Description of BOLeODOruS buShehreNSIS n. sp. (Rhabditida: Tylenchidae) from Southern Iran, and Observations on a Commonly Known Species Author Monemi, Somayeh Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran Author Atighi, Mohammad Reza Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran Author Abolafia, Joaquín Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus Las Lagunillas, s / n, 23071, Jaén, Spain Author Pourjam, Ebrahim Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran Author Pedram, Majid Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran majid.pedram@modares.ac.ir text Journal of Nematology 2022 2022-04-17 54 1 1 13 http://dx.doi.org/10.2478/jofnem-2022-0004 journal article 298701 10.2478/jofnem-2022-0004 48aee40b-1fb2-4b00-ad94-b6993a0b5faa 2640-396X 11644623 Boleodorus bushehrensis n. SP. The morphological characters of B. bushehrensis n. sp. are represented in Figures 1–3 . For the measurements of B. bushehrensis n. sp. , see Table 1 . Description Female Body is short, open C-shaped after fixation, gradually narrowing toward both extremities. Cuticle with fine, transverse annules, being stronger at the distal region of the tail. Lateral fields show four lines, sometimes with irregular additional lines under SEM; the outer lines are plain. Cephalic region low and continuous with the adjacent body. The SEM and light microscopic observations show that the oral aperture is placed in a depression. SEM images further revealed that the cephalic region is annulated; the amphidial openings are short crescent-shaped slits, and four large cephalic papillae are present. Stylet is fine, the conus is shorter than the shaft, and the knobs are small and posteriorly directed. The pharyngeal dorsal gland orifice (DGO) is positioned posterior to the knobs at less-than-half-stylet length. Pharyngeal procorpus is slender; metacorpus is slightly swollen, with vestigial-to-invisible valve plates in the shape of two small roads; isthmus is narrow and slender; and the pharyngeal bulb is pyriform, with usually one visible nucleus. The excretory pore is located at the middle of the pharyngeal bulb or in a position slightly anterior to it. The hemizonid is indistinct. The nerve ring surrounds the anterior part of the isthmus; the cardia is small and hemispherical. Intestine is simple; rectum and anus are functional. The reproductive system is monodelphic–prodelphic, composed of an outstretched ovary, oocytes arranged in a single row, a poorly discernible apparently tubular oviduct, and rounded, offset, and functional spermatheca filled with spheroid sperm cells. Crustaformeria is with unclear cell arrangement; the uterus is simple, and the vagina is perpendicular to the body axis, straight or slightly anteriorly sloping. The postvulval uterine sac (PUS) is about as long as the vulval body diameter. Tail is conical, and usually, its distal region is ventrally curved, forming a hook; in some specimens, it is concave on the ventral side, and its tip is finely or widely rounded. Figure 2: Light microphotographs of Boleodorus bushehrensis n. sp. (A, B, C, E, F, I, J, K: female; D, G, H: male). (A, B) Anterior region showing cephalic region and stylet, respectively. (C) Pharyngeal metacorpus. (D) Anterior body region showing the oral aperture in a depression. (E, J) Pharyngeal bulb region showing excretory pore. (F, G, I) Tail tip. (H) Bursa. (K) Lateral field at midbody. (All scale bars = 10 Mm). Figure 3: Scanning electron microphotographs of Boleodorus bushehrensis n. sp. (female). (A) Anterior body region showing beginning of lateral field (arrow showing the excretory pore). (B–D) Anterior end in ventrolateral, ventral, and en face views, respectively (arrows pointing to the amphidial openings). (E, F) Excretory pore in lateral and ventral views, respectively (arrow). (G) Lateral field at vulva. (H, I) Vulva in lateral and ventral views, respectively. (J) Anterior body region in ventral view showing excretory pore. (K,N) Lateral field at midbody showing unusual division and four incisures, respectively. (L,M) Anus in ventral and lateral views, respectively. Male Males are rare and functional (sperm observed inside females’ spermatheca). They are similar to females in general morphology, except in sexual characters. Spicules are tylenchoid, slender, and arcuate. Gubernaculum is simple and small. Tail is similar to that of females. Bursa is adcloacal and small, with smooth margin. Diagnosis and relationships The new species is mainly characterized by having a wide and low cephalic region continuous with the adjacent body, annulated in SEM observations; the oral aperture is in a depression in both light and SEM microscopy studies. Short crescent-like amphidial slits are found on SEM analysis; four lines are present in the lateral field, and tail is hooked, short, 26- to 38- µm long with a rounded tip. The new species is further characterized by having 334- to 464-Mm-long females, a weakly developed metacorpus with vestigial-to-invisible valve, and offset spherical spermatheca filled with spheroid sperm; males are present, having 11.5- to 12.0-Mm-long spicules and weakly developed bursa. By having a wide, continuous, and annulated cephalic region and short tail, the new species is unique in the genus. It is compared with seven known species of the genus having a conical tail and four lines in the lateral field, namely, B. acutus Thome and Malek, 1968 , B. azadkashmirensis Maqbool, Shahina, and Firoza, 1990 , B. citri Edward and Rai, 1970 , B. cynodoni Fotedar and Mahajan, 1974 , B. modicus Lal and Khan, 1988 , B. neosimilis Geraert, 1971 , and B. volutus Lima and Siddiqi, 1963 . The comparison of the new species with the aforementioned species is as follows. Table 1. Morphometrics of Boleodorus bushehrensis n. sp.

Characteristics	Holotype	Paratypes
Females	Males
n	13	3
L	449	412 ± 35.5	432 ± 17
		(334–464)	(417–450)
L’	413	379 ± 34	399 ± 16
		(298–432)	(385–416)
a	34	32.5 ± 3.0	39.8 ± 2.5
		(27.8–37.4)	(37.2–42.0)
b	4.8	4.8 ± 0.5	5.0 ± 0.5
		(4–6)	(4.3–5.4)
c	12	12.6 ± 1.5	13.1 ± 0.1
		(9.3–15.8)	(12.9–13.0)
c’	4.9	3.9 ± 0.5	4.1 ± 0.1
		(2.9–4.9)	(4.0–4.3)
V or T	79	74.8 ± 1.9	–
		(70.8–79.5)
V’	80.7	80.9 ± 1.2	–
		(77.5–82.0)
Cephalic region height	2.3	2.1 ± 0.3	2.1 ± 0.4
		(1.7–2.6)	(1.8–2.7)
Cephalic region width	6.5	5.9 ± 0.6	6.1 ± 0.4
		(5.0–6.7)	(5.6–6.5)
Stylet length	8	8.7 ± 0.4	9.7 ± 0.3
		(8.0–9.3)	(9.5–10.0)
Conus length	3	3.2 ± 0.2	3.3 ± 0.3
		(3.0–3.5)	(3.0–3.5)
m	37.5	36.8 ± 3.4	33.6 ± 2.3
		(32.3–42.7)	(31.5–36.0)
DGO	3	3.1 ± 0.2	3.0 ± 0.07
		(2.8–3.5)	(2.9–3.0)
Distance from excretory pore to anterior end	78.5	69.5 ± 5.2	63.9 ± 1.6
		(64–79)	(62–65)
MB	48.2	55.8 ± 12.1	57.8 (n=1)
		(43.0–82.3)
Pharynx length	91	87 ± 7	87.7 ± 7.5
		(73–99)	(80–95)
Distance from anterior end to vulva	333	307 ± 28	–
		(245–347)
BW	12	12.7 ± 1.3	10.8 ± 0.3
		(12–15)	(10.6–11.0)

(Continued) Table 1: Continued

Characteristics	Holotype	Paratypes
Females	Males
Anal BW	9	8.6 ± 0.6	8.7 ± 0.9
		(7–9)	(8–9)
Vulva–anus (V–A) distance	80	72.2 ± 8.5	–
		(54–84)
PUS length	11.5	10.6 ± 1.3	–
		(8.2–12.0)
Tail/V–A	0.5	0.5 ± 0.07	–
		(0.4–0.7)
PUS/BW	1	0.8 ± 0.07	–
		(0.7–1.0)
Tail length	36	33 ± 3	32.5 ± 0.7
		(26–38)	(32–33)
Spicules length	–	–	11.5 ± 0.4
			(11.5–12.0)
Gubernaculum length	–	–	4.5 ± 0.2
			(4–5)
Bursa length	–	–	6.3 ± 0.5
			(6–7)

All measurements are in micrometers and in the form: mean ± SD (range). BW, body width; DGO, dorsal gland opening; PUS, postvulval uterine sac; SD, standard deviation. Compared to B. acutus , it has a shorter body length (412 [334–464] Mm vs 500 Mm), wide and low cephalic region (vs narrower and higher,according to the original drawings), shorter stylet (8.7 [8.0–9.3] Mm vs 13 Mm), shorter pharynx (87 [73–99] Mm vs 114 Mm), greater c value (12.6 [9.3–15.8] vs 8), greater V value (74.8 [70.8–79.5] vs 67), and shorter tail (33 [26–38] Mm vs 63 Mm). It differs from B. azadkashmirensis by the presence of a continuous, wide, and low cephalic region (vs narrower and high), shorter stylet (8.7 [8.0–9.3] Mm vs 10.5–12.0 Mm), presence vs absence of a vestigial valve in the median bulb, a shorter pharynx (87 [73–99] Mm vs 96 Mm), greater V value (74.8 [70.8–79.5] vs 63.5–67.5), greater c value (12.6 [9.3–15.8] vs 7–8), and shorter tail (33 [26–38] Mm vs 60 Mm) not ending in a ventrally curved tip (vs ventrally curved). Figure 4: Bayesian 50% majority rule consensus tree inferred from the SSU rDNA of Boleodorus bushehrensis n. sp. under the GTR + G + I model. Bayesian posterior probability values are given for corresponding clades. The new species is in bold font. GTR, general time-reversible; G, gamma; I, invariant; rDNA, ribosomal DNA; SSU, small subunit. It can be distinguished from B. citri by having a C-shaped (vs spiral) longer body (412 [334–464] Mm vs 280–310 Mm), wide and lower cephalic region (vs narrow and higher, according to the original drawings), shorter stylet (8.7 [8.0–9.3] Mm vs 9.0–10.5 Mm), posteriorly located excretory pore (69.5 [64–79] Mm vs 55–61 Mm from anterior body end), greater a value (32.5 [27.8–37.4] vs 21–23), greater MB (55.8 [43.0– 82.3] vs 35), greater V value (74.8 [70.8–79.5] vs 64– 68), and shorter tail (33 [26–38] Mm vs 51–82 Mm). It differs from B. cynodoni by the shorter body length (412 [334–464] Mm vs 420–490 Mm), greater V value (74.8 [70.8– 79.5] vs 62–65), and shorter tail (33 [26–38] Mm vs 54 Mm). It differs from B. modicus by having a C-shaped (vs spiral) body, wide cephalic region at the apex (vs narrower), greater c value (12.6 [9.3–15.8] vs 7.7–9.8), greater V value (74.8 [70.8–79.5] vs 66–71), and shorter tail (33 [26–38] Mm vs 51 Mm). It is different from B. neosimilis by having a wider cephalic region at the apex (vs narrower), a greater V value (74.8 [70.8–79.5] vs 68), a shorter tail (33 [26–38] Mm vs 51 Mm), and shorter spicules (11.5 [11.5–12.0] Mm vs 14 Mm). The new species differs from B. volutus by having a C-shaped body (vs spiral), a slightly shorter body length (412 [334–464] Mm vs 390–510 Mm), a wider cephalic region at the apex (vs narrower), presence of a vestigial valve in the metacorpus (vs absence), shorter pharynx (87 [73–99] Mm vs 92–104 Mm), and shorter tail (33 [26–38] Mm vs 35–60 Mm). Type habitat and locality The new species was recovered from a soil sample collected from the rhizosphere of wheat in Shah Firouz village (south of Dashtestan), Bushehr Province , southern Iran , on 30 January 2021 . The global positioning system (GPS) coordinates are 29°32.316´N and 50°54.303´E . Figure 5: Continued Figure 5: Bayesian 50% majority rule consensus tree inferred from the LSU rDNA D2–D3 sequences of Boleodorus bushehrensis n. sp. under the GTR + G + I model. Bayesian posterior probability values are given for the corresponding clades. The new species is in bold font. GTR, general time-reversible; G, gamma; I, invariant; LSU, large subunit; rDNA, ribosomal DNA. Type material Holotype female , 10 to 13 paratype females , and three paratype males were deposited at the Nematology Collection of the Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. Etymology The specific epithet refers to the Bushehr Province , where the new species was found. Molecular phylogenetic analyses Sequencing of the SSU and LSU rDNA D2– D3 fragments of the new species yielded a single 1,242-nt-long SSU (accession number OK018183); and two 517- and 584-nt-long LSU sequences (accession numbers OK018176 and OK018177). The BLAST search using the newly generated SSU sequence revealed a 98.00%– 98.79% identity with nine sequences assigned to B. thylactus (KJ869348, KJ869350, AY993976, AY593915, KJ869349, MW716329, MK639397, MK639396, and MW716330). Its identity with the sequence assigned to B. volutus (FJ969117) was 98%. In the SSU phylogenetic tree ( Fig. 4 ), the DNA sequences of Boleodorinae formed a poorly supported clade (Clade A, 0.77 Bayesian posterior probability [BPP]). The DNA sequences representing Boleodorus formed a major clade, and the sequences assigned to B. thylactus occupied different placements in this tree. The new species formed a poorly supported clade (0.76 BPP) with five sequences assigned to B. thylactus (MW716330, MW716329, MZ081056, MZ081059, and MZ081057). The BLAST search using the LSU sequence of the new species (OK018177) revealed that its identity with all currently available LSU sequences of Tylenchidae is <96% (the highest identity was 95.02%, belonging to Boleodorus sp. [JQ005002]). The DNA sequences of Boleodorinae (for phylogenetic status of Atetylenchus Khan, 1973 , see the Discussion section) formed a maximally supported clade in the LSU tree ( Fig. 5 , clade B). Sequences of Boleodorus formed a clade; however, several sequences assigned to B. thylactus occupied different placements. The relationships of the new species with six sequences (JQ005002, MW056183, JQ005021, DQ328718, MK639377, and MK639378) have not been resolved due to polytomy.