Morphological and Molecular Characterization of Ottolenchus isfahanicus n. sp. (Tylenchomorpha: Tylenchoidea) from Rhizosphere of Gramineous Plants in Isfahan Province, Iran

Soil sampling, nematode extraction and morphological characterization: 180 soil samples were collected from the grasslands of Isfahan province, Iran, between 2019-2022. The samples were placed in plastic bags, transferred to the nematology laboratory of Tarbiat Modares University, and maintained at cool temperature conditions. Nematodes were extracted from soil samples using the tray method (Whitehead and Hemming, 1965); handpicked under a Nikon SMZ1000 (Nikon, Tokyo, Japan) dissecting microscope; heat-killed by adding boiling, 4% formaldehyde solution; and transferred to anhydrous glycerin according to de Grisse (1969). Drawings and morphological studies were performed using a drawing tube attached to a Nikon E600 light microscope, and digital drawings were completed using CorelDraw software version 2020. The light microphotographs of the fresh individuals and mounted specimens were prepared using an Olympus BX51 microscope equipped with a digital DP72 camera (Olympus) and differential interference contrast (DIC) optics. Siddiqi (2000) and original descriptions of Ottolenchus spp. were used as the taxonomic framework and morphological comparisons resources, respectively.

Scanning electron microscopy (SEM): Few specimens preserved in glycerine were selected for SEM observation according to Abolafia (2015). The nematodes were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).

DNA extraction, polymerase chain reaction (PCR) and sequencing: Seven specimens were examined on temporary slides to confirm their identity, and then individual DNA samples from each specimen were extracted in 15 μl of TE buffer (10mM Tris-Cl, 0.5mM EDTA; pH 9.0). DNA samples were stored at -20 °C until they could be used as PCR templates. The SSU rDNA was amplified using two pairs of primers which yielded overlapping fragments: forward 1096F (5´-GGTAATTCTGGAGCTAATAC-3´) and reverse 1912R (5´-TTTACGGTCAGAACTAGGG-3´) primers to amplify the first fragment, and forward 1813F (5´-CTGCGTGAGAGGTGAAAT-3´) and reverse 2646R (5´-GCTACCTTGTTACGACTTTT-3´) primers to amplify the second fragment (Holterman et al., 2006). Primers for the LSU D2-D3 amplification were the forward primer D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and the reverse primer D3B (5´-TCGGAAGGAACCAGCTA CTA-3´) (Nunn, 1992). The PCR mixture (35 ml) contained the following components: 17 μl Taq DNA Polymerase 2× Master Mix RED, 2-mM MgCl2 (Ampliqon, Odenese, Denmark), 10 μl distilled water, 1.5 μl of each primer, and 5 μl of DNA template. The thermocycling program was as follows: denaturation at 95°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 52°C for 40 sec, and extension at 72°C for 80 sec. A final extension was performed at 72°C for 10min. The PCR products were sequenced with a DNA multicapillary sequencer (Model 3130XL Genetic Analyzer; Applied Biosystems, Foster City, CA, USA), using the BigDye Terminator Sequencing Kit v.3.1 (Applied Bio-systems) at the Stab Vida sequencing facility (Caparica, Portugal) with the same primers used for amplification, and deposited into the GenBank database under the accession number OP972577 for SSU, and the accession numbers OQ025275, OQ025276 and OQ025277 for LSU rDNA D2-D3.

Phylogenetic analyses: The chromatogram files of the newly generated sequences were checked using Chromas Lite 2.1.1 (https://technelysium.com.au/wp/chromas/), edited, trimmed, and assembled manually. The SSU sequence of Ottolenchus isfahanicus n. sp. was compared with other available sequences in the GenBank database using the basic local alignment search tool (BLAST). Relevant sequences (mostly belonging to the subfamily Tylenchinae) used in SSU phylogeny in previous studies (Monemi et al., 2022) were selected for inferring the SSU tree. Two sequences of Aphelenchoidea Fuchs, 1937, namely Bursaphelenchus mucronatus Mamiya & Enda, 1979 (AY284648) and Aphelenchoides fragariae Ritzema Bos, 1890 (AY284645), were selected as outgroups. The LSU sequences of Ottolenchus isfahanicus n. sp. were compared with other available sequences in the GenBank database using BLAST homology search program. The maximal number of relevant sequences as already used in our previous studies (Monemi et al., 2022; Mortazavi et al., 2021) were included in LSU phylogeny.

The SSU and LSU datasets were aligned using the Q-INS-i algorithm of the online version of MAFFT (version 0.91b) (https://mafft.cbrc.jp/alignment/server) and the resulting alignments were edited manually using MEGA6 (Tamura et al., 2013). The best-fit model of nucleotide substitution was selected using PAUP*/MrModeltest.2 (Nylander, 2004). The Akaike-supported model, a general time-reversible (GTR) model including among-site rate heterogeneity and estimates of invariant sites (GTR + gamma [G] + invariant [I]), was selected and used in both phylogenies. Bayesian analyses were performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) by running the chains for 5 × 10⁶ generations. After discarding burn-in samples, the remaining samples were retained for further analyses. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) using the 50% majority rule. The convergence of model parameters and topology was assessed based on an average standard deviation of split frequencies and potential scales reduction factor values. Adequacy of the posterior sample size was evaluated using autocorrelation statistics as implemented in tracer v.1.6 (Rambaut and Drummond, 2009). The output files of the trees were visualized using Dendroscope v3.2.8 (Huson and Scornavacca, 2012) and digitally drawn in CorelDRAW software version 2020.

The genus Ottolenchus is now a valid and well-established genus (Qing and Bert, 2017; Hosseinvand et al., 2021). It is paraphyletic in SSU, and monophyletic in LSU phylogenies, after recent studies based on available sequences of its representatives (Hosseinvand et al., 2021). On the other hand, currently only few representatives of Ottolenchus have been sequenced and further information will better depict its phylogenetic nature in the future.

In the present study, the new species was compared with the original description of the morphologically closest species. The morphometrics of most species of Tylenchidae have been expanded following numerous reports (Geraert, 2008); however, most of the original and subsequent reports lack molecular sequences, making the identification of most of the species questionable. Monemi et al. (2022) stated that most of the sequences assigned to Boleodorus thylactus Thorne, 1941 probably belong to cryptic forms, and therefore, topotype sequences of the species are needed to decide on their identity. Likewise, topotype sequences have been used to prove the status of sequences assigned to Xiphinema brevicolle Lordello and Da Costa, 1961 (Lazarova et al., 2019) and X. robbinsi Pedram, Niknam & Decraemer, 2008 (Jahanshahi Afshar, 2019). For all of the above, the molecular data of the type series, e.g., topotypes, are needed for correct identification of given species in future. In the current study, the new species was separated from Ottolenchus fungivorus, the tentative closest species, based on the morphometric data, and their independent lineages were corroborated by their position on the SSU tree.

Figure 5