Morphological and molecular characterization of Ektaphelenchoides pini (Massey, 1966) Baujard, 1984 (Aphelenchoididae; Ektaphelenchinae) from Iran, with morphological and taxonomic observations on some species

Wood and bark samples (48 in total) were collected from natural forests of Golestan province in north of Iran, during 2019. The nematodes were extracted from the samples using the tray method (Whitehead and Hemming, 1965), handpicked under a Nikon stereomicroscope model SMZ1000, heat killed by adding boiling 4% formalin solution, transferred to anhydrous glycerin according to De Grisse (1969) and mounted on permanent slides. The slides were then studied and the nematodes were measured under a Nikon Eclipse E600 light microscope. Photographs were taken using an Olympus DP72 digital camera attached to an Olympus BX51 microscope equipped with differential interference contrast (DIC). The paratype specimens and the original descriptions of some previously described species of the genus were also studied in detail to amend their characterization or propose new taxonomic placements.

To prepare DNA samples, a single live nematode specimen of the recovered species was picked out, examined on a temporary slide and transferred to a small drop of TE buffer (10 mMTris-Cl, 0.5 mM EDTA, pH 9.0; Qiagen) on a clean slide and crushed using a cover slip. The suspension was collected by adding 20 μl TE buffer. The DNA sample was stored at −20°C until used as PCR template (two separate females were used for this purpose, and two DNA samples were prepared). The SSU rDNA was amplified using the forward primer F22 (5´-TCCAAGGAAGGCAGCAGGC-3´) (Dorris et al., 2002) and reverse primer 18S 1573R (5´-TACAAAGGGCAGGGACGTAAT-3´) (Mullin et al., 2005) (the several primers designed to amplify this fragment, e.g. two pairs designed by Holterman et al., 2006 did not yield on high-quality amplifications, or yield on no amplifications). The D2–D3 expansion segments of LSU rDNA were amplified using the forward D2A (5´-ACAAGTACCGTGAGGGAAAGT-3´) (Nunn, 1992) and reverse primer 1006R (5´-GTTCGATTAGTCTTTCGCCCCT-3´) (Holterman et al., 2008). The ITS1 region was amplified using the forward primer rDNA1 (5´-TTGATTACGTCCCTGCCCTTT-3´) and reverse primer rDNA1.58S (5´-ACGAGCCGAGTGATCCACCG-3´) (Subbotin et al., 2000). The polymerase chain reaction (PCR) cycles and sequencing of amplified fragments were according to Heydari et al. (2020). The newly obtained sequences of the species were deposited into GenBank database (accession number MT007525 for partial SSU and MT008125 for partial LSU rDAN D2-D3).

For phylogenetic studies, two separate SSU and LSU datasets were prepared. Several available ektaphelenchid and seinurid species were included in both datasets. The SSU dataset included 50 sequences and the LSU dataset totaled 56 sequences (including newly generated sequences and sequences of the outgroup taxa), their accession numbers being given in the SSU and LSU phylogenetic trees. Each dataset was aligned using the QINS-i algorithm of online version of MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley, 2013). The Gblocks program (version 0.91b) with all the three less stringent parameters, a server tool at the Castresana Lab (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) was used for post-editing of the alignments, i.e., to eliminate poorly aligned regions or divergent positions. The Akaike-supported model, a general time reversible model, including among-site rate heterogeneity and estimates of invariant sites (GTR + G + I), was used in both phylogenies. The Bayesian analyses were performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) with a random starting tree, running the chains for 4 × 10⁶ 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 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 the 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 phylogenetic programs were visualized using Dendroscope V.3.2.8 (Huson and Scornavacca, 2012) and both SSU and LSU Bayesian trees were redrawn in CorelDRAW software version 17.

Iranian population of Ektaphelenchoides pini (Massey, 1966; Baujard, 1984) is shown in Figure 1.

Figure 1:

Measurements of this population are given in Table 1.

Large nematodes. Body is slightly ventrally curved after fixation. Cuticle is finely annulated. Lateral fields have three incisures. Cephalic region is slightly set off from the body by a shallow depression. Stylet is well developed, its lumen is wide, and the conus is ca 38% of the total length with thick sclerotized walls, conophore, and knobs lacking. The protractor muscles are well developed, V-shaped, adjoining the stylet base to the cephalic framework. Procorpus is slender, wide, marked by a constriction at its junction with the median bulb; the latter is elongate ellipsoid, with anterior granular part occupying ca 30% of its length and post-central well-developed/sclerotized valves. Pharyngo-intestinal junction is immediately posterior to the base of median bulb. The dorsal lobe of pharyngeal glands forms a 101 to 148 μm long overlap. The intestine ends to a blind sac, rectum indistinct, and anus vestigial (in 11 females out of 14 examined individuals), which is presumably not functional. Nerve ring is located about one metacorpus length behind it. Hemizonid is immediately behind the E pore. The reproductive system is monodelphic-prodelphic, located on the right side of intestine, composing of an outstretched ovary with oocytes mostly at multiple rows behind germinal zone and tubular oviduct, axial spermatheca with small spheroid sperm in all examined females, the crustaformeria, uterus sometimes including sperm, anteriorly inclined 23 to 33 μm long vagina with sclerotized walls, vulva is a crescent-shaped slit without flap in ventral view and PUS ca 1.2 times the vulval body width long including sperm, apparently with no differentiation at the junction with uterus. Posterior body is elongated conoid, ending to a filiform tip.

Males are similar to females in anterior region morphology. Posterior body is strongly curved after fixation. The reproductive system is monarchic, located on the right side of intestine. Testis is outstretched, expanded anteriorly, not reflexed. Spermatocytes are in two to three rows in the germinal zone of the testis. Spicules are massive, wide, and their condylus is well-developed, marked by indentations at the apex, rostrum is developed with blunt tip, the lamina-calomus is complex and ventrally curved, and the distal tip of spicules is simple. Bursa and gubernaculum are absent. Two pairs of precloacal + caudal subventral papillae are present, the first pair (P2) is 5 to 6 μm anterior to cloacal opening, and the second pair (P3) is located at 30 to 41% of the tail, or 40 to 49 μm from the tail tip. The single precloacal papilla (P1) is lacking. Tail is dorsally convex, ventrally flat, with an 11 to 13 μm long spike ending.

Recovered from the wood and bark samples of a dead broadleaf forest tree, collected in Golestan province, north of Iran, during October 2019, with GPS coordinates 36°41´36.9˝ N, 54°04´49.8˝E.

Three slides of the studied population of this species including eight females and four males were deposited in the USDA Nematode Collection, Beltsville, MD, USA (slides numbers: T-7401p to T-7403p).

The detailed study of Ektaphelenchoides maafiae (Golhasan et al., 2019) revealed that it has close morphology with E. tonekabonensis (Aliramaji et al., 2019a) and has some minor morphometric differences, e.g. slightly longer stylet (13-15 vs 10.5-12.0 µm) in females and smaller c´ (3.4-4.5 vs 6.5-8.3) in males. However, according to light microphotographs (Fig. 2A, E in Golhasan et al., 2019), its stylet length is calculated 12 µm and the c´ value of male is calculated 5.85, and by identical 28S rDNA D2-D3 (few differences were observed that could be due to the quality of the reading of the sequence or its editing artefacts), and based on the Principle of Priority, is proposed as a junior synonym of E. tonekabonensis. The species lacks the cloacal pair of male caudal papillae (P2) (the observation of Aliramaji et al., 2019 is amended herein).

Figure 2:

A review of the species of the genus revealed that Ektaphelenchoides winteri (Hooper, 1995) better fits the diagnostics of the genus Ektaphelenchus (Fuchs, 1937) by the posterior body end morphology of females and is proposed to be transferred to the latter genus.

The close study of the paratype male of Ektaphelenchoides poinari (Aliramaji et al., 2014) revealed that a slightly projecting structure is present slightly anterior to the cloacal opening. This could be the single precloacal P1 papilla, or the paired papillae, similar to those in E. pini. The last pair is situated at 14.5 µm distance to the tail tip. The arrangement and number of papilla for this species are amended herein.

The repeated sequencing of D2 to D3 expansion segments using the second female revealed an identical sequence to the MT008125, and thus, only one sequence was used in LSU tree.