Rotylenchus castilloi, Talezari, Atefeh, Pourjam, Ebrahim, Kheiri, Ahmad, Liébanas, Gracia, Aliramaji, Farzad, Pedram, Majid, Rezaee, Saeed & Atighi, Mohammad Reza, 2015

Talezari, Atefeh, Pourjam, Ebrahim, Kheiri, Ahmad, Liébanas, Gracia, Aliramaji, Farzad, Pedram, Majid, Rezaee, Saeed & Atighi, Mohammad Reza, 2015, Rotylenchus castilloi n. sp. (Nematoda: Hoplolaimidae), a new species with long stylet from northern Iran, Zootaxa 3931 (1), pp. 88-100 : 90-95

Rotylenchus castilloi n. sp.

( Figs 1–4 )

Measurements. See Table 1.

Female: Body habitus open- to closed-C in shape, varying to closed-J or spiral after heat relaxation. Cuticle without longitudinal striations, annuli 2.6–3.3 (2.0±0.3) wide at mid-body. Lateral field with four smooth equidistant lines, beginning anteriorly at annuli 8–10 as three lines (forming two bands), after 8–11 annuli, central line dividing to form a third band, three bands 11–14 (14.0±1.5) wide at mid-body, approximately one-fifth as wide as body diameter, external bands regularly areolated in pharynx region, but not on rest of the body. Lip region hemispherical, continuous with body contour, with 7–8 annuli under LM and irregular corncob-like appearance under SEM, 13–16 (14.6±1.0) Μm wide, 10–12 (10.8±0.9) Μm high. Labial disc distinct, rounded to somewhat rectangular, separated from rest of lip region, but not elevated. Labial framework strong, outer margin extending 2–3 annuli posteriorly into the body. Stylet well developed, 4–5 times longer than labial region diameter, conus 51.5–54.0% of total length, basal knobs rounded, slightly flattened anteriorly, 9–11 Μm wide. Dorsal pharyngeal gland orifice at ca. one tenth stylet length behind the knobs. Procorpus cylindrical, 51–68 (58.7±5.4) Μm long, median pharyngeal bulb well developed, oval, 22–30 Μm long and 17–20 Μm diameter, located at 69.1–76.3 (71.6±2.2)% of pharynx length, pharyngeal glands sacciform, overlapping intestine dorsally. Nerve ring enveloping isthmus at mid-point, 152–184 (165.5±8.3) Μm from anterior end. Excretory pore distinct. Hemizonid 1.5–2.0 annuli long, 1–3 annuli anterior to excretory pore. Reproductive system with two genital branches equally developed, anterior branch 450–660 (576±91.5) Μm long, posterior branch 475–700 (569±84.2) Μm long, ovaries with single row of oocytes, spermatheca rounded, 22–33 Μm in diameter, functional, with spherical sperm. Vulva slightly posterior to mid-body, with protruding double epiptygma. Phasmid pore-like, its location varying from 2–12 Μm anterior, to 2–10 Μm posterior to anus or at the level of anus (1-6 annuli anterior to anus or 1-4 annuli posterior to it). Tail short, variable in shape from rounded to dorsally convex-conoid, rarely bi-lobed, with 8–12 annuli, 0.5–0.7 anal body diameter long, terminus annulated.

Male: Common, approximately as abundant as females. Morphology similar to that of female except for sexual dimorphism. Body habitus slightly curved ventrally after heat relaxation. Lip region 11–13 (12.0±0.7) Μm in diameter, 8–11 (9.4±0.8) Μm high. Lateral field 8–12 (10.2±1.0) Μm wide at mid-body, ca one-fifth as wide as body diameter. Stylet and stylet knobs less developed compared to those of female, knobs 6–9 Μm wide. Pharyngeal glands overlapping intestine dorsally. Testis single, anteriorly outstretched, 467–849 (713±127) Μm long. Phasmid distinct, its location varying from 4 Μm anterior to cloaca to 11 Μm posterior to it. Spicules slightly cephalated, ventrally arcuate. Gubernaculum non-protrusible, distal part bent backward. Tail conoid, nearly 1.3–1.8 times cloacal region diameter with hyaline terminus. Bursa well developed, 74–90 Μm long, crenate, enveloping tail terminus.

Type locality and habitat. The typel population of Rotylenchus castilloi n. sp. was recovered from a soil sample collected around the rhizosphere of oriental beech tree ( Fagus orientalis Lipsky ) in Asalem forests, Ardebil province, northwestern Iran (GPS coordinates: N: 37°42' 37.373", E: 48°53' 44.363") by Farzad Aliramaji in November, 2013. A second population was recovered from soil samples collected from the rhizosphere of forest trees in Golestan province, northern Iran, during 2014 by Atefeh Talezari.

Type specimens. Holotype female, four female, and six male paratypes deposited in the Nematode Collection of the Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. Two female and two male paratypes deposited at each of the following collections: CABI Europe-UK, Egham, Surrey, UK; USDA Nematode Collection, Beltsville, MD, USA; and WANECO collection, Wageningen (http://www.waneco.eu/).

Etymology. The new species is named after Prof. Pablo Castillo for his contribution to the taxonomy of the genus Rotylenchus .

Diagnosis and relationships. Rotylenchus castilloi n. sp. is a bisexual species assigned to the species group having a hemispherical lip region, lateral field areolated only in pharynx region, stylet longer than 41 µm and rounded female tail ( Castillo & Vovlas 2005). The new species is characterised by having a hemispherical and continuous lip region with irregular corncob-like appearance in SEM, very long stylet (62–68 Μm) vulva located at 49.7–62.2% with a protruding double epiptygma, rounded to convex-conoid, rarely bi-lobed tail with 8–12 annuli and specific sequences of D2-D3 segments of 28S and ITS1-rRNA genes.

Table 1. Morphometrics of Rotylenchus castilloi n. sp. from Iran. All measurements are in μm and in the form: mean ± s.d. (range) a. Abbreviations are defined in Siddiqi (2000). Morphologically, Rotylenchus castilloi n. sp. can be distinguished from all similar species within the genus by its matrix code (as described by Castillo & Vovlas 2005) as follows: A6, B1, C1, D4, E4, F2, G5, H2, I2, J1, K2. The new species is morphologically similar to four known species of the genus: R. mesorobustus Zancada, 1985 ; R. magnus Zancada 1985 ; R. cazorlaensis Castillo & Gómez Barcina, 1987 and R. jaeni (Castillo, Vovlas, Gómez Barcina & Lamberti, 1993) Vovlas, Subbotin, Troccoli, Liébanas & Castillo, 2008 .

It differs from R. mesorobustus by its longer body (2258–2919 vs 1300–1480 Μm), shape of the lip region (hemispherical and irregular corncob-like appearance in SEM vs rounded with regular blocks), longer stylet (62–68 vs 47.5–49.5 Μm) and tail (18–30 vs 9.0–15.5 Μm) and the location of phasmid (varying from anterior to posterior of anus vs 5–8 annuli anterior to anus).

The new species differs from R. magnus by its body length (2258–2919 vs 1520–2140 Μm), continuous lip region (vs set off), location of phasmid (varying from anterior to posterior to to anus vs always anterior to anus) and presence of male (vs absence).

R. castilloi n. sp. differs from R. cazorlaensis by its body length (2258–2919 vs 1440–2280 Μm), shape of labial region (hemispherical vs truncate), stylet length (62–68 vs 46.5–56.5 Μm), distance of dorsal pharyngeal gland orifice from stylet base (6–8 vs 8.5–11.5 Μm) and location of phasmid (varying from anterior to posterior to anus vs at the level of anus to posterior).

When compared with R. jaeni , the new species differs by its body length (2258–2919 vs 1500–2200 Μm), lip region characters (continuous vs set off) and location of phasmid (varying from anterior to posterior to anus vs 0–6 annuli anterior to anus).

Molecular phylogenetic relationships. Amplification of the partial D2-D3 expansion segments of 28S and ITS1-rRNA genes for the new species yielded two fragments of 721 and 750 bp long, respectively. The D2-D3 dataset consisted of 33 species/isolates with 577 total characters (after trimming); of these 320 characters were conserved and 253 characters were variable. The average nucleotide composition was as follows: 15.3% A, 26.2% C, 36.8% G and 21.7% T. The results (number and percentage of similarity and gaps) of pairwise alignment of the new species with the morphologically compared species using D2-D3 region are presented in Table 2. Fig. 5 presents the phylogenetic tree inferred using the D2-D3 dataset. Using Tylenchorhynchus claytoni ( EU368589 ) as outgroup, two major clades 1 and 2 were inferred. The major clade 1 consisted of several species of Helicotylenchus Steiner, 1945 in a highly supported clade (1.00/99) and the major clade 2 (with weak support 0.66/ -) which is divided into two subclades, A and B. The subclade 2A includes several Rotylenchus spp. and the new species. The species R. striaticeps (Vovlas, Castillo & Lamberti, 1993) Siddiqi, 2000 occupies the most basal position (supported only by Bayesian analysis), but the second most basal species within subgroup 2A cannot be inferred due to polytomy. In other words, we cannot determine which species is the most closely related to R. striaticeps . This observation can be compared to the phylogenetic relationship of the latter species in our ITS1 tree ( Fig. 6 ), in which the phylogenetic relation with Helicotylenchus spp. is well supported (1.00/96). The subclade 2B includes five species/isolates of Helicotylenchus , as a sister clade to Rotylenchus dalikhaniensi s. Although the new species and the three morphologically similar species ( R. cazorlaensis , R. jaeni and R. magnus ) are part of subclade 2A, the relationships among these four species are not fully resolved due to polytomy. Currently there are no molecular data for R. mesorobustus . Future sequencing of other morphologically similar species (for example, species with long stylet) may provide more insights on phylogenetic relationships of this group of species. The close relationship of the new species with R. eximius Siddiqi, 1964 (although with low branch supports) is surprising as these species differ in some morphological characters such as stylet length and lip region conformation.

New species Morphologically compared species/ Similarity Gaps

isolates

Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus cazorlaensis ( EU373669 ) 408/682 (59.8%) 193/682 (28.3%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus cazorlaensis ( EU373670 ) 412/682 (60.4%) 191/682 (28.0%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus cazorlaensis ( EU373671 ) 415/682 (60.9%) 191/682 (28.0%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus magnus ( EU373676 ) 376/675 (55.7%) 236/675 (35.0%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus magnus ( EU373659 ) 376/675 (55.7%) 236/675 (35.0%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus magnus ( EU373659 ) 392/671 (58.4%) 210/671 (31.3%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus magnus ( EU373660 ) 391/670 (58.4%) 209/670 (31.2%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus magnus ( EU373665 ) 391/670 (58.4%) 209/670 (31.2%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus jaeni ( EU373661 ) 379/687 (55.2%) 250/687 (36.4%) Rotylenchus castilloi n. sp. ( KM251416 ) Rotylenchus jaeni ( EU373662 ) 378/687 (55.0%) 250/687 (36.4%) The ITS1 dataset consisted of 43 species/isolates of Rotylenchus , three species of Helicotylenchus and one belonolaim outgroup taxon. The dataset had 836 total characters (after manually trimming) of which 138 characters were conserved and 693 characters were variable. The average nucleotide composition was as follows: 19.6% A, 26.6% C, 30.3% G and 23.8% T. The results (number and percent of similarity and gaps) of pairwise alignment of the new species with the morphologically compared species using ITS1 region are presented in Table 3 . Fig. 6 presents the phylogenetic tree inferred using the above mentioned dataset. Using Tylenchorhynchus zeae ( EF519711 ) as outgroup, three major clades were inferred. Clades 1 and 2 include several species of Rotylenchus spp., including the new species in major clade 1, forming a monophyletic group with R. unisexus Sher, 1965 with high (1.00/95) support. There are some common morphological characters like shape of lip region, tail in female and presence of double epiptygma which might support affinity of these two species, while both species have basic differences in some key characters like the length of the stylet. Based on current molecular knowledge, the paraphyly of the family Hoplolaimidae and the genus Rotylenchus is well documented ( Atighi et al. 2011, 2014; Vo v l a s et al. 2008; Cantalapiedra-Navarrete et al. 2013). Accordingly, we believe more species of the genus need to be sequenced before we can resolve the phylogenetic relationships among the species of the genus Rotylenchus and between the genera Rotylenchus and Helicotylenchus .

Figures

FIGURE 1. Line drawings of Rotylenchus castilloi n. sp. (type population). A, Details of pharynx; B, Anterior genital tract showing fine spherical sperm in spermatheca; C, D, Anterior end in detail; E, Vagina and protruding double epiptygma; F, G, I- K, Female tail; H, Male tail. (Scale-bars: A, 25 Μm; B, 50 Μm; C, 15 Μm; D, 30 Μm; E, 20 Μm; F-K, 30 Μm).

FIGURE 2. Light micrographs of Rotylenchus castilloi n. sp. (type population). A, B, Female anterior end; C, Male anterior end; D, Female mid body showing part of female reproductive system and fine spherical sperm in spermatheca; E, Lateral field at mid-body; F, Part of female reproductive system showing functional spermatheca; G, Details of pharyngeal glands; H, Exceretory pore and hemizonid (lower and upper arrow, respectively); I, Vulva region showing protruding double epiptygma (Scale bars: 10 µm, D; 20 µm).

FIGURE 3. Light micrographs of Rotylenchus castilloi n. sp. (type population). A – D, Variation in female tail morphology; E – H, Male tail region; I, Female tail showing phasmid. (Scale-bars: 10 µm).

FIGURE 4. Scanning electron microscope photographs of Rotylenchus castilloi n. sp. (type population). A – C, Female lip region in different views; D, Vulva region showing protruding double epiptygma; E, Lateral view of female anterior end showing details and beginning of lateral field; F, Lateral field at mid-body; G, Female tail region showing phasmid; H, Male tail region; I, Female tail region showing its tip; J, Phasmid in close view (Scale bars: A, B, F, I, 5 Μm; C, D, J, 2 Μm; E, G, H, 10 Μm).

FIGURE 5. Bayesian inference analysis 50 % majority rule consensus tree as inferred from D 2 – D 3 expansion segments of 28 S rDNA sequence alignment under the GTR + G + I model. Bayesian posterior probabilities and maximum likelihood bootstrap values more than 50 % are given for appropriate clades in the form: BPP / ML BS. New sequence is in bold font.

FIGURE 6. Bayesian inference analysis 50 % majority rule consensus tree as inferred from ITS 1 rDNA sequence alignment under the GTR + G + I model. Bayesian posterior probabilities and maximum likelihood bootstrap values more than 50 % are given for appropriate clades in the form: BPP / ML BS. New sequences are in bold font.

Tables

TABLE 3. Results of pairwise alignment of the new species with compared species / isolates for ITS 1.

New species	Morphologically compared species/ isolates	Similarity	Gaps
Rotylenchus castilloi n. sp. ( KM251415 )	Rotylenchus cazorlaensis ( EU280793 )	514/572 (89.9%)	12/572 (2.1%)
Rotylenchus castilloi n. sp. ( KM251415 )	Rotylenchus cazorlaensis ( EU280792 )	514/572 (89.9%)	12/572 (2.1%)
Rotylenchus castilloi n. sp. ( KM251415 )	Rotylenchus magnus ( EU280789 )	519/573 (90.6%)	12/573 (2.1%)
Rotylenchus castilloi n. sp. ( KM251415 )	Rotylenchus jaeni ( EU280791 )	518/577 (89.8%)	19/577 (3.3%)