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. 2018 Jun 3;50(2):219–228. doi: 10.21307/jofnem-2018-025

Nothotylenchus andrassy n. sp. (Nematoda: Anguinidae) from Northern Iran

Parisa Jalalinasab 1, Mohsen Nassaj Hosseini 2, Ramin Heydari 1,*
PMCID: PMC6909307  PMID: 30451439

Abstract

Nothotylenchus andrassy n. sp. is described and illustrated from moss (Sphagnum sp.) based on morphology and molecular analyses. Morphologically, this new species is characterized by a medium body size, six incisures in the lateral fields, and a delicate stylet (8–9 µm long) with clearly defined knobs. Pharynx with fusiform, valveless, non-muscular and sometimes indistinct median bulb. Basal pharyngeal bulb elongated and offset from the intestine; a long post-vulval uterine sac (55% of vulva to anus distance); and elongate, conical tail with pointed tip. Nothotylenchus andrassy n. sp. is morphologically similar to five known species of the genus, namely Nothotylenchus geraerti, Nothotylenchus medians, Nothotylenchus affinis, Nothotylenchus buckleyi, and Nothotylenchus persicus. The results of molecular analysis of rRNA gene sequences, including the D2–D3 expansion region of 28S rRNA, internal transcribed spacer (ITS) rRNA and partial 18S rRNA gene are provide for the new species.

Keywords: 18S rRNA, D2–D3 region, Molecular, Morphology, Moss, Sphagnum sp., ITS rRNA

The genus Nothotylenchus Thorne, 1941 belongs to subfamily Anguininae Nicoll, 1935 within the family Anguinidae Nicoll, 1935. Dong-Geun et al. (2005) showed that the type species of the genus, Nothotylenchus acris, is a parasite of strawberry (Fragaria × ananassa Duchesne). Nishizawa and Iyatomi (1955) also reported this nematode in association with strawberry diseases in Japan. Nothotylenchus species are morphologically closely related to members of Ditylenchus Filipjev, 1936, but they differ in morphology of the median pharyngeal bulb which is indistinct or non-muscular, non-valvate in Nothotylenchus. Furtuner and Maggenti (1987) and Sturhan and Brzeski (1991) did not give due taxonomic importance to the presence/absence of a muscular, valvate median pharyngeal bulb, even at the generic level, and considered Nothotylenchus, Diptenchus Khan et al., 1969, Safianema Siddiqi, 1981 and Orrina Brzeski, 1981 as synonyms of Ditylenchus. But some authors (Siddiqi, 2000; Andrássy, 2007) kept these genera valid. Relationships within Anguinidae were not well resolved in the phylogenetic analysis using the D2–D3 expansion segments of 28S, ITS, and partial 18S rRNA gene sequences (Subbotin et al., 2004). Here, we followed the classification scheme of Siddiqi (2000) and Andrássy (2007).

To date more than 48 species have been recognized for Nothotylenchus, although some species are considered as species inquirendae (Andrássy, 2007). Recently two new species of the genus, Nothotylenchus persicus Esmaeli et al., 2016 and Nothotylenchus phoenixae Esmaeli et al., 2017 were described in Iran from rhizosphere of grapevine (Vitis spp.) and date palm (Phoenix dactylifera L.), respectively. During a nematode survey on eastern forests of Guilan province, northern Iran, an unknown anguinid nematode population belonging to the genus Nothotylenchus was recovered from moss samples (Sphagnum sp.). Detailed observations using light microscopy and molecular assays indicated that this population differed from all previously described members of the genus and should be assigned to a new species. This publication includes a description of Nothotylenchus andrassy n. sp. through morphological observation and molecular characterization by the partial 18S rRNA, D2–D3 expansion region of 28S rRNA and ITS rRNA gene sequences.

Materials and methods

Sampling, extraction, mounting, and drawing

Soil, root, and moss samples, were randomly collected from different regions of eastern forests of Guilan province, northern Iran during 2015. Nematodes were extracted from sample materials by the tray method (Whitehead and Hemming, 1965) and were soaked in a small amount of water for 48 hr. The extracted nematodes were observed and hand-picked using a stereomicroscope. Adult specimens for microscopic observation were killed by gentle heat and fixed in a solution of FGA 4:1:1 (formaldehyde, glycerin, and acetic acid) and processed to anhydrous glycerin (De Grisse, 1969). Permanent slides were made and examined using an Olympus BH2 light microscope. Morphometric data were obtained using a drawing tube and photomicrographs were taken using a digital camera. Line drawings were redrawn using CorelDraw® software version 17.

DNA extraction, polymerase chain reaction, and sequencing

Single nematode specimens were handpicked and examined individually by light microscopy and transferred to 10 μl of distilled water on a glass microscope slide, crushed with a pipette tip and collected in 50 μl AE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0, Qiagen, Valencia, CA) by pipette. DNA extracts were stored at −20° C until used as template for polymerase chain reaction (PCR) amplification.

A volume of 1 μl of extracted DNA was transferred to an Eppendorf tube containing: 2.5 μl 10× NH4 reaction buffer, 0.75 μl MgCl2 (50 mM), 0.25 μl dNTPs mixture (10 mM each), 0.75 μl of each primer (10 mM), 0.2 μl BIOTAQ DNA Polymerase (Bioline, UK) and ddH2O to a final volume of 25 μl. The D2–D3 expansion segment of 28S rRNA gene was amplified using the forward D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and reverse D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers (Nunn, 1992). The ITS region was amplified using forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and reverse primer 5.8SM5 (5′-GGCGCAATGTGCATTCGA-3′) (Maafi et al., 2003; Vovlas et al., 2008), and the partial 18S was amplified using primers 1096F (5′-GGTAATTCTGGAGCTAATAC-3′), 1912R (5′-TTTACG GTCAGAACTAGGG-3′) (Holterman et al., 2006). PCR cycle conditions were as follows: one cycle of 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, annealing temperature of 55°C for 45 sec, 72°C for 3 min, and finally one cycle of 72°C for 10 min. PCR products were purified after amplification using ExoSAP-IT (Affmetrix, USB Products), quantified using a Nanodrop spectrophotometer (Nanodrop Technologies) and used for direct sequencing in both directions using the primers referred to above.

Phylogenetic analyses

Newly obtained sequence of the D2–D3 expansion region of 28S, ITS, and partial 18S rRNA and available sequences of anguinid nematodes obtained from GenBank were used for phylogenetic reconstructions. The newly obtained and published sequences were aligned using Muscle (Edgar, 2004) with default parameters implemented in MEGA 5.0 (Tamura et al., 2011). Sequence alignment was edited using MEGA 5.0. The most appropriate model was determined using the Bayesian Information Criterion (BIC) implemented in the jModelTest program (Posada, 2008). Phylogenetic analyses of the sequence data set was performed based on Bayesian inference (BI) using MRBAYES3.1.2 (Ronquist and Huelsenbeck, 2003). The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) were given on appropriate clades. Trees were visualized using TreeView (Page, 1996).

Results

Nothotylenchus andrassy n. sp.(Table 1; Figs. 1, 2)

Table 1.

Morphometrics of Nothotylenchus andrassy n. sp. All measurements in micrmeter and in the form: mean ± s.d. (range).

Female Male
Character Holotype Paratypes Paratypes
n ˋ 6 4
L 778 754.7 ± 98.6 (681–962) 640 ± 70.7 (574–738)
a 38.9 35.3 ± 3.6 (30.3–38.9) 34.1 ± 1.7 (31.9–35.6)
b 6.1 5.9 ± 0.4 (5.4–6.4) 5.4 ± 0.4 (5.2–6.0)
c 13.2 11.7 ± 1.3 (8.9–13.2) 10.6 ± 1.7 (8.4–12.5)
c¢ 4.5 5.0 ± 0.6 (4.4–6.0) 4.7 ± 0.9 (3.8–5.9)
V or T 81.6 80.7 ± 1.7 (77.1–82.4) 47.6 ± 1.8 (45.3–49.7)
Lip region height 2.0 2.0 2.3 ± 0.5 (2.0–3.0)
Lip region width 5.0 5.4 ± 0.5 (5.0–6.0) 5.3 ± 0.5 (5.0–6.0)
Stylet length 9.0 8.3 ± 0.5 (8.0–9.0) 8.1 ± 0.3 (8.0–8.5)
m a 44.4 45.7 ± 2.9 (43.8–50.0) 49.2 ± 3.9 (43.8–52.9)
E. pore from anterior end 103 99.0 ± 7.4 (92–112) 92 ± 3.9 (87–96)
Pharynx length 128 127.4 ± 16.2 (114–162) 117.3 ± 6.0 (109–123)
Max. body diam. 20 21.6 ± 3.7 (18–28) 18.8 ± 1.5 (18–21)
Vulval body diam. (VBD) 19 19.7 ± 3.3 (17–26) –
Vulva–anus distance (V–A) 84 80.3 ± 14.8 (70–112) –
Post-uterine sac (PUS) length 45 43.4 ± 5.6 (39–55) –
PUS/Vulval body diam. 2.4 2.2 ± 0.2 (2.0–2.4) –
PUS/V–A% 53.6 54.6 ± 4.3 (49.1–62.0) –
Ovary length or testis 410 358.7 ± 69.6 (275–457) 305 ± 42.1 (273–367)
Anal (cloacal) body diam. 13 13.0 ± 2.3 (11–18) 13.0 ± 1.4 (12.0–15.0)
Spicule length – – 18.0 ± 0.6 (18.0–19.0)
Gubernaculum length – – 5.3 ± 0.5 (5.0–6.0)
Tail length 59 66.1 ± 18.6 (55–108) 62.3 ± 18.0 (46–88)
Bursa (% of tail) – – 28.0 ± 3.4 (24.6–32.6)
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aLength of conus as percentage of total stylet length.

Fig. 1.

Fig. 1

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Line drawings of Nothotylenchus andrassy n. sp. A, Female body; B, Male body; C, Pharyngeal region of female; D, Female anterior end; E, Lateral field of female; F, Female posterior body; G, Male posterior body. (Scale bars: A and B = 20 µm, C–G = 10 µm).

Fig. 2.

Fig. 2

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Photomicrographs of Nothotylenchus andrassy n. sp. A, Female pharyngeal region showing fusiform median bulb; B, Female pharyngeal region showing indistinct median bulb; C, Basal pharyngeal bulb region; D, Female anterior end; E, Lateral field; F, Vulva region showing post-uterine sac; G, Male posterior body showing leptoderan bursa; H, Spicules lateral view; I, Male posterior body (tail); J, Female posterior body (tail). (All scale bars = 10 µm).

Description

Females

Body subcylindrical, tapering at both ends and almost straight upon fixation. Cuticle with transverse striae measuring ca 1.1 μm wide in mid-body region. Lateral fields with six incisures occupying a third of body diam. Lip region continuous with body 5 to 6 μm wide and 2 to 3 μm high. Stylet 8 to 9 μm long with well developed basal knobs, conical part occupying 44% to 50% of total stylet length. Orifice of dorsal pharyngeal gland at 1 to 1.5 μm posterior to stylet knobs. Pharynx with cylindrical corpus and valveless, non-muscular, fusiform median bulb which is indistinct in some specimens and connecting to an elongate pharyngeal basal bulb via a slender isthmus. Basal pharyngeal bulb is offset from intestine. Excretory pore 92 to 112 µm from anterior end of body. Hemizonid about two to three body annuli wide and situated just anterior to excretory pore. Reproductive system prodelphic, ovary outstretched and oocytes arranged in a single row. Uterine quadricolumellar consisting of four rows of four cells, followed by an elongated spermatheca. Spermatheca filled with large rounded sperm cells in some specimens. Vulva a transverse slit with slightly protuberant lips. Vagina straight and reaching almost halfway across body. Post vulval uterine sac well developed occupying 49–62% of the vulva anus distance. Tail elongate, conical, regularly tapering toward a pointed tip, 4.4 to 6 times the anal body diam.

Males

Similar to female in general morphology with usually shorter body size. Lip region is slightly higher than female, 2 to 3 μm high and 5 to 6 μm wide. Cuticle with transverse striae measuring ca 1.1 μm wide in mid-body region. Stylet delicate with rounded basal knobs. Testis single, sometimes reflexed at anterior end. Spicules curved ventrally, 18 to 19 μm long. Gubernaculum simple, 5 to 6 μm long and slightly less than one third the length of the spicules. Bursa short, leptoderan, beginning almost opposite the proximal end of the spicules and covering 25 to 33% of tail length. Tail elongate, conoid, usually straight, with pointed tip, 4 to 6 times the anal body diam.

Diagnosis and relationships

Nothotylenchus andrassy n. sp. is characterized by a medium body size, six incisures at the lateral fields, a delicate stylet (8–9 µm long) with clearly defined knobs; Pharynx with cylindrical corpus, fusiform, valveless and sometimes indistinct median bulb and elongated basal pharyngeal bulb. Vulva at 77% to 82% of body length; a long post-vulval uterine sac (55% of the vulva–anus distance) and elongate, conical tail with pointed tip. Nothotylenchus andrassy n. sp. is morphologically and morphometrically similar to Nothotylenchus geraerti Kheiri, 1971, Nothotylenchus medians Thorne and Malek, 1968, Nothotylenchus affinis Thorne, 1941, Nothotylenchus buckleyi Das, 1960, and N. persicus. The new species differs from N. geraerti mainly by more elongate basal pharyngeal bulb, longer spicules (18–19 vs. 16 µm) and tail tip pointed vs. rounded. It differs from N. medians by slightly longer stylet (8–9 vs. 6.5–8 µm), slightly longer spicules (18–19 vs. 15–18 µm), shorter bursa as percentage of tail length (25–33 vs. 27–84%) and tail tip pointed vs. rounded. It can be distinguished from N. affinis by longer post-vulval uterine sac (2–2.4 vs. 1.1–1.3 times vulval body diam.), slightly longer spicules (18–19 vs. 15–17 µm); slightly shorter bursa length as percentage of tail length (25–33 vs. 50%) and tail tip pointed versus rounded. Nothotylenchus andrassy n. sp. can be distinguished from N. buckleyi by longer body length (0.68–0.96 vs. 0.43 mm in females and 0.57–0.74 vs. 0.45 mm in males); shorter stylet (8–9 vs. 11 µm), posterior position of the excretory pore (opposite posterior one third of basal pharyngeal bulb vs. opposite middle of isthmus), more posteriorly located vulva (V = 77–82% vs. 71%); and longer spicules (18–19 vs. 15 µm). Finally it can be distinguished from N. persicus by longer stylet (8–9 vs. 5–6 µm), anterior position of the excretory pore (opposite posterior one third of basal pharyngeal bulb vs. posterior to basal bulb), longer post-vulval uterine sac (2–2.4 vs. 0.4–0.6 times vulval body diam.) and slightly shorter spicules (18–19 vs. 21–22 µm).

Type habitat and locality

The new species was recovered from moss samples (Sphagnum sp.) in Leila koh region, Langarud, Guilan province (GPS coordinates: 37° 10′ 29″ N, 50° 7′ 19″ E), northern Iran.

Type material

Holotype female (slide ANA001) together with four paratype specimens: Two females, two males (slides ANA001, ANA002) deposited in the Nematode Collection of the Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. Two female and two male paratypes deposited at Royal Belgian Institute of Natural Sciences, Brussels, Belgium. Paratype females deposited in the National Nematode Collection of the Department of Nematology, Iranian Research Institute of Plant protection, Tehran, Iran.

Etymology

The new species is named in honor of Dr István Andrássy, a pioneering scientist in the systematics of nematodes.

Molecular phylogeny

Amplification of D2–D3 expansion segments of 28S, ITS, and the partial 18S rRNA yielded a single fragment of 615 bp, 430 bp, and 775 bp, respectively.

The molecular phylogenetic trees were obtained from Bayesian analysis under the GTR + I + G model (Tavaré, 1986) to infer the relative placement of the new species among other species of Nothotylenchus and other genera of Anguinidae. The trees inferred by partial 18S, D2–D3 segments of 28S and ITS rRNA are shown in Figs. 3–5, respectively. In these trees, N. andrassy n. sp. is shown in bold font.

Fig. 3.

Fig. 3

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The molecular phylogenetic tree generated from the partial 18S rRNA inferred from Bayesian analyses under GTR + G + I model. Posterior probability values exceeding 50% are given on appropriate clades. The new species is in bold font.

Fig. 5.

Fig. 5

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The molecular phylogenetic tree generated from the ITS rRNA inferred from Bayesian analyses under GTR + G + I model. Posterior probability values exceeding 50% are given on appropriate clades. The new species is in bold font.

The BlastN search of partial 18S rRNA gene sequence of N. andrassy n. sp. (GenBank MG025825) revealed the highest match with sequences of Ditylenchus dipsaci (Kühn, 1857) Filipjev, 1936 (KJ636298 and HQ219210) with 97% identity. The 28S rRNA D2–D3 sequence of N. andrassy n. sp. (GenBank MG025824) revealed the highest match with sequences of Dyschirius persicus Esmaeili et al., 2017 (KX463285) with 98% identity. The ITS rRNA sequence of N. andrassy n. sp. (GenBank MG025826) revealed the highest match with sequences of Dosidicus gigas Vovlas et al., 2011 (JN376074, HQ219239, HQ219236, KC310732) with 85% identity and less than 85% homology with other available DNA sequences from GenBank.

The molecular phylogenetic tree generated from the partial 18S rRNA included 32 in-group and three outgroup taxa. In this tree N. andrassy n. sp. clustered with Nothotylenchus adasi Sykes, 1980 (EU669909, KJ636375) and also a clade containing some anguind species, i.e., D. dipsaci, D. gigas, Subanguina radicicola (Greef, 1872) Paramanov, 1967, Litylenchus coprosma Zhao et al., 2011, Anguina tritici (Steinbuch, 1799) Chitwood, 1935 and Anguina agrostis (Steinbuch, 1799) Filipjev, 1936. Nothotylenchus andrassy n. sp. differs from N. adasi by having morphological and morphometric differences such as six versus four lines in lateral fields, shorter stylet (8–9 vs. 11–13 µm), shorter spicules (18–19 vs. 22–24 µm), more posteriorly located vulva (77%–82% vs. 68%–76%) and sharply pointed tail tip versus rounded or dull tail tip. The other Nothotylenchus species with available 18S rRNA sequence, N. acris Thorne, 1941 (AY593914) is in a monophyletic clade having posterior probability support of 100% with Sphaerularia vespae Kanzaki et al., 2007 (AB300595), and Sphaerularia bombi Dufour, 1837 (AB250212).

The molecular phylogenetic tree generated from D2–D3 expansion segments of 28S rRNA included 36 in-group and three outgroup taxa. In this tree N. andrassy n. sp. clustered with D. persicus (KX463285) in a clade with 73% posterior probability. It differs morphologically from that species by having valveless fusiform, sometimes indistinct versus valvate, well developed median pharyngeal bulb and some other characters. The other Nothotylenchus species with available 28S rRNA sequence, N. persicus (KT149799), clustered with Neotylenchus sp. and Ficotylus congestae.

The molecular phylogenetic tree generated from ITS rRNA included 27 in-group and two outgroup taxa. In this tree all species of Anguinidae grouped in a 100% supported monophyletic clade and N. andrassy n. sp. was separated from other anguinid species as the only species in the genus in this tree.

Fig. 4.

Fig. 4

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The molecular phylogenetic tree generated from the D2–D3 of 28S rRNA inferred from Bayesian analyses under GTR + G + I model. Posterior probability values exceeding 50% are given on appropriate clades. The new species is in bold font.

Acknowledgments

This research was financially supported by University of Tehran. The authors would like to thank Mehrab Esmaeili (University of Tehran, Iran) for his technical help.

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