Cryptaphelenchus iranicus, Esmaeili, Mehrab, Heydari, Ramin, Taheri, Zahra Majd, Fang, Yiwu & Li, Hongmei, 2016

Esmaeili, Mehrab, Heydari, Ramin, Taheri, Zahra Majd, Fang, Yiwu & Li, Hongmei, 2016, Description of Cryptaphelenchus iranicus n. sp. (Nematoda: Ektaphelenchinae) recovered from bark samples of Pinus nigra from Iran, Zootaxa 4139 (1), pp. 117-127 : 118-124

Cryptaphelenchus iranicus n. sp.

Figs 1–3

Measurements. See Table 1 .

Description. Male. Body slender, arcuate to J-shaped after heat relaxation, posterior end more ventrally curved. Anterior region similar to that of female. Testis single, expanded anteriorly, not reflexed. Vas deferens composed of small rounded cells, not clearly differentiated from other parts of gonad. Spicules arcuate, separate, 11–13 µm long along the arc line, ca. 2.2 times capitulum width, lamina/calamus smoothly and symmetrically curved, rostrum short, moderately developed with pointed tip. Condylus distinct with round tip. Cucullus, gubernaculum and bursa absent. The posterior body end (tail) conical, bearing a short sharp mucro ca. 3.0–3.5 µm long. Seven (a single ventral and three subventral pairs) genital papillae present: single midventral precloacal papilla (P1), located ca. 1.3 times cloacal body diameters anterior to cloacal aperture or opposite to level of condylus; first paired papillae (P2) subventral located at level of or just posterior to cloacal aperture; second postcloacal paired papillae (P3) subventral located at ca. 65–67% of tail length posterior to cloacal aperture; and third paired small papillae (P4) subventral located at ca. 85% of tail length posterior to cloacal aperture.

Female. Body cylindrical, slightly ventrally curved when heat-killed. Cuticle less than 1 µ m thick, finely annulated. Lateral fields with three incisures. Head distinctly offset from body and separated by a clear constriction ca 2–3 µm high and 4–5 µm broad, separated in six equal-sized lips or having small lateral sectors (en face view). Stylet 7–8 µm long, with distinct basal swellings, conus delicate and occupying ca. two-fifths of the total stylet length. Procorpus tubular, ending in an oval median bulb (metacorpus) with central valve plates. Dorsal pharyngeal gland orifice opening into lumen of metacorpus midway between anterior end of metacorpal valve and anterior end of metacorpus. Pharyngo-intestinal junction immediately posterior to metacorpus. Nerve ring situated just posterior to metacorpus. Dorsal pharyngeal glands well-developed, overlapping intestine dorsally, 2.5–4.0 times as long as body diameter at level of median bulb and the third dorsal pharyngeal gland nuclei was observed. Excretory pore located ca. 1.5–2.0 body diameters posterior to median bulb or about two median bulb diameters posterior to nerve ring. Hemizonid ca. two body annuli long, situated just posterior to excretory pore. Reproductive tract located on ventral side of body, including an ovary, oviduct, spermatheca, crustaformeria, uterus, vagina + vulva, and postvulval uterine sac. Ovary single, anteriorly outstretched, located on the dorsal side of intestine. Developing oocytes mostly in two rows. Oviduct small, tube-like, horizontal wrinkle-like pattern sometimes appearing on its surface, constructed of oval-shaped cells, connecting ovary. Spermatheca oval to irregular, filled with rounded sperm. Crustaformeria obscure. Uterus thick-walled, vagina slightly sclerotized, straight. Vulva located posteriorly at ca. 74% of the body length. Vulval lips distinct, vulval flaps absent. Post-vulval uterine sac short, ca. 0.6–1.0 body diameters at vulva region in length. Intestine apparently ending in a blind sac ca. one third to one half of the distance from vulva to posterior end, rectum or functional anus absent. Posterior body end (tail) conical, ending in a pointed tip.

Type locality and habitat. Isolated directly from bark and wood samples of a weakened tree of Pinus nigra in the locality Cheshmeh-e-Nezamei of Gilan-e-Gharb city in Kermanshah Province, western Iran (GPS coordinates: N 33˚59', E 46˚12', 1248 meters above sea level).

Type material. Holotype male (Slide ACI001), four paratype females and two paratype males (Slides ACI001 and ACI002) deposited in nematode collection of the Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Iran. Three paratype females and two paratype males deposited at the nematode collection of the Royal Belgian Institute of Natural Sciences, Brussels. Two paratype females and two paratype males deposited at the WANECO collection, Wageningen. Two paratype females and one paratype male deposited at the National Nematode Collection of the Department of Nematology, Iranian Research Institute of Plant Protection, Tehran, Iran.

Remarks. PCR on the ITS1/2 and 18S of rDNA sequences was not successful despite many attempts. Cryptaphelenchus iranicus n. sp. was successfully cultured on Botryotinia fuckeliana growing on Potato Dextrose Agar (PDA).

Diagnosis and relationships. Cryptaphelenchus iranicus n. sp. is characterized by body lengths of 250–330 µm (females) and 230–275 µm (males). The cuticle is weakly annulated, stylet 7–8 µm long with distinct basal knobs, lateral fields with three incisures. The excretory pore is located ca. 1.5–2.0 body diameters posterior to metacorpus. Post-vulval uterine sac short with 7–10 µm long. Intestine apparently ending in a blind sac at ca. one third to one half of distance from vulva to posterior body end, female rectum or functional anus absent. Posterior body conical, ending to a pointed tip. The male spicules are 11–13 Μ m long in arc line, with sharply pointed rostrum and rounded condylus. The male tail is conical with a sharply pointed terminus. The male genital papillae comprise a single precloacal papilla and an adcloacal and two post-cloacal pairs.

Based on morphological and morphometrics characters, C. iranicus n. sp. is differentiated from all other species in genus of Cryptaphelenchus by the cephalic region (offset vs. non-offset), shape of median bulb (oval vs spherical) and post-vulval uterine sac (presence vs. absence) ( Kurashvili et al., 1980; Korentchenko 1987). Furthermore, the new species differs from C. cirrus Massey, 1974 by the stylet basal swelling (present vs. absent); it differs from C. latus Massey, 1974 by the presence of the subventral pair of postcloacal papillae (P4) vs. absence; and it differs from C. leptocaudus Rühm, 1956 by the body length (250–330 vs 325–382 µm), the position of P1 (opposite rostrum vs. condylus) and the presence of P4 vs. absence.

1Length of conus as percentage of total stylet length.

2Distance between anterior end of body and centre of metacorpus as percentage of pharyngeal length. 3Distance from pharyngo-intestinal junction to posterior end of dorsal gland.

Molecular characterisation and phylogeny. The sequence of the partial 28S D2-D3 region of Cryptaphelenchus iranicus n. sp. was deposited in GenBank with accession number KT895255 (691 bp, isolate KER). A total of 70 aphelenchid taxa were selected to construct the phylogenetic tree using Aphelenchus avenae Bastian, 1865 ( AB368536 ) as outgroup. The dataset was composed of 905 total characters after aligning and manually editing. Phylogenetic relationships among the isolates for the dataset were determined using Bayesian inference (BI) and maximum likelihood (ML) methods ( Fig. 4 ). The trees produced using each method show Cryptaphelenchus iranicus n. sp. clustered with two unnamed Cryptaphelenchus species in an independent clade among other Ektaphelenchinae species. This clade occupies a basal position with a posterior probability (PP) support of 91% with a clade of some members of other genera of the subfamily Ektaphelenchinae such as Devibursaphelenchus , Ektaphelenchus , Ektaphelenchoides and a member of subfamily Acugutturinae, Noctuidonema Remillet & Silvain, 1988 . The new species shares a sequence similarity with Cryptaphelenchus sp. ( AB597985 ) of 77.1% and with Cryptaphelenchus sp. ( EU287596 ) of 79.2%. However, the new species is separate from other species in the phylogenetic trees and when compared against other sequences deposited in GenBank.

Etymology. The new species epithet refers to the country where the species was found.

Figures

FIGURE 1. Line drawing of Cryptaphelenchus iranicus n. sp. Female entire body; B: Male entire body; C: Female anterior end; D: Metacorpus region; E: Female pharynx region; F: Male posterior body and showing caudal papillae in lateral view (arrowhead); G: Male posterior body and showing caudal papillae in ventral view (arrowhead); H: Cross section of female showing lateral field; I: Spicule in detail; J: Vulva region showing post-uterin sac (PUS); K, L: Female posterior body end. (A, B, E, J, K & L = 20 µm, C, D, F, G & H = 10 µm, I = 5 µm).

FIGURE 2. Light micrographs of Cryptaphelenchus iranicus n. sp. female. A: Anterior body; B: Head region; C: Cross section of female showing lateral field; D: Posterior region of pharynx showing excretory pore and hemizonid by arrow; E: Anterior genital tract; F: Genital tract with spermatheca and PUS indicated by arrow; G: Spermatheca; H: Vulval region showing PUS by arrow; I & J: Posterior body. (A, D & E 15 = µm, B, F - J = 10 µm, C = 5 µ m).

FIGURE 3. Light micrographs of Cryptaphelenchus iranicus n. sp. A: Male entire body; B: Female entire body; C-F: Male posterior body with cloacal region (cl), spicules (sp) and papillae (P) arrangement indicated by arrows. (A & B = 40 µm, C = 20 µm, D, E & F = 15 µm).

FIGURE 4. Phylogenetic relationships of Cryptaphelenchus iranicus n. sp and aphelenchid nematodes based on 28 S rDNA sequences. The 10001 st Bayesian tree inferred under the GTR + I + G model (ln L = - 24880.1105; freqA = 0.1808; freqC = 0.1650; freqG = 0.3292; freqT = 0.3250; R (a) = 1.0714; R (b) = 4.9532; R (c) = 2.2091; R (d) = 0.7280; R (e) = 5.6708; R (f) = 1.0000; Pinvar = 0.1970; Shape = 0.7650). Aphelenchus avenae served as the outgroup species. Posterior probability and bootstrap values are given on appropriate clades.

Tables

TABLE 1. Morphometric data of Cryptaphelenchus iranicus n. sp. All measurements are in µ m and in the form: mean ± s. d. (range).

	Male		Female
Character	Holotype	Paratypes	Paratypes
n	–	1 5	1 6
L	240	257 ± 14.9 (230-275)	286.8 ± 31.7 (250–330)
a	24.0	24.3 ± 1.5 (22.3–27.0)	25.1 ± 3.2 (20.8–30.0)
b	6.9	6.9 ± 0.5 (6.3–8.1)	7.3 ± 1.2 (5.2–9.1)
b’	2.5	2.8 ± 0.3 (2.5–3.4)	3.3 ± 0.4 (2.7–3.8)
c	14.0	13.0 ± 1.1 (11.4–15.0)	–
c’	1.9	2.1 ± 0.2 (1.8–2.4)	–
V or T	48.0	47.7 ± 6 (37.7–53.8)	74.2 ± 1.6 (71.9–76.7)
Lip region height	2.5	2.8 ± 0.3 (2.5–3.0)	2.9 ± 0.2 (2.5–3.0)
Lip region width	4.0	3.8 ± 0.3 (3.5–4.0)	4.3 ± 0.4 (4.0–5.0)
Stylet length	7.0	6.8 ± 0.4 (6.0–7.0)	7.3 ± 0.5 (7.0–8.0)
Conus length	2.5	2.8 ± 0.3 (2.5–3.0)	2.9 ± 0.2 (2.5–3.0)
m 1	35.7	41.2 ± 2.9 (35.7–42.9)	40.1 ± 3.8 (31.3–42.9)
MB2	85.5	91.2 ± 4.1 (82.9–94.7)	88.4 ± 5.5 (81.4–95.2)
Body diam. at median bulb	9.0	9.4 ± 0.5 (9.0–10.0)	10.8 ± 0.4 (10.0–11.0)