Description of Rotylenchus rhomboides n. sp. and a Belgian population of Rotylenchus buxophilus (Tylenchomorpha: Hoplolaimidae)

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).

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).

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.

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 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).

Name of the new species regarded from rhomboideus (modern Latin), which refers to typical rhomboid enlargement in the lateral field at vulva level.

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″).

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.

Rotylenchus buxophilus (Golden, 1956)

Figures 3–5, 7, Tables 4, 5.

All the molecular analyses of the Belgian population of Rotylenchus buxophilus were executed together with the sequences of Rotylenchus rhomboides n. sp. as above.

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).

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″).

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.