Laimaphelenchus hyrcanus, Miraeiz, Esmaeil, Heydari, Ramin, Maafi, Zahra Tanha & Bert, Wim, 2015

Miraeiz, Esmaeil, Heydari, Ramin, Maafi, Zahra Tanha & Bert, Wim, 2015, Laimaphelenchus hyrcanus n. sp. (Nematoda: Aphelenchoididae), a new species from northern Iran, Zootaxa 3915 (4), pp. 591-600 : 592-597

Laimaphelenchus hyrcanus n. sp.

Figs 1 , 2

Measurements. See Table 1 .

Material examined. Holotype, male, slide ALH001 together with eight paratype specimens: five males, three females (Slides ALH001, ALH002) deposited in the Nematode Collection of the Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. Additional paratypes distributed as follows: two males and two females at the Museum voor Dierkunde (collection number UGMD 104294), Ghent University, Ghent, Belgium, and two females at the National Nematode Collection of the Department of Nematology, Iranian Research Institute of Plant Protection, Tehran, Iran.

Description. Female. Medium sized nematodes, 0.67–0.87 mm long. The body ventrally arcuate and Cshaped upon fixation. Cuticle with indistinct transverse annulations, about 1.5–2.1 Μm wide at mid-body. Lateral field ca 4–5 µm wide, occupying 19–22% of body diameter at mid-body, and marked by three incisures; the inner one less apparent than the outer ones and difficult to observe by light microscopy, two outer lines are slightly areolated at posterior region and extending to origin of tubercle.

Head region hemispherical, heavily sclerotized, clearly wider than body at base, 3–3.5 µm high and 7–8 µm wide. SEM shows sixed-lobed cephalic region with equal width of labial sectors, not separated by ribs and without clear labial disc. The rounded oral aperture is surrounded by a rim. The pore-like amphidial apertures are positioned in the lateral sectors, just posterior to labial disc. Cephalids not seen. Stylet slender, with conical anterior part and tubular shaft with three small basal swellings. Procorpus cylindrical, 50–58 Μm long, metacorpus (median bulb) ovoid with centrally-located crescentic valves, 16–18 Μm long, 11–12 Μm wide, located 58–66 Μm from the anterior end; pharyngeal glands well-developed and clearly visible, overlapping intestine dorsally, extending for 81–135 Μm. The nerve ring is located posterior to the pharyngo-intestinal junction, at 77–98 µm from the body end. Excretory pore somewhat variable in position but always located posterior to nerve ring; hemizonid not seen. Intestine is a visibly straight tube, rectum straight.

1Length of conus as percentage of total stylet length

2Distance between anterior end of body and center of median pharyngeal bulb as percentage of pharyngeal length 3Distance from pharyngeal-intestine junction to end of dorsal gland tip

Genital system mono-prodelphic with outstretched ovary with oocytes in single line; spermatheca oblong to irregular in shape and filled with spheroid sperm cells. Uterus thick-walled; post-uterine sac (PUS) occupying 49–71% of distance from vulva to anus and ca 5.5 times the corresponding body diameter long, usually containing sperm cells. Vagina straight, sloping anteriorly, with one pair of oval sclerotizations as it joins the uterus. Vulva with well-developed anterior flap, overlapping the vulval slit. Tail ventrally curved, subcylindrical, ending in a single stalk-like terminus with four pedunculate tubercles bearing each 9–10 finger-like projections.

Male. Morphology similar to that of female but more curved, especially in tail region. Genital system monorchic, testis outstretched with developing germ cells in single line. Spicule rose-thorn shaped, 22–24 µm long, with prominent capitulum and broad rostrum with rounded tip; no gubernaculum observed. Two pairs of papillae present: first pair of adanal subventral papillae (P2) at level of or just anterior to cloacal aperture, second pair of post-cloacal subventral papillae (P3) clearly visible at about 54–60% of distance between cloacal aperture to tail tip. Tail subcylindroid and morphologically similar to that of female but more ventrally curved.

Type habitat and locality. The specimens were recovered from bark samples of cypress trees ( Cupressus sp.) in Naharkhoran National Park in the south of Gorgan (GPS coordinates: N 36° 78', E 54° 46'). The species was also found in jujube trees ( Ziziphus jujube Miller ) in Heidar Abad, a village in the west of Gorgan (GPS coordinates: N 36° 85', E 54° 29'), Golestan province, northern Iran.

Diagnosis and Relationships. Laimaphelenchus hyrcanus n. sp. is characterized by the presence of an anterior vulval flap, cephalic region with six labial sectors of equal width not divided by ribs and without clear labial disc, a tail with 4 clearly pedunculate tubercles ending in 9–10 finger-like projections, a lateral field with three lines of which the two outer lines are slightly areolated in the posterior region, and two pairs of subventral caudal papillae, adanal and post-cloacal, in the male.

The species in the genus Laimaphelenchus are divided into those with and without a vulval flap ( Baujard 1981, Hunt 1993). Since L. hyrcanus n. sp. has a vulval flap, it should be compared with members of the genus Laimaphelenchus also possessing vulval flaps, i.e. L. persicus , L. penardi , L. deconincki , L. pensobrinus Massey, 1966 , L. cocuccii , L. unituberculus Bajaj & Walia, 2000, L. helicosoma Peneva & Chipev, 1999 , L. preissii Zhao, Davies, Riley & Nobbs, 2006 and L. simlaensis . Based on the morphology of the tail tip (tail ending in a single stalk with 4 tubercles) in both sexes and having a vulval flap in females, the new species is morphologically closer to L. persicus , L. penardi , L. deconincki , L. cocuccii and L. pensobrinus . L. hyrcanus n. sp. resembles L. persicus in its general aspects, but differs in the number of lateral lines (3 vs 4) and number of caudal papillae (two pairs vs three pairs). L. hyrcanus n. sp. can be distinguished from L. penardi by having a slightly shorter stylet (11–12 vs 13–14 Μm), a higher number of lateral lines (3 vs 2), and number of caudal papillae (two pairs vs three pairs). It differs from L. deconincki by the greater length of the post uterine sac (97–152 vs 36–42 µm), the shape of cuticle surrounding the vagina (oval vs more angular), and the presence of males. The new species can be separated from L. cocuccii by having a different shape of cephalic region (six labial sectors not separated by ribs vs six labial sectors separated by pairs of well-marked ribs), greater length of the post uterine sac (97–152 vs 40–63 µm), areolated outer lateral lines in the posterior region and the presence of males. It can be differentiated from L. pensobrinus by greater length of the post uterine sac (97–152 vs 48–86 µm), shorter spicule length (22–24 vs 33– 37 µm) and number of lateral lines (3 vs 4).

The new species is clearly separated from L. helicosoma , L. unituberculus , L. preissii and L. simlaensis by having tail ending in a single stalk with 4 tubercles vs one broad tubercle.

Etymology. The species epithet refers to Hyrcania, the ancient Greek name of Gorgan, the capital city of Golestan province, from which the new species was recovered.

Molecular phylogenetic relationships. For molecular analysis, a 771 bp fragment of ribosomal DNA was sequenced (GenBank accession number KJ567061 ). There are only six LSU sequences of Laimaphelenchus species available in the GenBank; these species are: L. penardi , L. deconincki , L. persicus , L. australis , L. preissii and L. heidelbergi Zhao, Davies, Riley & Nobbs, 2007 . According to our molecular analysis L. hyrcanus sp. n. is sister taxon to a clade containing L. deconincki ( KF998578 .) and L. penardi (KJ472144). L. heidelbergi shows a distinct position from other Laimaphelenchus species in the phylogenetic tree ( Fig 4 ). It is morphologically different from all other Laimaphelenchus species based on its characteristic tail shape with a single offset terminal tubercle covered by 20–30 tiny knob-like appendages. L. hyrcanus sp. n. is molecularly most similar to L. deconincki but differs in 84 (ca 11.8%) positions including 5 insertions/deletions, it differs from L. penardi in 86 (ca 12.2%) positions including 5 insertions/deletions. Our phylogenetic analyses indicate that Laimaphelenchus species form a polyphyletic group.

Figures

FIGURE 1. Laimaphelenchus hyrcanus n. sp. A: Female pharyngeal region; B: Female anterior region; C, D: mail tail; E: Cross section of female at mid body; F, G: Vulval region; H: Entire body of male; I: Entire body of female. Scale bars: A – G: 10 µm, H, I: 20 µm.

FIGURE 2. Light microscopy observation of Laimaphelenchus hyrcanus n. sp. Female (A – F). A: Pharyngeal region; B: Genital system showing post-uterine sac; C: Anterior end; D: Vulval region; E: Position of excretory pore; F: Female tail. Male (G – I). G: Posterior body region showing the genital papillae (arrows); H: Cloacal region; I: Spicule. Scale bars: A, B = 20 µm, C – I = 10 µm.

FIGURE 4. Bayesian tree inferred from LSU gene DNA sequences. Posterior probabilities exceeding 50 % are given on appropriate clades. Nematode species and GenBank accession numbers are listed for each taxon.

Tables

TABLE 1. Morphometric data for Laimaphelenchus hyrcanus n. sp. All measurements are in Μm and in the form: mean ± s. d. (range).

Population Characters	Holotype female	Paratype females	Paratype males
n		10	5
L	760	763 ±66 (671–878)	834±27 (809–874)
a	30.4	33 ±1.8 (30– 36)	43.1±2.7 (38.5–45.2)
b	10	10 ±0.7 (9.1–11)	10.6±0.4 (10.1–11.2)
b'	4.8	4.1 ±0.4 (3.4–4.8)	4.6±0.1 (4.3–4.7)
c	17.3	18.3 ±1.5 (16.4– 21.4)	18±0.3 (17.6–18.5)
c'	3.1	3.3 ±0.1 (3.1–3.5)	3±0.1 (2.8–3.1)
V or T	65	66 ±1.5 (63.2–67.8)	71.4±7.7 (63–79)
Head height	3	3.2 ±0.3 (3–3.5)	3.5±0.4 (3–4)
Head width	7	7.5 ±0.4 (7–8)	7.2±0.3 (7–7.5)
Stylet	12	11.4 ±0.5 (11–12)	11.2±0.4 (11–12)
m¹	40	37 ±2.4 (33.3– 42)	45±0.0
Anterior end to valves of median bulb	63	64 ±2.5 (58–66)	66±2 (63–68)
MB²	82.9	83 ±2.4 (79.2–88)	84±1.5 (82.5–86)
Nerve ring from anterior body	85	86 ±5.9 (77–98)	87±1.7 (84–88)
Excretory pore	98	101 ±7 (86–112)	108±6.2 (98–113)
Maximum body width	25	23 ±1.5 (21–26)	19.4±1.1 (18–21)
PUS length	120	127 ±18.4 (97–152)	_
PUS/maximum body width	4.8	5.5 ±0.8 (4.3–6.6)	_
PUS/V–A	54.1	58 ±7 (50–71)	_
Vulva –anus	222	219 ±25.3 (175–260)	_
Pharynx	76	76.6 ±2 (72–80)	79±1.6 (76–80)
Overlap³	81	108.5 ±14.2 (81–135)	104±7.3 (96–113)
Testis or ovary length	339	278 ±62.2 (187–370)	596±74.7 (510–672)
Anal (cloacal) body width	14	12.5 ±1.1 (11–14)	16±0.7 (15–17)
Tail length	44	42 ±3.6 (36–48)	46.6±1.3 (46–49)
Spicule length (arc line)	_	_	23±0.9 (22–24)