Morphological and Molecular Characterization of Tylenchorhynchus Clarus Allen, 1955 and T. Zeae Sethi & Swarup, 1968 (Rhabditida: Telotylenchidae) from Iraq

Abstract During a survey on the biodiversity of plant-parasitic nematodes in Misan province (southeast Iraq), Tylenchorhynchus clarus and T. zeae were discovered around the rhizosphere of sugarcane and pumpkin, respectively. The morphological and morphometric data were provided for the recovered species. The morphological characters of both populations are in agreement with the type populations and other populations of them. To the best of our knowledge, this is the first report of these two species from Iraq, and a first report of the association of T. zeae with pumpkin. Molecular phylogenetic analyses of the Iraqi populations of T. clarus and T. zeae using the D2–D3 expansion segments of 28S rDNA and internal transcribed spacer (ITS) rDNA sequences using Bayesian inference (BI), showed they form maximally supported clades with other sequences of both species.

The cosmopolitan nematode genus Tylenchorhynchus Cobb, 1913, is one of specious genera. Its representatives are migratory ectoparasites of a wide range of plant species (Siddiqi, 2000). It is morphologically similar to the genus Bitylenchus Filipjev, 1934. According to Gómez- Barcina et al. (1992), genus Bitylenchus is differentiated from Tylenchorhynchus in having areolated outer bands of lateral fields, a large post-anal intestinal sac containing intestinal granules and fasciculi, relatively more thickened cuticle at the female tail tip, and gubernaculum lacking a crest. These characters are described for several species belonging to both Tylenchorhyncus and Bitylenchus (Handoo, 2000). Some nematologists considered Bitylenchus as a junior synonym of Tylenchorhynchus. Siddiqi (2000) listed 105 valid species under the genus Tylenchorhynchus. Geraert (2011) considered Bitylenchus as a junior synonym of Tylenchorhynchus and placed 133 species under the genus. Subsequently, T. bambusi Singh, Lal, Rathour & Ganguly, 2010, T. mediterraneus Handoo, Palomares-Rius, Cantalapiedra-Navarrete, Liébanas, Subbotin & Castillo, 2014, and T. iranensis Azimi, Mahdikhani-Moghadam, Rouhani & Rajabi Memari, 2016 were described from India, Spain, and Iran, respectively. In studies on integrative taxonomy of the genera Bitylenchus and Tylenchorhynchus, these two genera were separated from each other and considered as valid genera (Ghaderi et al., 2014;Handoo et al., 2014;Hosseinvand et al., 2020;Azizi et al., 2022). Stephan et al. (1985) have reported 20 genera and 43 species of plant-parasitic nematodes from vineyard soils of Iraq. Morphological and morphometric data were however not provided for any of the reported species in previous study. Tylenchorhynchus brassicae Siddiqi, 1961 andT. mashhoodi Siddiqi andBasir, 1959 are two species of the genus Tylenchorhynchus that were included in this report.
During a survey on the plant-parasitic nematodes of the Misan province, southeast Iraq, T. clarus Allen, 1955 andT. zeae Sethi andSwarup, 1968 were recovered. According to published literature, this is the first report of these two species from Iraq. Thus, the present study aims to characterize the Iraqi populations of the two recovered species based on morphological and morphometric characteristics. Furthermore, molecular data of two D2-D3 expansion segments of 28S rDNA and ITS rDNA were used to study the phylogenetic relationships of the recovered species with other relevant species and genera.

Nematode extraction and morphological observations
Several soil samples were collected from the rhizosphere of sugarcane (Saccharum officinarum L.) and pumpkin (Cucurbita moschate L.) in Misan province, Iraq. The centrifugal flotation technique (Jenkins, 1964) and the tray method (Whitehead and Hemming, 1965) were used to extract the nematodes from soil samples. The collected specimens were killed in a hot 4% formaldehyde solution and transferred to anhydrous glycerin according to the method discussed in the study of De Grisse (1969). Observations and measurements were conducted using a Leitz SM-LUX light microscope (Leitz Corporation, wetzlar, Germany) equipped with a drawing tube. Some of the specimens were photographed using an Olympus Corporation, Tokyo, Japan DP12 digital camera attached to an Olympus BX51 light microscope (Olympus Corporation, Tokyo, Japan).

Taxonomic framework
The taxonomic framework proposed by Siddiqi (2000) was followed in this study, which considered Bitylenchus, Sauertylenchus Sher, 1974, and Tylenchorhynchus as valid genera.

DNA extraction, PCR and sequencing
For molecular analyses, single female specimens were picked out, examined in a drop of distilled water on a temporary slide under the light microscope, transferred to 5 µl of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) on a clean slide, and then crushed using a cover slip. Each suspension was collected by adding 15 µl TE buffer. The DNA samples were stored at -20°C until they were used as PCR templates. Primers for LSU rDNA D2-D3 amplification were forward primer D2A (5'-ACAAGTACCGTGAGGGAAAGT-3') and reverse primer D3B (5'-TCGGAAGGAACCAGCTACTA-3') (Nunn, 1992). Primers for amplification of ITS rDNA were forward primer rDNA1 (5'-TTGATTACGTCCCTGCCCTTT-3') and reverse primer rDNA1.58S (5'-ACGAGCCGAGTGATCCACCG-3') (Subbotin et al., 2000). The 30 µl PCR mixture contained 10 µl of distilled water, 15 µl of Master Mix 2X (Ampliqon, Odenese, Denmark), 1 µl of each primer (10 pmol/µl), and 3 µl of DNA template. The thermal cycling program for amplification of both markers was as follows: denaturation at 95°C for 6 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52.5°C (LSU D2-D3 primers) / 54.7°C (ITS rDNA primers) for 30 s, and extension at 72°C for 60 s. A final extension was performed at 72°C for 10 min. Amplification success was evaluated by electrophoresis on 1% agarose gel. The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) following the manufacturer's protocol and sequenced directly using the PCR primers with an ABI 3730XL sequencer (Bioneer Corporation, Daejeon, South Korea). The newly obtained sequences of the studied species were deposited into the GenBank database (accession numbers ON651683, ON651684, ON651685, ON651686 for LSU D2-D3, and ON667938, ON667939 for ITS rDNA).

Phylogenetic analyses
The newly obtained sequences of the D2-D3 fragments of LSU rDNA and ITS rDNA and additional sequences of relevant species were selected after the nucleotide basic local alignment search tool (BLASTn, National Center for Biotechnology Information, Bethesda, Maryland, United States) search. The sequences were aligned by Clustal X version 2 using the default parameters (Larkin et al., 2007). The outgroup taxa were chosen according to a previous study (Abdolkhani and Azimi, 2021). The editing of both alignments was performed manually in the MEGA7 program (Kumar et al., 2016). The base substitution model was selected using MrModeltest2 (Nylander, 2004) based on the Akaike information criteria. A general time reversible model, including among-site rate heterogeneity and estimates of invariant sites (GTR + G + I), was used in both phylogenies. The Bayesian analysis was performed to infer the phylogenetic trees using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), running the chains for four million generations. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to determine equilibrium distribution and help estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) using the 50% majority rule. Bayesian posterior probability (BPP) values >0.50 are given on appropriate clades. The output files of the phylogenetic program were visualized using Dendroscope v3.2.8 (Huson and Scornavacca, 2012) and were digitally drawn in CorelDRAW software version 17 (Corel Corporation, Ottawa, Canada).

Description
Female Body arcuate ventrally to open C shaped after heat fixation. Cuticle annuli 1.6-2.3 µm wide at mid-body. Lateral field 6.0-7.8 μm wide, with four incisures along the body, outer lines slightly crenate. Lip region continuous to slightly offset from the body, bearing four to five annuli, cephalic framework weak. Stylet about 2.5 times lip region width, knobs rounded, their anterior surfaces flattened to slightly concave, 3-4 µm across. Median pharyngeal bulb elliptical, basal bulb elongate pyriform, overlapping intestine for 2-3 µm in most specimens. Hemizonid usually one to three annuli anterior to excretory pore. Cardia well developed. Intestinal fasciculi present in the intestinal region. Post-anal intestinal sac absent. Reproductive system didelphic-amphidelphic, both genital branches almost equally developed, ovaries straight, vagina about half corresponding body diameter long, vulva a transverse slit, spermatheca underdeveloped. Tail conoid, its terminus smooth, slightly narrowing, hyaline portion 3.5-5.0 µm thick. Phasmids 8-12 μm behind the anus.

Male
Not found.

Remarks
The general morphology of the recovered population of the species closely resembles that of the type population (Allen, 1955) and other populations from Jordan (Hashim, 1983), southern Spain (Castillo et al., 1991), Spain and USA (Handoo et al., 2014), and Iran (Ghaderi and Karegar, 2016). Compared with the Egyptian population reported by Elmiligy (1969), the body length is shorter (414-523 vs 565-610 µm) and the number of tail annuli is less (14-16 vs 19-20 µm). Compared to the data given by Geraert (2011), no remarkable differences were observed. The presently studied species was recovered from the rhizosphere of sugarcane in Misan province, southeast Iraq (GPS coordinates: 31°35'22.48"N, 47°12'31.98"E). The present study is the first to report T. clarus from Iraq.

Description Female
Body arcuate ventrally after heat fixation. Cuticle annuli 1.1-1.4 µm wide at mid-body. Lateral field with four incisures along the body, outer lines slightly crenate. Lip region slightly offset from the body, bearing four annuli, cephalic framework slightly sclerotized. Stylet about six times length of lip region, knobs rounded, their anterior surfaces flattened, 3.1-3.7 µm across. Median pharyngeal bulb elliptical, basal bulb pyriform to slightly cylindrical. Excretory pore about at level with the pharyngeal bulb. Cardia distinct. Post-anal intestinal sac absent. Reproductive system didelphicamphidelphic, both genital branches almost equally developed, ovaries straight, spermatheca rounded, filled with rounded sperm, vagina about half body diameter long, vulva a transverse slit. Tail conoid to sub-cylindrical, its terminus widely rounded, smooth, hyaline portion 2.5-3.6 µm. Phasmids in the middle of the tail.

Male
General morphology is similar to that of female except for character states associated with sexual differences. Tail conoid, its tip pointed, enveloped with bursa. Spicules slightly curved ventrally. Gubernaculum well developed, about half the length of the spicules. Bursa 36.8-51.9 μm long.

Remarks
The general morphology of the recovered population of the species closely resembles that of the type population (Sethi and Swarup, 1968). However, the length of the tail is shorter (25.2-38.2 vs 46 µm). This species has already been reported from Spain (Handoo et al., 2014), Iran (Alvani et al., 2017), and China (Xu et al., 2020). There is no significant difference between these populations and the population recovered from Misan province (southeast Iraq) as part of the current study. Compared to the morphometric data ranges given by Geraert (2011), the Iraqi population has a slightly shorter tail (25.2-38.2 vs 46 mm).
The presently studied species was recovered from the rhizosphere of pumpkin in Misan province, southeast Iraq (GPS coordinates: 32°07'56.81"N, 46°43'05.75"E). The present study is the first to report the discovery of T. zeae in Iraq, and additionally represents the first report of its association with pumpkin.

D2-D3 fragments of 28S rDNA phylogeny
Three 718 nt long D2-D3 expansion segments of LSU rDNA (ON651683, ON651684, ON651685) were obtained for the Iraqi population of T. clarus. The BLAST search using these sequences revealed that they have 99.58% identity with another sequence of the same species (KJ461536). The sequence variation between the Iraqi population and this sequence amounted to five mismatches and one gap.
A 692 nt long partial sequence of the D2-D3 region with accession number ON651686 was obtained for the Iraqi population of T. zeae. The BLAST search using this sequence revealed that it has 99.86% identity with another sequence of the same species (KJ461565). The sequence variation between the Iraqi population and this sequence amounted to one mismatch.
A total of 74 sequences of Tylenchoidea Örley, 1880 and two sequences of Aphelenchoidea (Fuchs, 1937) Thorne, 1949 as outgroup taxa (DQ328683 and JX683686) were selected for the LSU phylogeny. This dataset comprised 797 total characters. The phylogenetic tree inferred using this dataset is presented in Figure 3. The newly generated sequences of the Iraqi populations of Tylenchorhynchus clarus and T. zeae have formed a maximally supported clade with other sequences of both species in this tree.

Partial ITS rDNA phylogeny
The sequencing of the ITS rDNA of the Iraqi population of Tylenchorhynchus clarus yielded one fragment with a 662 nt long sequence (ON667938). The BLAST search using this sequence revealed it has 99.80% identity (one mismatch and no gap) with another ITS sequence of the species (KJ461575).
A newly obtained 649 nt long partial sequence of the ITS rDNA with accession number ON667939 was generated for the Iraqi population of T. zeae. The BLAST search using this sequence revealed it has 99.69% identity with another ITS sequence of the species (MN757910). The sequence variation between the Iraqi population and this sequence amounted to two mismatches and no gap.
A total of 46 sequences of Tylenchoidea and two sequences of Aphelenchoidea as outgroup taxa (JX683685 and KX856336) were selected for ITS phylogeny. This dataset comprised 1,285 total characters. The phylogenetic tree inferred using this dataset is presented in Figure 4. The sequences of the Iraqi populations of Tylenchorhynchus clarus and T. zeae formed a maximally supported clade with other sequences of both species in this tree.  Allen, 1955 and T. zeae Sethi and Swarup, 1968 under the GTR + G + I model. Bayesian posterior probability values of >0.50 are given for appropriate clades. New sequences are indicated in bold.

Discussion
The objectives of this study were the morphological and molecular characterization of recovered populations of Tylenchorhynchus clarus and T. zeae from Iraq. In general, there are currently poor data on diversity of plant-parasitic nematodes in Iraq, and both reported species represent new records for the country.
Phylogenetic analyses and the subsequent alternative hypothesis tests based on the 28S rDNA by Handoo et al. (2014) showed the monophyly of the genus Tylenchorhynchus sensu Siddiqi (2000), although a representative of the genus Telotylenchus Siddiqi, 1960 was embedded inside the clade of Tylenchorhynchus species. Similar results have been achieved in some recent molecular phylogenies (Azimi et al., 2016;Abdolkhani and Azimi, 2021;Azizi et al., 2022). In the present 28S phylogeny, most of the included Tylenchorhynchus species + Telotylenchus sp. formed a clade and Tylenchorhynchus mediterraneus occupied a distant placement. This situation showed that the genus Tylenchorhynchus is polyphyletic.
The alternative hypothesis test by Handoo et al. (2014) using the ITS rDNA marker again corroborated Figure 4: Bayesian 50% majority rule consensus tree inferred from analysis of the ITS rDNA of Iraqi populations of Tylenchorhynchus clarus Allen, 1950 andT. zeae Sethi andSwarup, 1968 under the GTR + G + I model. Bayesian posterior probability values of >0.50 are given for appropriate clades. New sequences are indicated in bold. the monophyly of the genus Tylenchorhynchus. In their original ITS tree, T. mediterraneus had occupied a distant placement. In the present study, the Tylenchorhynchus species were separated into different clades and T. mediterraneus formed a separate clade with other genera. This result represents that the genus is not monophyletic. Similar results were obtained in some recent phylogenies (Abdolkhani and Azimi, 2021;Sheikhzadeh et al., 2022).
A molecular phylogenetic study using the higher number of species of the closely related genera like Bitylenchus, Paratrophurus Arias, 1970, Sauertylenchus, and Tylenchorhynchus could result in a better phylogeny of these genera in future.