Hemicycliophora ahvasiensis n. sp. (Nematoda: Hemicycliophoridae), and data on a known species, from Iran

Abstract Hemicycliophora ahvasiensis n. sp., recovered from the rhizospheric soil of date palm in Khuzestan province, southwest Iran, is described and illustrated based upon morphological, morphometric and molecular data. The new species is characterized by its sheath, closely fitting most of the body, cuticle with or without numerous irregular lines, sometimes appearing as blocks in distal body region. Lateral field without discrete longitudinal lines, but often with continuous broken striae or anastomoses. Continuous lip region with single annulus, slightly elevated labial disc, stylet with posteriorly sloping knobs. Vulva with or without slightly modified lips, spermatheca with sperm and tail conoid, symmetrically narrowing at distal region to form a narrow conical region. Morphologically, the new species looks similar to H. indica, H. labiata, H. siddiqii, H. tenuistriata and H. typica. The latter species appears more similar to the new species under light microscopy, but could be separated using the scanning electron microscopy and molecular data. The new species was also compared with H. epicharoides and H. dulli, two species with close phylogenetic affinities to it. The phylogenetic relationships of the new species were reconstructed and discussed using partial sequences of the D2-D3 expansion segments of large subunit, and internal transcribed spacer regions (LSU D2-D3 and ITS rDNA). Hemicycliophora conida, the second studied species, was recovered from north Iran and characterized by morphological and molecular data.

In their excellent contribution to the systematics of the superfamily Hemicycliophoroidea Skarbilovich, 1959 (Siddiqi, 1980), Chitambar and Subbotin (2014) reviewed the taxonomy of the genus Hemicycliophora (De Man, 1921) and updated data of the currently valid species. In the same year, Subbotin et al. (2014) addressed aspects of the pathogenicity of Hemi cycliophora species on associated host plants, the difficulties of morphological identifications due to morphological plasticity, and the lack of scanning electron microscopic (SEM) and molecular data.
Currently the genus contains 133 species (132 listed in Chitambar and Subbotin, 2014 and one in Maria et al., 2018).
There are 12 species of Hemicycliophora have been reported from different provinces in Iran. They are H. belemnis Germani & Luc, 1973, H. chilensis Brzeski, 1974 Thorne, 1955, H. iranica Loof, 1984, H. lutosa Loof & Heyns, 1969, H. megalodiscus Loof, 1984, H. poranga Monteiro & Lordello, 1978 ripa Van den Berg, 1981, H. sculpturata Loof, 1984, H. spinituberculata Loof, 1984, H. sturhani Loof, 1984 Reed & Jenkins, 1963. All of these species were characterized using traditional taxonomic methods (Eskandari, 2018). In an effort to document Hemicycliophora species occurring in Iran, two populations were recovered from soil samples obtained from different geographical locations in northern and southern regions. The preliminary morphological studies revealed the population recovered from south Iran resembled H. typica de Man, 1921 under light microscope (LM), but further studies using SEM and molecular data, and comparisons with all known species of the genus, revealed it to be an unknown species, described herein as H. ahvasiensis n. sp. The second species recovered from north Iran belonged to H. conida Thorne, 1955.

Nematode extraction and morphological observations
Several soil samples were collected from date palm and fruit tree gardens in Khuzestan and Gilan provinces, Iran. The relevant information of the presently studied nematode populations, and those included in phylogenetic analyses, are given in Table 1. Jenkins' method (Jenkins, 1964) was used to extract the nematodes from soil samples. The collected specimens were killed in hot 4% formaldehyde solution and transferred to anhydrous glycerin according to De Grisse (1969). Observations and measurements were conducted using a Leitz SM-LUX light microscope equipped with a drawing tube. Some of the specimens were photographed using an Olympus DP72 digital camera attached to an Olympus BX51 light microscope equipped with differential interference contrast (DIC).

Scanning electron microscopy (SEM)
Specimens preserved in glycerin were selected for observation according to Abolafia (2015). They were hydrated in distilled water, dehydrated in a graded mixture of ethanol-acetone series, critical pointdried with liquid carbon dioxide, and coated with gold. The mounts were examined with a Zeiss Merlin microscope (5 kV).

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 3 μ l 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. The suspension was collected by adding 20 μ l TE buffer. One DNA sample for the Gilan population and two DNA samples for the Khuzestan population were prepared in this manner. The DNA samples were stored at -20°C until used as a PCR template. 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 TW81 (5'-GTTTCCGTA GGTGAACCTGC-3') and reverse primer AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3') as described in Vovlas et al. (2008). The 25 μ l PCR mixture contained 14.5 μ l of distilled water, 3 μ l of 10 × PCR buffer, 0.5 μ l of 10 mM dNTP mixture, 1.5 μ l of 50 mM MgCl2, 1 μ l of each primer (10 pmol/μ l), 0.5 μ l of Taq DNA polymerase (Cinna Gen, Tehran, Iran, 5 U/μ l), and 3 μ l of DNA template. The thermal cycling program was as follows: denaturation at 95°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 40 s, and extension at 72°C for 80 s. A final extension was performed at 72°C for 10 min. Amplification success was evaluated by electrophoresis on 1% agarose gel (Aliramaji et al., , 2020. The PCR products were purified using the QIAquick PCR purification kit (Qiagen ® ) following the manufacturer's protocol and sequenced directly using the PCR primers with an ABI 3730XL sequencer (Bioneer Corporation, South Korea). The newly obtained sequences of the studied species were deposited into the GenBank database (accession numbers LSU D2-D3 MT901580/MT901581 and ITS rDNA MT901582 /MT901583 for the new species and MT901584 for ITS rDNA of H. conida, as indicated in Table 1).

Phylogenetic analyses
The newly obtained sequences of the D2-D3 fragments of LSU rDNA of the both populations, and the selected sequences from GenBank, were aligned by Clustal X2 (http://www.clustal.org/) using the default parameters. The ITS dataset was aligned using MUSCLE as implemented in MEGA6 (Tamura et al., 2013). The editing of both alignments was performed manually. The outgroup taxa were chosen according to previous studies Van den Berg et al., 2018;Maria et al., 2018). The base substitution model was selected using MrModeltest 2 (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 selected for the 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 two 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 the Bayesian framework were 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 higher than 0.50 are given on appropriate clades. The output files of the phylogenetic program was visualized using Dendroscope v3.2.8 (Huson and Scornavacca, 2012) and re-drawn in CorelDRAW software version 17.

Results
Systematics Hemicycliophora ahvasiensis n. sp. (Figures 1-4; Table 2). with spheroid sperm cells, vulva with not or slightly modified lips, vulval sleeve slightly elongate, one to two annuli long. Body portion behind vulva slightly narrowing towards distal region. Distance between vulva to anus about five anal body diam. Tail conoid, symmetrically narrowing at about 35% of its length at distal region to form a narrower conical section ending to a finely rounded to sharp terminus.

Male
Not found.

Juvenile
One juvenile specimen was found in the population that is similar to female except by a smaller body size and undeveloped sexual organs.

Type host and locality
This population was recovered from the rhizospheric soil of date palm (Phoenix dactylifera L.) collected from Ahvaz city in Khuzestan province, southwest Iran. The GPS information of the sampling site is 31°18´11.1˝N, 48°39´10.1˝E.

Etymology
The specific epithet of the new species refers to the original city name in Latin where it was discovered.

Type material
The holotype and 12 paratype females were deposited into the nematology laboratory of the Department of Plant Protection, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Three paratype females deposited at the Wageningen Nematode Collection (WaNeCo), Wageningen, The Netherlands. Two paratype females deposited at the Nematode Collection of the Department of Animal Biology, Plant Biology and Ecology of the University of Jaén, Jaén, Spain. The ZooBank Life Science Identifier (LSID) for this publication is as follows: http://zoobank.org/ urn:lsid:zoobank.org:pub:EEF9C9E9-90B8-4EC1-8BD9-A403FD8D58E4.

Diagnosis and relationships
Hemicycliophora ahvasiensis n. sp. is mainly characterized by a cuticle with or without longitudinal lines on annuli. Instead of lateral lines there may be broken or continuous striae or anastomoses on lateral sides of the body. The lip region is continuous with body contour and has a single annulus, slightly elevated labial disc, and plugged amphidial openings. Other characters include posteriorly sloping stylet knobs, vulva with or without slightly modified lips and short vulval sleeve, spermatheca full of sperm and conoid tail, symmetrically narrowing at about 35% of its length at the distal region to form a narrower conical region. The polytomous identification codes of the new species from Chitambar and Subbotin (2014) are: A4, B2, C3, D1, E1, F1, G23, H1, I12, J1, K23, L3, M2, N1, O1, P1, Q2, R2, S3, T1, U2, V1, W1, X1, Y-.
From H. labiata, by annuli with or without longitudinal lines (vs not), lateral field lacking line(s) (vs having one line), lip region with one annulus (vs two or three annuli), short vulval sleeve (vs moderately long) and body not constricted immediately posterior to vulva (vs constricted).
From H. typica, by cuticle lacking distinct blocks (vs having blocks), lateral field lacking line(s) (vs having two lines), body not constricted immediately posterior to vulva (vs constricted) and short vulval sleeve (vs moderately elongate).
From H. epicharoides Loof, 1968, a species with close phylogenetic affinities in both LSU and ITS phylogenies, by higher R, RV(ant) and respectively), lip region with one annulus (vs two or three annuli), lower St%L (7.5-8.4 vs 9-11), excretory pore located posterior to pharynx base (vs anterior or posterior) and tail symmetrically narrowing at about 35% of its length at distal region to form a narrower conical section (vs cylindroid anteriorly, mostly narrowing to a bluntly triangular or wedgeshaped posterior part).
From H. dulli Van den Berg & Tiedt, 2001, a species with close phylogenetic affinities in ITS phylogeny, by shorter stylet (63.3-71.0 vs 73-79 μ m), lower Rst (18-21 vs 21-25), lateral field lacking line(s) (vs having one or two lines), lip region continuous with one annulus (vs set off with two annuli), excretory pore located posterior to pharynx base (vs anterior or posterior) and vulva with not or slightly modified lips (vs vulval lips elongated).

Description
Female Body straight or ventrally arcuate. Cuticular sheath fitting closely to loosely to body. Lateral fields with two distinct longitudinal lines forming a band, with irregularities and breaks of striae or with anastomosis, an additional central line appears due to four ellipsoid markings on each annulus forming four blocks. Annuli outside lateral field with scratches. Labial region broad, anterior margins rounded, with two distinct annuli and elevated labial disc. Stylet long and slender, knobs posteriorly directed. Pharynx typical of the genus. Nerve ring encircling isthmus. Excretory pore, four annuli posterior and opposite to pharynx base. Reproductive system monodelphic-prodelphic, outstretched, spermatheca rounded to ovate, filled with spheroid sperm cells, vulval lips modified, vulval sleeve absent. Tail conical, symmetrically narrowing at distal region, tip rounded.

Host and locality
This population was recovered from the rhizospheric soil of pomegranate (Punica granatum L.) collected from Rasht city, Gilan province, in north Iran. The geographical position of the sampling site is N36°54´1.687˝, E49°28´37.923˝.

Remarks
H. conida was originally described by Thorne (1955) from a sugar beet field in Ireland. It was later reported from several countries . In the report of Loof (1984), the species was recovered from East Azarbaijan province of Iran. Males, however, were not recovered in this study. Later, the species was again recovered from Azarbaijan province, but no morphometric or morphological data were provided (Barooti, 1998). The presently recovered population agreed well with other populations of the species that have been reported from different regions, based upon the morphometric data and morphology . The spicules length in The Netherlands populations was measured as 18-29 μ m by Loof (1968)  , but it was calculated about 55 μ m after the drawings, which is in accordance with the presently studied population.

Molecular characterization and phylogenetic relationships
Two 673 and 682 nt long D2-D3 expansion segments of LSU (MT901580, MT901581), one from each female specimen, were generated for the new species. A BLAST search using these sequences revealed they have 99.34% identity with Hemicycliophora sp. 9 and Hemicycliophora sp. 13 (KF430509 and KF430508, respectively). The efforts to get the LSU Table 3. Morphometrics of Hemicycliophora conida Thorne, 1955 from Gilan province, Iran, and comparison with other population from East Azarbaijan province, Iran.

Reference
Present study Loof (1984) Province A total of 70 sequences of Hemicycliophora spp. and three sequences of Paratylenchus minutus Linford in Linford, Oliveira & Ishii, 1949, Trophotylenchulus floridensis Raski, 1957and Gracilacus bilineata Brzeski, 1995 as outgroup taxa (EF126180, JN112261 and EU247525, respectively) were selected for an ITS phylogeny. This dataset comprised 1164 total characters. The phylogenetic tree inferred using this dataset is presented in Figure 7. The major clade including the new species, also includes Hemicycliophora sp. 9 (KF430604, KF430605) that represents the putative closest relative of the new species, H. epicharoides (KF430606, KF430608) and H. labiata (MK305973, MK305974). The clade including two species H. typica (GQ406238, GQ406239, KF430603) and H. dulli (MT329671, MT329672) is in sister relation to the aforementioned clade. The ITS sequence of the Iranian isolate of H. conida formed a clade with two previously available sequences (KF430579, KF430580) of the species.

Discussion
The objectives of this study were to characterize one new and one known species of the genus Hemicycliophora from Iran. As common in reliable identifications of Hemicycliophora spp., the new species was studied using an integrative approach exploiting both morphological (including SEM) and molecular data .
The newly described species in present study appeared similar to H. typica under LM, however, the SEM and molecular data revealed they differ. Sequences of LSU D2-D3, and ITS rDNA sequences of H. ahvasiensis n. sp. differed from those of H. typica by 6 bp (1.4%) and 31 bp (1.5%), respectively. In the inferred phylogenies, it formed a subclade separate from H. typica and other Hemicycliophora species.
The new species was isolated from the rhizosphere of date palm tree, that is a major food source for local populations in the Middle East, and plays important roles in their culture and economy (Chao and Krueger, 2007). Additional study is required to clarify if the parasitism of high nematode populations of H. ahvasiensis n. sp. can cause damages to this plant.