A new needle nematode, Longidorus zanjanensis sp. nov. (Nematoda: Longidoridae) from north-western Iran

Abstract During a survey of soil nematodes in 2022, a new species of the genus Longidorus, described here as Longidorus zanjanensis sp. nov., was discovered in the rhizosphere of Astragalus sp. in Zanjan Province, Iran. The new needle nematode is described and illustrated based on morphological, morphometric, and molecular traits. Further, its females are characterized by having a long body ranging 5.6–7.7 mm long, lip region anteriorly flattened and almost continuous or slightly offset by a depression with body contour, ca 16.5–18.5 μm wide, amphidial fovea pouch-like without basal lobes, guiding ring at 35–41 μm distance from the anterior end, and an odontostyle and odontophore ranging 102–115 and 47–75 μm long, respectively. The pharyngeal bulb is 123–153 μm long, female reproductive system didelphic–amphidelphic containing sperm, vulva almost equatorial, located at 46.7–51.4% of body length, tail short, rounded to bluntly conoid, bearing two pairs of caudal pores and terminus widely rounded with distinct radial lines in hyaline region (39–50 μm long, c = 122.4–189.4, c’ = 0.6–0.8). Males are common, making up to 60% of the adults, and are functional, with spicules 68.0–80.0 μm long, as well as having 8–14 ventromedian copulatory supplements. All four juvenile life developmental stages were present, with the tail of first-stage juvenile conoid shape, dorso-ventrally curved with rounded terminus. The polytomous codes delimiting the new species are: A4-B3-C3-D3-E1-F34-G12-H1-I2-J1-K6. Morphologically, the new species comes close to eight known species of the genus, namely L. apulus, L. armeniacae, L. crassus, L. kheirii, L. soosanae, L. proximus, L. pauli, and L. ferrisi. The morphological differences between the new species and the aforementioned species are discussed. Molecular phylogenetic analyses based on D2-D3 of large subunit (LSU) and internal transcribed spacer 1 (ITS1) rRNA sequences indicate that Longidorus zanjanensis sp. nov. is closely related to L. hyrcanus, L. soosanae, and L. elongatus.


Nematode population sampling, extraction, and morphological identification
About 100 soil samples were collected from the rhizosphere of different plants at a depth of 10-50 cm, in the Zanjan province, north-western Iran.Specimens of an unidentified Longidorus sp.nov.were obtained from the rhizosphere of Astragalus sp. in Zanjan province.Nematodes were extracted using the tray method (Whitehead & Hemming 1965), the magnesium sulphate (MgSO4) centrifugal flotation method (Coolen 1979), and a modification of Cobb's decanting and sieving method (Flegg 1967).Nematodes were handpicked under a stereomicroscope, killed by adding hot FPG (4:1:1, formaldehyde: propionic acid: glycerin) solution, transferred to anhydrous glycerine according to De Grisse (1969), and mounted on permanent glass slides to allow handling and observation.Morphometric values and photomicrographs were taken using a Dino-Eye digital eyepiece camera (Model AM7023, bundled with DinoCapture 2.0 software; AnMo Electronics Corporation, New Taipei City, Taiwan) attached to a Leitz Dialux 22 light microscope.Line drawings were first made using a drawing tube, then re-drawn and prepared for publication using CorelDRAW software version 16 (Corel Corp, Canada).Morphological comparisons were performed using the polytomous identification keys for the identification of Longidorus species (Chen et al. 1997;Loof & Chen 1999) and with the descriptions of all other characterized species up to the present.The position of pharyngeal gland nuclei was calculated according to Loof & Coomans (1972), and the juvenile developmental stages were identified according to Robbins et al. (1995).All measurements were recorded in micrometres (μm), except for body length in millimetres (mm) and ratios.Ratios are defined in Jairajpuri & Ahmad (1992).

Phylogenetic analyses
The newly obtained sequences of L. zanjanensis sp.nov.(D2-D3 expansion segments of 28S rRNA, and ITS1 rRNA) and other sequences of different Longidorus spp.from GenBank were used for phylogenetic analyses.ITS1 rRNA did not have enough similarity with other sequences deposited in the GenBank, and for this reason, sequence similarity comparisons were only made with the closest phylogenetically related species.Outgroup taxa for each dataset were chosen following previously published studies (He et al. 2005;Holterman et al. 2006;Palomares-Rius et al. 2008;Gutiérrez-Gutiérrez et al. 2013;Archidona-Yuste et al. 2019b;Cai et al. 2020a, b).Multiple sequence alignments for each gene were made using the FFT-NS-2 algorithm of MAFFT V.7.450 (Katoh et al. 2019).Sequence alignments were visualized using BioEdit (Hall 1999) and manually edited and trimmed of poorly aligned positions using a light filtering strategy (up to 20% of alignment positions), which has little impact on tree accuracy and may save some computation time, as suggested by Tan et al. (2015).Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2(Ronquist & Huelsenbeck 2003).The best-fit model of DNA evolution was obtained using JModelTest V.2.1.7 (Darriba et al. 2012) with the Akaike Information Criterion (AIC).The best-fit model, base frequency, proportion of invariable sites, and gamma distribution shape parameters and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses.The general timereversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for the D2-D3 segments of 28S rRNA and the general time-reversible model and a gamma-shaped distribution (GTR + G) for ITS1 rRNA were run with four chains for 4 × 10 6 generations, respectively.The Markov chains were sampled at intervals of 100 generations.Two runs were conducted for each analysis.After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for in-depth analyses.The topologies were used to generate a 50% majorityrule consensus tree.Posterior probabilities (PP) were given on appropriate clades.Trees from all analyses were visualised using FigTree software version 1.4.4 (Rambaut 2018).

Results and Discussion
The integration of nematode morphology with the morphometric analysis and molecular data using ribosomal sequences allowed us to describe herein a new species of the genus as L. zanjanensis sp.nov.
Female.Body ventrally bent varying from J to G shape when heat-relaxed.Cuticle appearing smooth under light microscope; its thickness varies over the body, 4-5 μm at guiding ring level, to 3-4 μm at mid-body, 7-8 μm at anterior lip of anus, and 14-23 μm at tail end (the hyaline part of tail tip), and marked by very fine superficial transverse striae mainly in tail region.Lateral chord 23-30% of corresponding body diameter.Lip region anteriorly flattened, continuous with the adjacent body (Figures 1, 2).Amphidial fovea is pouch-like without lobes at base.Stylet guiding ring located at ca. two times lip region diameter from anterior end.Odontostyle long and narrow, approximately 1.9 times as long as odontophore (Figures 1, 2).Nerve ring surrounding the slender portion of the pharynx posterior to the odontophore base, located at 247-280 μm from anterior end.Pharynx dorylaimoid, anterior slender part flexible, posteriorly expanding to a muscular terminal bulb occupying about 24.1 ± 1.7 (20.7-27.4)% of the total pharynx (neck region).The dorsal gland nucleus (DN) smaller, at 26.1-36.4%,and the two ventrosublateral nuclei (S1N) at about the same level and at 51.0-61.5% of the pharyngeal bulb length (location of glands nuclei according to Loof & Coomans (1972)).Cardia conoid to rounded, 5.0-7.0 μm long.Intestine with prerectum.The reproductive system didelphic-amphidelphic, with both branches almost equally developed, each branch 500-1100 μm long, with reflexed ovaries highly variable in length, anterior ovary (173-324 μm long), and posterior ovary (157-390 μm long).Oviducts slightly longer than ovaries.Uterus bipartite, quite variable in length, anterior uteri (230-276) μm long, and posterior uteri (208-286) μm long; sphincter well developed, between uterus and oviduct.Sperm commonly found in the uteri.Vagina 50-74 μm long or ca.66% of corresponding body width; pars distalis 11-16 μm long, pars proximalis vaginae measuring 20-34 μm long; vulva a transverse slit.Prerectum variable in length, 3.9-11.4times longer than anal body width and rectum simple, 0.8-0.1 times as long as tail length.Tail bearing two caudal pores, conoid, convex dorsally, and ventrally almost straight or slightly concave with rounded terminus.
Male.Common (about 60% of the population) and functional.Similar to females in general morphology, except for the reproductive system and posterior end more ventrally curved Male genital reproductive system diorchic.Spicules arcuate, robust, about 1.5 times longer than tail length, lateral guiding piece more or less straight.Adanal supplements paired, preceded anteriorly by a row of 8-14 irregularly spaced ventromedian supplements.Tail bluntly conoid, dorsally convex and ventrally concave, terminus widely rounded, with distinct radial lines in hyaline region.Tail length almost equivalent to cloacal body width.
Juveniles.Morphologically similar to adults in most respects except for size and development of reproductive system.All juvenile developmental stages were detected and distinguished by relative lengths of body and functional and replacement odontostyle (Figure 4).J1 characterized by a conoid tail, dorso-ventrally curved with rounded terminus, and slight depression at hyaline region level, with a c´ratio average of 2.3, odontostyle length ca.66.3 μm, and shorter distance from anterior end to stylet guiding-ring than that in adult stages.For the rest of the juvenile stages (J2, J3, J4), the replacement odontostyle were located at some distance posterior to the odontophore base and morphology of tail were similar to females (bluntly conoid with a rounded terminus, dorsally convex and ventrally almost straight or slightly concave), becoming stouter after each moult (Figure 3).
According to the body and odontostyle length, shape of amphidial fovea, distance of guiding ring from anterior body end, lip region and tail shape, a and c' ratios, and frequency of males, the new species is close to eight known Longidorus species, namely L. apulus; L. armeniacae; L. crassus; L. ferrisi Robbins et al., 2009;L. kheirii;L. pauli Lamberti et al., 1999;L.proximus; and L. soosanae.In addition, L. zanjanensis sp.nov. is closely related molecularly to L. hyrcanus; L. elongatus; and also L. soosanae.

Etymology
The specific epithet refers to the province of Zanjan, north-western Iran where the new species was collected.

Figure 4 .
Figure 4. Relationship between body length and functional and replacement odontostyle length in all developmental juvenile life stages and mature adults of Longidorus zanjanensis sp.nov.
n = number of specimens on which measurements are based; L = overall body length; a = body length/greatest body diameter; b = body length/distance from anterior end to pharyngo-intestinal junction; c = body length/tail length; c' = tail length/tail diameter at anus or cloaca; V = distance from body anterior end to vulva expressed as percentage (%) of the body length; T = distance from cloacal aperture to anterior end of testis expressed as percentage (%) of the body length.8M.Asgari et al.

Figure 6 .
Figure 6.Phylogenetic relationships of Longidorus zanjanensis sp.nov.within the genus Longidorus.Bayesian 50% majority rule consensus tree as inferred from ITS1 region sequence alignment under the GTR + G model.Posterior probabilities more than 0.70 are given for appropriate clades.Newly obtained sequences in this study are shown in boldface type, and coloured box indicates clade association of the new species.Scale bar = expected changes per site.

Figure 5 .
Figure 5. Phylogenetic relationships of Longidorus zanjanensis sp.nov.within the genus Longidorus.Bayesian 50% majority rule consensus tree as inferred from D2 and D3 expansion domains of 28S rRNA sequence alignment under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I+ G).Posterior probabilities more than 0.70 are given for appropriate clades.Newly obtained sequences in this study are shown in boldface type, and coloured box indicates clade association of the new species.Scale bar = expected changes per site.

Table 1 .
Morphometrics of Longidorus zanjanensis sp.nov.from Zanjan, Iran.All measurements are in μm (except L, in mm) and in the form: mean ± standard deviation (range)