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

Abstract During a survey in the Botanical garden of Ghent University, a new species Rotylenchus rhomboides n. sp. and a population of Rotylenchus buxophilus were found. Rotylenchus rhomboides n. sp. is characterized by the presence of a rhomboid-like widening of the mid-ridge of lateral field at the level of vulva, a feature previously unknown within the genus. The population of the new species, composed only by females, has a rounded labial region with 4 to 5 annuli, robust stylet (31–37 μm long), short dorsal esophageal gland (9–19 μm) overlap of the intestine, vulva located slightly posterior to mid-body, and hemispherical or rounded tail shape with large phasmids located 3 to 5 annuli anterior to the level of anus. The hierarchical cluster analysis based on morphological features indicated that the new species closely resembles R. corsicus, R. gracilidens, and R. rugatocuticulatus. The DNA analyses of the D2-D3 of 28S rDNA, ITS rDNA, and COI mtDNA sequences of Rotylenchus rhomboides n. sp. show a close relationship with R. buxophilus, R. goodeyi, R. laurentinus, R. pumilus, and R. incultus, all of which can also be differentiated from the new species by morphological features. The combination of morphological, morphometric, and molecular characteristics confirmed the new species and the first report of R. buxophilus on yam (Dioscorea tokoro) in Belgium.

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).
The genus Rotylenchus is widely distributed all over the world and has been recorded from all continents with 103 valid species to date (Castillo and Vovlas, 2005;Vovlas et al., 2008;Atighi et al., 2011;Cantalapiedra-Navarrete et al., 2012, 2013Atighi et al., 2014;Aliramaji et al., 2015;Noruzi et al., 2015;Talezari et al., 2015;Golhasan et al., 2016). According to Castillo and Vovlas (2005), the number of Rotylenchus species is second highest in Europe, after Asia. Several species of Rotylenchus are of economic importance in agriculture, among them, only R. robustus, R. buxophilus (Golden, 1956), R. uniformis (Thorne, 1949;Loof and Oostenbrink, 1958), and R. goodeyi (Loof and Oostenbrink, 1958) have been reported in Belgium (Steel et al., 2014;Viaene et al., 2017). They are the main cause of yield losses in many agricultural crops such as carrot, olive, orange, mango, soybean, broccoli, cabbage, tomatoes, etc. Rotylenchus robustus is considered as the most common species worldwide and has been reported in 25 countries and islands on all continents except Antarctica. Rotylenchus buxophilus is also a widely distributed species, occurring in Europe, Asia, North America, and New Zealand. Rotylenchus uniformis was found in the Netherlands and New Zealand. Rotylenchus goodeyi was recorded from several localities in the UK, the Netherlands, Belgium, Bulgaria, Slovakia, Spain, Switzerland, Luxemburg, and Uzbekistan (Castillo and Vovlas, 2005).
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.

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 μ l 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 × 10 6 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.
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/.

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 μ m 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 μ m high. Dorsal esophageal gland opening 2 to 5 μ m 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) μ m, pore-like, located 3 to 5 annuli anterior to level of anus (Figs 1G, H, 2H, I, K, L).

Male
Not found.

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 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. 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 midbody, 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 μ m); 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.
Description of Rotylenchus rhomboides n. sp. and a Belgian population of Rotylenchus buxophilus 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.
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 μ m 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)    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 μ m vs between 21 and 30.9 μ m); 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. 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 μ m vs between 21 and 30.9 μ m); 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.

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

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 μ m vs 33.5 μ m), 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).
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 μ m vs 23-28 μ m); 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 o 2′6.9′′, E: 3 o 43′22.6′′).

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).