Discopersicus hexagrammatus n. sp. (Rhabditida: Tylenchidae), the second species of the genus

Abstract Discopersicus hexagrammatus n. sp., is described and illustrated from a population associated with the rhizosphere of carrot (Daucus carota L.) in Dezful, Khuzestan province, south-western Iran. Based on morphological and morphometric data, the new species is characterized by a 601-734 µm long body, a prominent perioral labial disc and oblique amphidial slits, 10-11 µm long stylet, vulva at 65-67% of body length, 15.5-17 µm long spicules, and an elongate conoid tail with a pointed terminus. It is the second species of the genus Discopersicus and differs from its counterpart D. iranicus, by its anterior position of vulva, shorter stylet, lower M.B. ratio, different shape of tail tip, and shorter spicules in male specimens. A molecular phylogenetic analysis using the D2-D3 expansion segments of 28S rDNA sequences, placed the new species in close relationship with five sequences of the genus Boleodorus.

Khuzestan province is located in southwest of Iran, and with arid and warm climate, has an important role for production of agricultural products in Iran. A large number of nematodes have been described from this province (Azimi and Pedram, 2020;Eisvand et al., 2019;Ghaderi and Karegar, 2016;Hosseinvand et al., 2019Hosseinvand et al., , 2020aPanahandeh et al., 2019). Family Tylenchidae Örley, 1880 is one of the most plentiful and diverse nematode groups recovered in soil habitats, where they may represent up to 30% of the nematode abundance in any given soil sample . According to Karegar (2018), 115 species of this family have been reported from Iran that descriptions, morphometric data, and illustrations have been provided for 92 species of them. Afterward, 24 further species from four genera have been described (Hosseinvand, 2020). Hosseinipour (1992) found a population of the family Tylenchidae showing a perioral labial disc and slit-like amphidial apertures, obliquely placed on lateral sides of the head. The well-known nematologist, Dr. Etienne Geraert identified it as a new genus and tentatively named it as Discobasiria, but the relevant paper has never been published.  found two species of this genus in his M.S. thesis (Karegar, 2018). Ghaemi et al. (2012) introduced the new species Discotylenchus iranicus (Ghaemi et al., 2012) and described it with a minute amphidial aperture, appearing as longitudinal slits in lateral view. Yaghoubi et al. (2016) restudied this species and proposed the new genus Discopersicus, with D. iranicus (Yaghoubi et al., 2016) as its type species. In this study, we describe the second species of the genus based on morphological and molecular characters.

Materials and methods
Nematode sampling, extracting, mounting and morphological characterization Soil samples were collected from the rhizosphere of carrot (Daucus carota L.) in Dezful, Khuzestan province, southwestern Iran. Nematodes were extracted using the tray method (Whitehead and Hemming, 1965), killed and fixed by adding hot FPG (4:1:1, formaldehyde: propionic acid: glycerin), then transferred to anhydrous glycerin following the method of De Grisse (1969). To prepare nematodes for morphological observations, fixed specimens of the new species were handpicked under a Olympus SZH stereo microscope, and mounted in a small drop of pure glycerin supported with paraffin on permanent glass slides. Morphological characters were examined using a Leitz Dialux 22 light microscope. Morphometric characters and photographs were taken using a Dino-Eye digital eyepiece camera (Model AM7023, bundled with the DinoCapture 2.0 software; AnMo Electronics Corporation; New Taipei City; Taiwan) adjoined to the aforementioned microscope. Line drawings were first made using a drawing tube attached to the microscope, then redrawn and prepared for publication using CorelDRAW ® software version 16.

Scanning electron microscopy
For the scanning electron microscopy, specimens preserved in glycerin were selected for observation under SEM following the protocol by Abolafia (2015). The nematodes were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried with liquid carbon dioxide, mounted on SEM stubs, coated with gold, and observed with a Zeiss Merlin microscope (5 kv) (Zeiss, Oberkochen, Germany).

DNA extraction, PCR, and sequencing
Nematode DNA was extracted from single live female individuals of the new species, as described by Tanha Maafi et al. (2003), and used as template for polymerase chain reaction (PCR). The D2-D3 expansion segments of 28S rDNA were amplified using the forward D2A (5′-ACAAGTACC GTGAGGGAAAGTTG-3′) and reverse D3B (5′-TCG GAAGGAACCAGCTACTA-3′) primers (Nunn, 1992). Each PCR reaction mixure with a final volume of 30 μl, contained: 15 μ l Taq DNA Polymerase 2x Master Mix RED (Ampliqon, Denmark), 1 μ l (10 pmol μ l −1 ) of each forward and reverse primers, 2 μ l of DNA template and 11 μ l deionised water. Reactions were carried out in a Thermal Cycler (Hybaid, Ashford, Middlesex, UK) with an initial denaturation step of 95°C for 4 min followed by 33 denaturation cycles of 94°C for 30sec, annealing for 30 sec at 57°C, extension at 72°C for 90sec and a final extension at 72°C for 10 min. The quality of DNA targets were checked by electrophoresis of 4 μ l from each of PCR products in 1% agarose gel containing ethidium bromide. The PCR products were visualized and photographed under UV light and the length of each PCR product was measured by comparison with the Low DNA Mass Ladder (Invitrogen, Carlsbad, CA, USA). The PCR products were purified and sequenced directly for both strands using the same primers with an ABI 3730XL sequencer (Bioneer Corporation, Seoul, South Korea). The newly obtained sequences were submitted to GenBank database under ac cession numbers MW202233 and MW202234 as indicated on the 28S phylogenetic tree.

Phylogenetic analyses
Sequences of D2-D3 expansion segments of 28S rDNA of the new species and several representatives of the family Tylenchidae available in GenBank, were used for phylogenetic reconstruction. The newly obtained sequences were edited and aligned with another sequences available in GenBank using Muscle alignment tool implemented in the MEGA7 (Kumar et al., 2016). The ambiguously aligned parts and divergent regions were known using the online version of Gblocks 0.91b (Castresana, 2000) and were removed from the alignments using MEGA7. The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically selected using jModelTest 2.1.10 (Darriba et al., 2012). Phylogenetic tree was generated with Bayesian inference (BI) method using MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001;Ronquist et al., 2012). Atetylenchus longilabiatus (Hosseinvand et al., 2020a) (MN807620) and Psilenchus hilarulus (De Man, 1921) (EU915489) were chosen as outgroups for the tree. The analysis under general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G) model was initiated with a random starting tree and run with the Markov Chain Monte Carlo (MCMC) for 1 × 10 6 generations. The tree was visualized and saved with FigTree 1.4.3 (Rambaut, 2014) and edited with Adobe ® Acrobat ® XI Pro 11.0.1.

Type material
Holotype, five paratype females and four paratype males were deposited in the nematode collection of the Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran. Three paratype females and three paratype males were deposited in the nematode collection of the Department of Animal Biology, Plant Biology and Ecology of the University of Jaén, Spain.

Etymology
The species epithet, hexagrammatus, refers to the hexagram pattern of six labial papillae around the oral aperture.

Discussion
Our 28S rDNA tree supported the position of Discopersicus in the subfamily Boleodorinae (Khan, 1964) in close affinity with the genera Basiria and Boleodorus. The monophyly of the subfamily Boleodorinae was confirmed as concluded in former researches (Eisvand et al., 2019;Hosseinvand et al., 2020a, b, c;Panahandeh et al., 2019;Yaghoubi et al., 2016). The new species formed a strongly supported clade with Boleodorus spp., while, in Yaghoubi et al. (2016) study, D. iranicus was placed inside Neopsilenchus spp. According to the shape of sper matheca in the subfamily Boleodorinae (except for two isolates of N. magnidens MK639379 and MK639380 that they have different spermatheca from other members), we could observe two major clades A and B. The clade A containing species with nonoffset spermatheca except D. iranicus, and the clade B including species with offset spermatheca. Adding the sequences of species having offset spermatheca from the genus Basiria can be used for testing this result. Figure 4: Bayesian 50% majority rule consensus tree inferred from D2-D3 expansion segments of 28S rDNA sequences of Discopersicus hexagrammatus n. sp. under the GTR + I + G model. Posterior probabilities more than 50% are given for appropriate clades. Newly obtained sequences in this study are in bold letters.