 3 slides) are available in the UGent Nematode Collection (slide number: UGnem-191–193) of the Nematology Research Unit, Department of Biology, Ghent University, Ghent, Belgium.
Discussion
The identification keys for Rotylenchus spp. used to be dichotomous (Sher, 1965; Geraert and Barooti, 1996). However, according to Geraert and Barooti (1996), the overlapping of various character states makes species differentiation difficult, something that represents an especially great challenge with genera with a large number of species (104 valid species at present for the genus Rotylenchus). Based on the principle of polytomous keys, Castillo and Vovlas (2005) developed a tabular or matrix key for the genus Rotylenchus, which facilitates the identification process and includes both major and supplementary characters for the identification of Rotylenchus species, currently in use. In this study, the utilization of hierarchical cluster analysis using the software Primer 6 with the data set based on the tabular key principle was successful in separating the 104 species including Rotylenchus rhomboides n. sp. into small groups that shared the highest number of similarities. This application can speed up the identification process, avoid mistakes made in manual comparisons (especially for a large data set), and avoid biases in the selection of closely related species to compare with. Furthermore, our web-based key facilitates the cluster analysis and is freely available at: http://nematodeidentification.mypressonline.com/category/identification-tool/. A similar web-based key was also created for Pratylenchus spp. (Nguyen et al., unpublished).
In this study, morphological and molecular characterizations were straightforward; however, molecular data are not available for every plant-parasitic nematode species on GenBank. In the literature, several examples of cryptic species are known, including Rotylenchus species (Palomares-Rius et al., 2014). For example, Vovlas et al. (2008) have recognized the subspecies Rotylenchus magnus jaeni as a separate (cryptic) species and have proposed the new name Rotylenchus jaeni. Cantalapiedra-Navarrete et al. (2013) also described Rotylenchus paravitis as a clear example of a cryptic species. Therefore, the combination of morphological and molecular approaches is warranted in order to address the limitations of each approach in the identification of Rotylenchus species, or plant-parasitic nematodes in general.
Rotylenchus rhomboides n. sp. has relatively large phasmid opening (1.2–2 µm) (see Fig. 1). The size of the phasmid is important as the large phasmid (scutellum) is the only feature to separate Scutellonema spp. from Rotylenchus spp. According to Germani et al. (1985), Rotylenchus species possess pore-like phasmids while Scutellonema species have scutella (external opening ca 3–9 µm); although Phillips (1971) described four Scutellonema species with a small scutellum (1.2–2.1 µm) in Australia. Germani et al. (1985) transferred these species to the genus Rotylenchus after consideration of all morphological and morphometric characteristics, but unfortunately, no molecular data are available for these species. In this study, despite the relatively large phasmids, Rotylenchus rhomboides n. sp. is clearly imbedded within the genus Rotylenchus according to molecular data.
Rotylenchus rhomboides n. sp. was found on banana (Musa basjoo) from Japan and R. buxophilus was found associated with yam (Dioscorea tokoro) from China. Both plants were imported in Belgium but survived for a decade outside under the Belgian weather conditions. Interestingly, R. buxophilus has been reported in many countries on many different hosts, including English boxwood (Buxus sempervirens var. suffruticosa L.), lima beans (Phaseolus lunatus L.), rye (Secale cereale L.), strawberry (Fragaria × ananassa Duchesne), tomato, and yew (Taxus canadensis Marsh.) in USA; Cupressus sempervirens L. and pea in Spain; Hydrangea macrophylla (Thunb.) Ser. in Italy; camellia (Camellia japonica L.) in France; unknown grass soils in Austria and Poland; vineyards in Switzerland; bilberries (Vaccinium myrtillus L.) in Slovakia; grassland in Romania; bilberry plants in Russia; sugarcane in Taiwan; elder, lilac, and Taraxacum F. H. Wigg. in Hungary; beech forest humus in Bulgaria; in Pakistan; olive in Turkey; Magnolia sp. L. in Iran; cabbage tree (Cordyline sp. Comm, ex R.Br.), Erica arborea L., fig (Ficus carica L.), and soil from a flower garden in New Zealand (Castillo and Vovlas, 2005). However, R. buxophilus has never been reported on yam (Dioscorea tokoro). In this study, although the host plant of R. buxophilus was imported from China, the above-listed distribution of R. buxophilus indicates that this species is more prevalent in Europe compared to Asia, as only one population was recorded from Taiwan.