Morphological and Molecular Characterization of Filenchus multistriatus n. sp. (Tylenchomorpha: Tylenchidae) and Data on a Known Species of the Genus from Bushehr Province, Southern Iran

A total of 40 soil samples were collected from different gardens and fields and natural regions of the east Bushehr province, in southern Iran, in 2021. The nematodes were extracted from soil using the tray method (Whitehead and Hemming, 1965) and the nematodes of interest were handpicked under a Nikon SMZ1000 dissecting microscope. The specimens were heat-killed by adding hot 4% formaldehyde solution and then, transferred to anhydrous glycerine according to De Grisse (1969) and mounted on permanent slides. Measurements and drawings were performed using a drawing tube attached to a Nikon E600 light microscope and were digitally drawn using CorelDraw software version 2020. Light microphotographs were taken with a Nikon Eclipse 80i microscope (Nikon, Tokio, Japan) powered with differential interference contrast (DIC) and equipped with a Euromex sCEMX-6 digital camera (Euromex Microscopen BV, Arnhem, The Netherlands) (Abolafia, 2023). Indices and ratios were calculated according to Siddiqi (2000).

Three females of each species preserved in glycerin were selected for SEM observations. 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 scanning electron microscope (5 kV) (Zeiss, Oberkochen, Germany) (Abolafia, 2015).

DNA was extracted from three individual female specimens of the new species separately. Each specimen was squashed in 15 μl Tris EDTA buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) after examination on a temporary slide. The DNA samples were prepared and stored at -20°C until used as PCR templates. The SSU rDNA was amplified using forward primer 1096F (5′-GGTAATTCTGGAGCTAATAC-3′) and reverse primer 1912R (5′-TTTACGGTCAGAACTAG GG-3′); and forward primer 1813F (5′-CTGCGTG AGAGGTGAAAT-3′) and reverse primer 2426R (5′-GC TACCTTGTTACGACTTTT-3′) (Holterman et al., 2006). Primers for LSU rDNA D2-D3 amplification were forward primer D2Tyl (5′-GAGAGAGTTAAA NAGBACGTG-3′) (Oliveira et al., 2013) and reverse primer 1006R (5′-GTTCGATTAGTCTTTCG CCCCT-3′) (Holterman et al., 2008). The 30 μl PCR mixture contained: 15 μl Taq DNA Polymerase 2x Master Mix RED, 2mM MgCl2 (Ampliqon-Denmark), 8 μl distilled water, 1 μl of each primer, and 5 μl of DNA template. The thermocycling program for amplification of both loci 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 10 min. The PCR products were sequenced with an ABI 3730XL sequencer (Bioneer Corporation, South Korea) in both directions using the same primers applied for amplification of that segment. The newly generated sequences were deposited into the GenBank database under the accession numbers OM914650 for SSU, and OM914648 and OM914649 for LSU rDNA D2-D3 of the new species.

The quality of the newly obtained sequences was manually checked using Chromas software (http://technelysium.com.au/wp/chromas), edited and assembled. Each of the SSU and LSU sequences was compared with other available sequences in the GenBank database using the basic local alignment search tool (BLAST) homology search program. Several relevant sequences of representatives of the family Tylenchidae were selected for both SSU and LSU phylogenies. Representatives of Aphelenchoidea Fuchs, 1937, including Aphelenchoides fragariae (Ritzema Bos, 1890), Christie, 1932 (AY284645), and Bursaphelenchus mucronatus Mamiya & Enda, 1979 (AY284648) for SSU, as well as B. acalolepta Kanzaki, Ekino, Maehara, Aikawa & Giblin-Davis 2020 (AB650013) and B. luxuriosae Kanzaki & Futai, 2003 (AB299228) for LSU, were used as outgroups. Both datasets were aligned using MUSCLE (Edgar, 2004) and manually edited using MEGA6 (Tamura et al., 2013). The best-fitting substitution model for both datasets was selected using PAUP*/MrModeltest2 (Nylander, 2004). The Akaike supported model, a general time reversible model, including among site rate heterogeneity and estimates of invariant sites (GTR + G + I) was selected and used in both phylogenies. Bayesian analysis was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) with running the chains for 5×10⁶ generations for both datasets. 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 were assessed based on average standard deviation of split frequencies and potential scale 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 new species was recovered from a soil sample collected from a tomato farm in Serajabad village (south of Dashtestan), Bushehr province, southern Iran, in 23 December 2020. GPS coordinates: 29°29.609′N, 050°56.803′E.

Holotype female and eleven paratype females were deposited into the WaNeCo nematode collection (http://www.waneco.eu/), The Netherlands.

B706568E-35F1-4719-A03C-5520FD2FDF7C

The specific epithet refers to the numerous short longitudinal and transversal striations or incisures appearing at the inner band of the lateral fields.

The new species is mainly characterized by having a wide and low lip region continuous with adjacent body, annulated in SEM observations, amphidial apertures as short slits confined to the labial plate under SEM, four lines in lateral fields forming three bands, the two outer bands broken by transverse, and inner one broken by both transverse and longitudinal lines and an elongate conoid tail with wide rounded tip. The new species was morphologically compared with three species Filenchus crassus Siddiqui & Khan, 1983, F. hamatus and F. sandneri as follow (morphological and morphometric data of F. hamatus after Brzeski (1997); and those of the two latter species according to their original descriptions):

From F. crassus by having a longer body (500-590 vs 350-400 μm) and longer pharynx (94-107 vs 74.5 μm in holotype).

From F. hamatus by having a wider and lower lip region (vs high, trapezoid) and straight vs ventrally bent (hooked) tail.

From F. sandneri by having a wider and lower lip region (vs high), longer body (500-590 vs 370-470 μm), smaller V (63.2-73.8 vs 73-75 μm) and straight tail (vs ventrally bent).

(Figs. 4,5; Table 1).

Figure 4:

The Dashtestan population of this species is in full morphological and morphometric agreement with the type population. It has been reported several times from Iran (Karegar, 2018). The range of morphometric data of this species has been expanded after several reports (Geraert, 2008). According to our SEM data (Fig. 5), the lip region is annulated and amphidial openings are small pores close to the labial plate. In the previous SEM study of species (Karegar and Geraert 1998), the amphidial openings were not seen. The identity of the population(s) reported as Filenchus cf. sandneri in the latter study, however, needs further validation, as there are remarkable differences between their morphometric data compared with those of the type population.

Figure 5:

Sequencing of SSU and LSU rDNA D2-D3 fragments of the new species yielded a single 1592 nucleotide long SSU (accession number OM914650); and two 605 nucleotide long LSU sequences (accession numbers OM914648 and OM914649).

The BLAST search using the newly generated SSU sequence, revealed it has a 96.87-97.00% identity with six sequences assigned to Tylenchus arcuatus Siddiqi, 1963 (MW716338, MW716337, EU306348, KJ869322, KJ869304, MN542209). Its identity with the sequence assigned to Filenchus vulgaris Brzeski, 1963 (KJ869307) was 96.87%. Its identity with all other SSU sequences was less than 96.87%. A number of 93 SSU sequences (including newly generated sequence of the new species and two aphelenchoidid sequences as outgroups) were included in SSU phylogeny (for accession numbers, see the SSU tree). The SSU phylogenetic tree in Fig. 6 includes sequences of Filenchus which have occupied different placements, corroborating its non-monophyletic condition according with this marker. The SSU sequence of the new species has formed a maximally supported clade with an SSU sequence (KJ869334) assigned to Filenchus sp. This clade is in sister relation with a clade including several sequences of Filenchus spp. (AY284592, JQ814877, KJ869307, JQ814879, KJ869336, JQ814880, JQ814876, KX156304, JQ814875, JQ814878, KJ869412) and the sequences assigned to Tylenchus arcuatus (EU306348, MW716338, MW716337, KJ869322, KJ869304, MN542209) and Tylenchus sp. (AY284589).

Figure 6:

The BLAST search using the LSU sequences of the new species revealed that their identity with all currently available LSU sequences of Tylenchidae is less than 91% (the highest identity was 90.76 %, belonging to Filenchus sp. (KX156330)). A number of 102 sequences including the newly generated sequences and two sequences of outgroup taxa were used for LSU phylogeny. In this tree (Fig. 7), several species of Filenchus have occupied different placements. The two LSU sequences of the new species have formed a clade with sequences assigned to Filenchus spp. (JQ005017, KX156337, JQ005014, MW346649) and Tylenchus sp. (KM047508, KM058573).

Figure 7: