Morphological and molecular characterization of Bitylenchus hispaniensis (Nematoda: Telotylenchidae) from Iran

(Figures 1–2; Table 1)

Figure 1:

Figure 2:

Body arcuate ventrally to open C shape after heat fixation. Cuticle annuli 1–1.4 µm wide at mid-body. Lateral field with four incisures along the body, including the tail region, outer two incisures areolated. Lip region rounded, bearing 5–6 fine annuli, continuous to slightly offset from the body, cephalic framework slightly sclerotized. Stylet knobs rounded, laterally to posteriorly directed, 3–4  µm across. Median esophageal bulb elliptical to slightly oblong, 14.2 ± 1.2 (13.0–16.2)  µm long and 12.0 ± 1.1 (11.7–13.1)  µm wide, hemizonid usually two to three annuli anterior to excretory pore, almost 1.5 annuli wide, basal bulb pyriform, 25.0 ± 2.8 (20.0–29.2)  µm long and 14.8 ± 1.3 (14.0–16.5)  µm wide. Cardia well developed. Intestinal fasciculi present in the intestinal region. Reproductive system didelphic-amphidelphic, vagina 9–11  µm long, epiptygma absent, vulva a transverse slit, spermatheca rounded, filled with rounded sperm. Tail rounded hemispherical, tail terminus annulated, hyaline portion 6–8  µm. Phasmids located almost at the middle of the tail, at 22.4 ± 0.5 (21.5–23.1)  µm distance behind the anus. Post-anal intestinal sac absent.

General morphology is similar to that of female except for character states associated with sexual differences. Tail conoid and pointed, enveloped by bursa. Spicules slightly curved ventrally. Gubernaculum well developed, half of the spicule length. The bursa is 61.5 ± 3.0 (52–65)  µm long.

Among the Bitylenchus species that have been characterized molecularly, those that are close to B. hispaniensis include B. dubius (Bütschli, 1873) Filipjev, 1934, B. huesingi (Paetzold, 1958) Jairajpuri, 1982 , B. parvulus (Hosseinvand, Eskandari, Ganjkhanloo, Ghaderi, Castillo & Palomares-Rius, 2020), B. parvus (Allen, 1955) Jairajpuri, 1982 and B. serranus (Gómez Barcina, Siddiqi & Castillo, 1992). Some characters separate B. hispaniensis from these species.

From B. dubius, by lip region continuous to slightly offset from the body bearing 5 to 7 annuli vs usually sharply offset from the body bearing 6 to 9 annuli and the absence of post-anal intestinal sac vs present. From B. huesingi, by tail bluntly rounded with a hemispherical to clavate terminus vs tail straight, cylindrical; and the absence of post-anal intestinal sac vs present. From B. parvulus, by cuticle anterior to vulva normal vs with irregular undulations (wrinkling) at its ventral side, intestinal fasciculi present vs absent, the absence of post-anal intestinal sac vs present, epiptygma absent vs present and tail bluntly rounded with a hemispherical to clavate terminus vs tail sub-cylindrical, abruptly narrowing near terminus giving a bluntly digitate appearance to its terminus. From B. parvus, it differs in the shape of the female tail being bluntly rounded with a hemispherical to clavate terminus bearing 40 to 66 annuli vs cylindrical tail with hemispherical terminus bearing 35 to 43 annuli; and in the absence of post-anal intestinal sac vs present. From B. serranus, by shorter stylet (15.5–18.0 vs 19–22  μm) and the absence of post-anal intestinal sac vs present.

The general morphology of the recovered population of the species closely resembles the characters given for the type population (Handoo et al., 2014) and another population from Greece (Tzortzakakis et al., 2018). The type population from Spain and another population from Greece were extracted from the rhizosphere of olive. The presently studied species was recovered from the rhizosphere of euphrates poplar tree in the Karkheh protected area (GPS coordinates: 31°53ʹ49.308ʺN, 48°15ʹ33.3ʺE), Khuzestan province, southwest Iran. B. hispaniensis is herein reported for the first time in Iran and for the first time in association with euphrates poplar worldwide.

Two 713 nt long D2-D3 expansion segments of LSU rDNA (MZ725030, MZ725031) were obtained for the Iranian population of this species. The BLAST search using these sequences revealed they have 99.85% identity with other sequences of the same species (MG770479, KJ461545 and KJ461547). A total of 74 sequences of Tylenchoidea Örley, 1880 and two sequences of Aphelenchoidea (Fuchs, 1937) Thorne, 1949 as outgroup taxa (LC583316 and DQ328683), were selected for the LSU phylogeny. This dataset comprised 796 total characters. The phylogenetic tree inferred using this dataset is presented in Figure 3. The newly generated sequences of the Iranian population of B. hispaniensis have formed a maximally supported clade with other sequences of the species in this tree.

Figure 3:

The amplification and sequencing of the ITS rDNA of the Iranian population of B. hispaniensis yielded one fragment with 532 nt long (MZ725020). The BLAST search using this sequence revealed it has 98.84% identity with another ITS sequence of the species (KJ461578). A total of 48 sequences of Tylenchoidea and two sequences of Aphelenchoidea as outgroup taxa (JX683685 and KX856336), were selected for ITS phylogeny. This dataset comprised 1140 total characters. The phylogenetic tree inferred using this dataset is presented in Figure 4. The sequence of the Iranian population of B. hispaniensis formed a maximally supported clade with other sequences of the species in this tree.

Figure 4: