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

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.

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.

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).

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.

Fig. 1

Fig. 2

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.

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.

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

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.

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. 3

Fig. 4

Fig. 5