Deladenus bonabensis n. sp. (Tylenchomorpha: Neotylenchidae) from East Azarbaijan province, northwestern Iran: A morphological and molecular phylogenetic study

Abstract A population of Deladenus, representing a new species, was recovered from Bonab’s Ghara-Gheshlagh lagoon. It is mainly characterized by having a long body (1051–1185 μm), long distance of anterior end to pharyngeal glands end (270–312 μm), six lines in lateral fields, and a short mucro-like differentiation on the tail tip. Furthermore, it has a small-sized stylet (8.5–11.0 μm) with three knobs, no postvulval uterine sac, and males with 24- to 28-μm-long tylenchoid spicules and penial tube. With six lateral lines, the new species is comparable with seven species of the genus: D. apopkaetus, D. brevis, D. cocophilus, D. durus, D. obtusicaudatus, D. processus, and D. ulani. It was furthermore compared with D. oryzae with an unknown number of lateral lines and D. aridus, D. obesus, and D. parvus having a different number of lateral lines but similar morphology. In the molecular phylogenic analyses using small and large subunit ribosomal DNA (SSU and LSU D2-D3 rDNA) sequences, the relationships of the new species with other species and genera were not resolved in SSU phylogeny. However, it formed a clade with Deladenus sp. and D. brevis in LSU phylogeny.

The genus Deladenus was established by Thorne (1941), describing D. durus (Cobb, 1922) Thorne, 1941 as its type species. It was placed in the subfamily Neotylenchinae Thorne, 1941 (it is now under the family Neotylenchidae Thorne, 1941sensu Siddiqi, 2000. Compared to other genera in the family Neotylenchidae, it is characterized by low lip reign, small stylet, anteriorly located pharyngo-intestinal junction to the nerve ring, secretory-excretory pore (S-E pore) at the level or slightly behind the nerve ring, no median bulb in pharynx, long dorsal overlap of pharyngeal glands over intestine, no postvulval uterine sac (PUS), and males with bursa. The genus has two free-living mycetophagous and entomoparasitic generations (Siddiqi, 2000). The free-living phase has a typical neotylenchoid life cycle, and the entomoparasitic phase has a typical sphaerularid cycle (Miraeiz et al., 2017). Currently, around 14 species of the genus are known by their mycetophagous phase. The list of valid species of the genus has already been given by several authors (Siddiqi, 2000;Miraeiz et al., 2017;Yu et al., 2017;Morris et al., 2018). The two species D. gilanica Esmaeili, Jalalinasab, Ye, & Heydari, 2020 and D. brevis Heydari, Abolafia, & Pedram, 2020 are the most recent species added to the genus (Esmaeili et al., 2020;Heydari et al., 2020).

Materials and Methods
Soil sampling, nematode extraction, and morphological identification Several soil samples were collected from different parts of the city of Bonab, East Azarbaijan province, northwestern Iran. The tray method (Whitehead and Hemming, 1965) was employed to extract nematodes from soil samples. The nematodes of interest were hand-picked under a Nikon SMZ1000 stereomicroscope, heat-killed by adding boiling 4% formaldehyde solution, transferred to anhydrous glycerin (De Grisse, 1969), mounted on permanent slides, and examined using a Nikon Eclipse E600 light microscope. Photographs were taken using an Olympus DP72 digital camera attached to an Olympus BX51 microscope powered with differential interference contrast. Drawings were made using a drawing tube attached to the microscope and were redrawn using CorelDRAW ® software version 16.

DNA extraction, PCR, and sequencing
A single nematode specimen of the new species was picked out and transferred to a small drop of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0, Qiagen) on a clean slide and squashed using a clean coverslip. The suspension was collected by adding 35 μl of TE buffer. DNA sample was stored at −20°C until used as PCR template. Primers for partial amplification of small subunit (SSU) rDNA were forward primer F22 (5´-TCCAAGGAAGGCAGCAGGC-3´) (Dorris et al., 2002) and reverse primer 1573R (5´-TACAAAGGGCAGGGACGTAAT-3´) (Mullin et al., 2005). The large subunit (LSU) rDNA D2-D3 expansion segments were amplified using the forward D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and reverse D3B (5´-TCGGAAGGAACCAGCTACTA-3´) primer pairs (Nunn, 1992). The PCR products were sequenced in both directions using the same primers used in PCR with an ABI3730XL sequencer. The newly obtained sequences were deposited into the GenBank database under the accession numbers OP250137 for SSU and OP250136 for LSU rDNA sequences.
The newly obtained sequences were compared with those of other nematode species available in GenBank using the BLAST homology search program. For reconstruction of the phylogenetic relationships, two independent SSU and LSU data sets were prepared. The selected SSU sequences were aligned using the Q-INS-i algorithm of the online version of MAFFT (version 0.91b) (see http://mafft. cbrc.jp/aligment/seaver/; Katoh and Standley, 2013). Clustal X2 was used to align the LSU sequences (Larkin et al., 2007). The poorly aligned positions and divergent regions of SSU and LSU data sets were eliminated using Gblocks (see http://phylogeny. lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks) and selecting all three less stringent options. The model of base substitution was selected using MrModeltest.2 (Nylander, 2004). The Bayesian analyses were performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) running the chains for 5 million generations for both data sets. After discarding burn-in samples, the remaining samples were retained for further analyses. The Markov chain Monte Carlo method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees using the 50% consensus majority rule (Larget and Simon, 1999). Adequacy of the posterior sample size was evaluated using autocorrelation statistics as implemented in Tracer v1.6 (Rambaut and Drummond, 2009). The species Acrobeloides maximus Thorne, 1925, Acrobeles ciliatus von Linstow, 1877, and Pseudacrobels sp. were used as outgroup species in SSU; and Poikilolaimus oxycerca de Man, 1895, P. piniperdae Fuchs, 1930, andOscheius myriophilus Poinar, 1986 were used as outgroups in LSU phylogenies. 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 16.
Parasitic female: A parasitic female specimen was not found.
Free-living mycetophagus male: These were present as frequently as females. General morphology spermatheca oblong-oval, filled with large sperm cells, crustoformeria with more than four cells in each row, vagina modernity sclerotized, vulva with elevated lips, no PUS, and no vulval flap. The tail is conical, uniformly narrowing to the end, with a small mucro-like differentiation at the tip.  and pharynx are similar to those of females. The reproductive system is monorchic. Spicules are tylenchiod. Gubernaculum is small and thin. A penial tube is present. Bursa well developed, enveloping the entire tail.

Type habitat and locality
Specimens were recovered from a soil sample collected in Ghara-Gheshlagh lagoon, Bonab, East Azarbaijan province, northwestern Iran, in December 2019. The Global Positioning System coordinate for the locality is 37°12´29˝N 45°57´57˝E.

Etymology
The specific name refers to the city of Bonab, where the new species was recovered.

Type material
Holotype female, four paratype females, and four paratype males were deposited at the WaNeCo nematode collection of the Wageningen University, The Netherlands. Four paratype females and four paratype males were deposited at the nematode collection of Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.

Diagnosis and relationships
Deladenus bonabensis n. sp. is mainly delimited by 1051-to 1185-µm-long females, with six lines in the lateral fields, vulva with no lateral flaps, conical tail, and penial tube present. It is further characterized by a stylet 8.5-to 11.0-µm-long with three basal knobs. By having six lateral lines, the new species is comparable to seven species of the genus, namely D. apopkaetus Chitambar, 1991;D. brevis;D. cocophilus Nasira, Shahina & Firoza, 2013;D. durus;D. obtusicaudatus Bajaj, 2015;D. processus Tomar, Somvanshi & Bajaj, 2015;and D. ulani Sultanalieva, 1983. It was further compared with D. oryzae Bajaj, 2015, with an unknown number of lateral lines, and D. aridus Andrássy, 1957;D. obesus Thorne, 1941;and D. parvus Zell, 1985, having a different number of lateral lines, but similar morphology. The comparisons with the aforementioned species follow.
It is distinct from D. durus (data according to Chitambar, 1991) by six vs. six or seven lines in lateral field, absence of pharyngeal chamber (vs. presence), weakly deveopled metacorpus (vs. developed) and S-E pore far from hemizonid (vs. close).
Deladenus bonabensis n. sp. is distinguished from D. obtusicaudatus mainly by its tail shape (conical, having a mucro-like differentiation at the tip vs. short and cylindrical with a broadly rounded to truncated terminus), S-E pore posterior to hemizonid (vs. anterior), and longer females (1051-1185 vs. 730 µm).

Molecular profiles and phylogenetic status
Sequencing of SSU and LSU rDNA D2-D3 fragments of the new species yielded a single partial SSU 908 nucleotides long, and an LSU D2-D3 sequence 674 nucleotides long. The BLAST search using the  SSU sequence revealed that it has 98.79% (three mismatches) and 98.68% (12 mismatches and nine gaps) identity with two sequences assigned to Tylenchidae sp. (LC382046 and LC382045, respectively). Its identity with all other SSU sequences was less than 98%. The BLAST search using the LSU sequence revealed it has a 99% (six mismatches and two gaps) identity with a sequence assigned to Sphaerularioidea sp. (MW577121). The identity with currently available LSU sequences of relevant genera was less than 90%.
Seventy-one sequences (including the newly generated sequence of the new species and three sequences of classic rhabditids as outgroups) were used in SSU phylogeny (species names and accession numbers are available in the SSU tree). Their alignment included 1,761 characters, 658 of which were variable. Fig. 3 represents the phylogenetic tree reconstructed using this data set. The sequences of Deladenus species occupied different placements in this tree and the relationships of the newly generated SSU sequence of the new species with other sequences were not resolved due to polytomy.
Seventy sequences (including the newly generated sequence of the new species and three sequences of classic rhabditids as outgroups) were used in LSU phylogeny (species names and accession numbers are available in the LSU tree). The LSU alignment was composed of 1,023 total characters, of which 796 were variable. Fig. 4 represents the phylogenetic tree inferred using this data set. In this tree, the LSU sequences of Deladenus spp. have occupied different placements in the tree, and the the new species has formed a clade with an unidentified sequence (Sphaerularioidea sp., MW577121). The former clade is in sister relation with the LSU sequence of D. brevis (MT009494).

Discussion
During this study, a population of the genus Deladenus was recovered from Ghara-Gheshlagh lagoon, Bonab, northwestern Iran. Previously, five species of this genus have been described or reported from Iran (Jahanshahi Afshar et al., 2014;Miraeiz et al., 2017;Esmaeili et al., 2020;Heydari et al., 2020;Jalalinasab et al., 2020). The currently described new species could be conspecific with the previously sequenced species recovered from West Azarbaijan province, of which only three specimens were found (the isolate west.az) and sequenced in our lab (LSU accession numbers MW577121 and MW577120).
Two life cycles including insect parasitic phase and free-living mycetophagous phase are known for Deladenus (Siddiqi, 2000). The former species originally added to the genus from Iran were described using their free-living; and in case of D. gilanica, two free-living and infective stages. This was the case for the new species, too. A penial tube was observed in males of the new species. This structure is seen in light microphotographs of Deladenus nitobei Kanzaki, Tanaka Fitza, Kosaka, Slippers, Kimura, Tsuchiya & Tabata, 2016 as well.
Based on available data, it seems that the genus is not monophylic in both SSU and LSU phylogenies, which is in agreement with previous studies (e.g., Heydari et al., 2020).