Longidorus carniolensis, Sirca, Sasa, Urek, Gregor, Lazarova, Stela, Elshishka, Milka & Peneva, Vlada, 2011

Sirca, Sasa, Urek, Gregor, Lazarova, Stela, Elshishka, Milka & Peneva, Vlada, 2011, Longidorus carniolensis sp. n. (Nematoda, Longidoridae) from vineyard soil in Slovenia, ZooKeys 141, pp. 1-27 : 2-11

Longidorus carniolensis sp. n. Figs 112

Measurements.

See Table 2.

Description.

Female. Body moderately long (L=5.6-8.2 mm) and plump (a=51-72.4), assuming a spiral to C shape when heat relaxed. Cuticle consisting of several layers under light microscope: 11-14 µm thick at guiding ring level; 7-8 µm along the body; 13-15 µm on tail posterior to anus. Lateral pores number 10-14 in pharyngeal region: a single pore in front of guide ring, rarely two or none; 3-5 in odontostyle and 1-3 in odontophore regions; 3-4 dorsal pores and 7-10 ventral pores; numerous lateral body pores. Usually the fifth ventral pore (sometimes the fourth) differs in size (Figs 1A, 4F and 6H) compared to the other ventral pores. Lip region continuous, anteriorly almost flat, 7-9 μm high. Labial papillae prominent. Amphid aperture assumed to be a minute pore, difficult to be observed under light microscope. Pouch-like amphidial fovea with convoluted fine dendritic branches (receptors), extending to 1/2 - 2/3 the distance between anterior end and guiding ring, fovea slightly longer (15-18 μm, n=5) than wide (14-16 μm, n=4) with no distinct margins. Fusus (sensillium pouch) at 57 ± 1.9 (55-60) μm from anterior end. Guiding ring 7-9 μm wide. Odontostyle long and very slender, 2 μm wide at the base. Odontophore with weakly developed flanges. In all females a small (2-3 μm long) rudimentary odontostyle tip (vestigium) present, directed forward, and observed in the slender pharynx at 300.5 ± 40.3 (224-350) μm from anterior end; in two specimens the vestigium located in odontophore area. Slender pharynx often coiled in its posterior part. In this region 5-7 glandular bodies are observed in all females. Nerve ring surrounding odontophore base, rarely surrounding mid-odontophore, or just behind it, second nerve ring at a distance of 85.2 ± 6.6 (78-98) μm behind the first one. Hemizonid flat, 10-11 μm long. Pharyngeal bulb about 1/4 of the neck length. Normal arrangement of pharyngeal glands, the nuclei of dorsal and ventrosublateral glands approximately the same size, their diameters 3.4 ± 0.4 (3-4) μm, n=7 and 3.9 ± 0.2 (3.5-4) μm, n=11, respectively. Cardia small, broadly rounded, wider than long, variable in size: 20.1 ± 1.8 (10-23) × 10.1 ± 1.8 (7-12) μm. Reproductive system amphidelphic, varying in dimensions due to the stage of maturity of female. Vagina extending about half body width. Pars distalis vaginae with characteristic shape (Fig. 2F, G), 26-28 µm and pars proximalis vaginae 32-38 µm long, respectively; muscular walls of the latter almost parallel. Uteri very long, anterior uterus 494.6 ± 52 (430-563) µm long, posterior uterus 510.0 ± 88.7 (357-643) µm long, differentiated, filled with sperm cells in all females examined; well developed sphincter between uterus and pars dilatata oviductus also containing numerous sperm cells. Anterior and posterior oviduct of similar size, measured in four specimens: 275-348 μm, and 283-330 μm. Anterior ovarium 263.4 ± 51.8 (210-347) μm long, n=7, posterior ovarium 234.3 ± 35.8 (183-309) μm long, n=5; in older mature specimens the length is about 3 times greater (1055-1060 μm for anterior and 1020 μm for posterior ovary). One egg in anterior pars dilatata oviductus measuring 227 × 87.5 μm and one uterine egg measuring 225 × 77.5 μm. A weakly developed ovijector present, 112.0 ± 12 (95-125) μm long. In one female a rudimentary adanal pair of supplementary papillae was observed (Fig. 9E). Prerectum variable in length; rectum 0.7 ± 0.1 (0.6-0.8) body width at anus. A short post-intestinal sac present. Tail bluntly conoidal, rounded to almost hemispherical; ventral side straight or slightly convex, the dorsal curvature greater. Two pairs of lateral pores.

Male. Body C shaped when heat relaxed, posterior part more strongly coiled ventrally. Similar to females in general morphology except for genital system. Lateral pores number 10-15 in pharyngeal region: a single pore in front of guide ring, 3-5 in odontostyle and 1-2 in odontophore regions; 2-5 dorsal pores, mostly 3-4, and 7-10 ventral pores. Cuticle in post-labial region at the guiding ring level 10.5-13.5 µm thick, 6.5-9 µm along body, 9-10 µm in post-cloacal area. Second nerve ring at 80.7 ± 14 (50-100) μm behind the first one (n=14). In all males a small vestigium (2-3 μm, in one specimen 6 μm long), directed forward (in two specimens directed rearward), is observed in the slender pharynx at 300.5 ± 40.3 (224-350) μm from anterior end; in two specimens the vestigium detected in odontophore area. Two to eleven glandular bodies observed in all males in posterior part of the slender pharynx and pharyngeal bulb. In two specimens lens-like hemizonion at a distance of 242 and 271 μm from anterior end observed. Pharyngeal bulb slightly less than 1/4 of neck length (22.9 ± 1.6 (20.9-26.7%). Ventromedian supplements composed of one adanal pair and a row of 13-17 irregularly spaced single ones, the first three appear as double in some specimens. Spicules comparatively slender, of almost equal width along the length, curved to almost at right angle. Lateral guiding piece not bifid, with uneven internal walls. Post-cloacal papilla well developed. Tail short, bluntly conoidal, ventral side almost straight, dorsal side convex. Two or three pairs of lateral caudal pores.

Juveniles. Four developmental stages clearly present (Fig. 11) as determined from the position of the replacement odontostyle and the principal morphometric characters of body, odontostyle and replacement odontostyle lengths, and developing gonad (genital primordium) size. The habitus of juveniles not changing considerably during successive stages, assuming J or C shape. In first stage juvenile, lip region somewhat different from the next stages, it is rounded with a very weak depression after the second circle of labial papillae, the latter slightly protruding and changing the lip region outline. Amphidial fovea in first two stages has no clearly visible receptors, only small refractive elements discernable. Both the tail and body width at anus is increasing in length and c’ ratio is decreasing. Tail shape in J1 is conoidal, ventrally almost straight or slightly concave, dorsally convex, which gives asymmetrical appearance, in successive stages it gradually becomes rounded but always with the dorsal curvature more strongly expressed.

Differential diagnosis and relationships.

Longidorus carniolensis is an amphimictic species, characterized by females with a moderately long (L=5.6-8.2 mm) and plump (a=51-72) body, assuming a spiral to C-shape when heat relaxed; head region continuous, anteriorly almost flat, lip region 23-25 µm wide, guiding ring situated posteriorly (42-47 μm, 43-50 μm in males), long odontostyle (146.6 (136-157) μm), distribution of pharyngeal glands normal, nuclei of approximately equal size, tail bluntly conoid to hemispherical. Males abundant, spicules slender and long (122-145 μm), ventromedian supplements 13-17, irregularly spaced and preceded by an adanal pair. Postembrional development through four juvenile stages.

The codes for identifying the new species when using the polytomous key by Chen et al. (1997) are: A 56, B 4, C 4, D 1, E 4, F 35, G 1, H 1, I 2. The species belongs to the group of species with long odontostyle - over 100 μm and bluntly conoid to hemispherical tail: Longidorus poessneckensis , Longidorus macrosoma , Longidorus caespiticola , Longidorus helveticus , Longidorus macroteromucronatus Altherr, 1974, Longidorus raskii Lamberti & Agostinelli, 1993, Longidorus kheirii Pedram, Niknam, Robbins, Ye & Karegar, 2008, Longidorus pius Barsi & Lamberti, 2001, L. nevesi Macara, 1986, Longidorus major Roca & d’Erico, 1987, Longidorus carpathicus , Longidorus piceicola , Longidorus vinearum Bravo & Roca, 1995, Longidorus pauli Lamberti, Molinari,De Luca, Agostinelli & Di Vito, 1999, Longidorus arthensis , Longidorus iuglandis Roca, Lamberti & Agostinelli, 1985, Longidorus picenus Roca, Lamberti & Agostinelli, 1985, Longidorus silvae Roca, 1993, Longidorus uroshis , Longidorus saginus Khan, Seshardi, Weischer & Mathen, 1971, Longidorus orongorongensis Yeates & Van Etteger, 1992, Longidorus cretensis Tzortzakakis, Peneva, Terzakis, Neilson & Brown, 2001, Longidorus cylindricaudatus Krnjaić, Roca, Krnjaic & Agostinelli, 2005, Longidorus fasciatus Roca & Lamberti, 1981 and Longidorus litchii . Longidorus carniolensis sp. n. can be differentiated from all these species either by morphometrics or/and quantitative characters. It differs from:

Longidorus poessneckensis - by its somewhat longer odontostyle (ave. 147.5 (136-157) vs ave. 133 (122-142), ave. 126 (122-130) and ave. 140.2 (132-148) μm); more posteriorly sitated guiding ring (ave. 44.6 (42-47) vs ave. 40 (36-43) and 39 ave. (37-40) μm); tail short conoidal vs elongate conoid in J1 ( c’=1.2– 1.5 vs 1.8-2.2 and 1.8-2.5); males abundant vs males very rare ( Sturhan and Loof 2001, Lišková and Kumari 2010, Kornobis and Peneva 2011);

Longidorus macrosoma - by its shorter (5.6-8.2 vs 8.4-11.9 mm) and more plump (a=51-72.4 vs 77-113 and 105-126) body; differently shaped lip region (laterally rounded vs truncated, flattened); somewhat longer odontostyle (136-157 vs 113-148 μm); different tail shape in J1, bluntly conoidal vs mucronate; longer spicules in males 122-145 vs 105 μm and ave. 116.2 (112-121) μm ( Hooper 1961, Lamberti et al. 2001);

Longidorus caespiticola - by its longer odontostyle (136-157 vs 100-120, 96-109 μm); different numbers of dorsal (2-5 vs a single pore) and ventral (7-10 vs 5-6) pores; more posteriorly sitated guiding ring (42-47 vs 30-41, 37, 42.5 μm); longer spicules in males (122-145 vs 90, 80-95 μm), smaller c’ value in J1 (1.2-1.5 vs almost 2) ( Hooper 1961, Širca and Urek 2009);

Longidorus helveticus - by different tail shape of J1 being bluntly conoid vs mucronated; longer (122-145 vs 104-118 μm) and differently shaped spicules ( Lamberti et al. 2001);

Longidorus macroteromucronatus - by having more posteriorly situated guide ring (42-47 vs 38 μm); thicker cuticle along the body (6-7 vs 4 μm) and on tail region (9-10.5 vs 13-15 μm); longer odontostyle (136-157 vs 133 μm) ( Altherr 1974);

Longidorus raskii - by its wider lip region (23-25 vs 15-19 and 14-16 μm); more posteriorly situated guiding ring (42-47 vs 33-38 and 33-43 μm); longer odontostyle (136-157 vs 90-103 and 98-100 μm);longer spicules (122-145 vs 82-103 and 79-90 μm) ( Lamberti and Agostinelli 1993, Lamberti et al. 2001, Krnjaić et al. 2002);

Longidorus kheirii - by itslonger odontostyle (136-157 vs 111-130 μm); different tail shape in J1 (bluntly conoidal vs mucronated); males abundant vs males rare; longer spicules (122-145 vs 80 μm) ( Pedram et al. 2008);

Longidorus pius - by its more posterior position of the guiding ring (42-47 vs 35-42 and 37-42.5 μm); different tail shape in J1 (bluntly conoidal vs mucronated); males abundant vs males absent ( Barsi and Lamberti 2001a, Barsi and De Luca 2008)

Longidorus nevesi - by having wider lip region (23-25 vs 16-22 μm), different amphidial fovea shape (pouch like, not bilobed vs bilobed); differently shaped and longer spicules in males (122-145 vs 87-100 μm) ( Macara 1986);

Longidorus major - by having shorter body (L=5.6-8.2 vs 8.5-12 mm); somewhat narrower lip region (23-25 vs 22-27 μm); different tail shape in J1 (bluntly conoidal vs mucronate) and amphidial fovea (pouch like, not bilobed vs bilobed), males abundant vs males absent ( Roca and d’Erico 1987);

Longidorus carpathicus - by its longer body (L=5.6-8.2 mm vs 6.2-6.5 mm); wider (23-25 vs 16-18 μm) and differently shaped lip region; lower c’ value ( c’ = ave. 0.6 (0.5-1.0) vsc’ = 0.8); different shape in J1 (bluntly conoidal vs mucronated with a rather long mucro); males abundant vs males absent ( Lišková et al. 1997);

Longidorus piceicola - by having longer body (L=5.6-8.2 vs 4.2-6.5, 4.4-8.0 and 5.2-7.9 mm); wider (23-25 vs 14-17 μm) and differently shaped lip region (continious, almost flat vs broadly rounded); lower c’ value ( c’ =ave. 0.6 (0.5-1.0) vsc’=0.9– 1.3); differently shaped tail in J1 (bluntly conoidal vs elongate conoid) ( Lišková et al. 1997, Barsi and Lamberti 2001b);

Longidorus vinearum - by having different lip region shape (abruptly vs gradually tapering), different shape of amphidial fovea (pouch like not bilobed vs irregularly bilobed); longer odontostyle (136-157 vs 105.5-132.5 μm); different tail shape in J1 (bluntly conoidal vs conical, c’=1.2– 1.5 vs 1.9-2.8) ( Bravo and Roca 1995);

Longidorus pauli - by having different (continious vs slightly offset) and wider (23-25 vs 14-17 μm) lip region, amphidial fovea pouch like, not bilobed vs bilobed; longer odontostyle (136-157 vs 102-118 μm); lower a and c’ values (a=51.0-72.4 vsa=120.3-143.5; c’ = ave. 0.6 (0.5-1.0) vsc’ =0.9 (0.8-1.0), respectively); more posteriorly situated guiding ring (42-47 vs 27-36 μm); longer spicules (122-145 vs 61-69 μm); different tail shape in J1 (bluntly conoidal vs subdigitate) ( Lamberti et al. 1999);

Longidorus arthensis - by itswider (23-25 vs 14-17 μm) lip region, amphidial fovea pouch like not bilobed vs bilobed; longer odontostyle (136-157 vs 102-111 μm); lower c’ values ( c’ =av 0.6 (0.5-0.1) vsc’ =av 0.9 (0.8-1.1); more posteriorly sitated guiding ring (42-47 vs 30-38 μm); longer spicules (122-145 vs 60-66 μm); different tail shape in J1 (bluntly conoidal vs mucronated) ( Brown et al. 1994);

Longidorus iuglandis - by its wider lip region (23-25 vs 14-16 μm); amphidial pouches not bilobed vs bilobed; longer odonostyle (136-157 vs 112-128 μm); more posterior position of the guiding ring (42-47 vs 31-41 μm); longer tail (34-69 vs 33-41 μm); longer spicules (122-145 vs 93-99 μm); different tail shape in J1 (bluntly conoidal vs mucronated) ( Roca et al. 1985);

Longidorus picenus - by its wider lip region (23-25 vs 19-22 μm); amphidial fovea not bilobed vs bilobed; more posterior position of the guiding ring (42-47 vs 31-41 μm); longer spicules (122-145 vs 103-112 μm); different tail shape in J1 (bluntly conoidal vs mucronated) ( Roca et al. 1985);

Longidorus silvae - by its more plump body (a=51.0-72.4 vsa=87.5-137.5 in Italian population and a=87.4-116 in Serbian populations), wider lip region (23-25 vs 14-17 μm); amphidial fovea not bilobed vs bilobed; longer odontostyle (136-157 vs 113.5-133 μm (Italian population) and 108-136 μm (Serbian populations)); different tail shape in J1 (bluntly conoidal vs mucronated); males abundant vs males rare; longer spicules (122-145 vs 77-78 μm) ( Roca 1993, Barsi and Lamberti 2004, Barsi et al. 2007);

Longidorus uroshis - by having wider (23-25 vs 15-20.5 μm) lip region; lower a values (a=51.0-72.4 vs a=96.9-108.9); different tail shape in J1 (bluntly conoidal vs mucronated); longer spicules (122-145 vs 59-72 μm) ( Krnjaić et al. 2000);

Longidorus saginus - by having longer body (L=5.6-9.5 vs 4.8-6.4 mm); amphidial fovea pouch shaped not bilobed vs asymetrically bilobed; longer tail (34-69 vs 21-33 μm) ( Khan et al. 1971);

Longidorus orongorongensis - by its more anterior position of the guiding ring (42 -47 vs 63-73 μm); shorter odontostyle (136-157 vs 152-166 μm); longer spicules (122-145 vs 84-87 μm) ( Yeates et al. 1992);

Longidorus cretensis - by having normal vs abnormal location of pharyngeal glands; wider lip region (23-25 vs 17-21 μm); longer spicules (122-145 vs 71-91 μm); different tail shape in J1 (bluntly conoidal vs conoid pointed) ( Tzortzakakis et al. 2001);

Longidorus cylindricaudatus - by having lip region abruptly vs gradually tapering; amphidial fovea not bilobed vs bilobed; shorter odontostyle (136-157 vs 164-178 μm); lower a values (a=51-72.4 vsa=94.4-113.4); males abundant vs males absent ( Krnjaić et al. 2005);

Longidorus fasciatus - by its wider lip region (23-25 vs 12-14 μm); different amphidial poches (not bilobed vs asymmetrically bilobed); longer odontostyle (136-157 vs 102-119 μm; male abundant vs males absent ( Roca and Lamberti 1981);

Longidorus litchii - by its somewhat shorter odontostyle (136-157 vs 138-171 mm); different amphidial poches (not bilobed vs bilobed); more anterior postion of the guiding ring (42-47 vs 82.5-96.5 μm); different tail shape in J1 (bluntly conoidal vs elongate conoid with long digitate tip, c’=1.2– 1.5 vsc’=2.7– 3.4); longer spicules (122-145 vs 68.5-71 μm) ( Xu and Cheng 1992).

Type-locality and plant association.

An old vineyard with roots of several Vitis vinifera varieties close to Drašiči village in southern part of Slovenia (45°39'N; 15°23'E), 229 m above sea-level.

Other-locality: a vineyard close to Krmačina village in southern part of Slovenia.

Distribution notes.

Longidorus carniolensis n. sp were detected in 6 out of 10 soil samples from locations of Drašiči and Krmačina. Population density was 4-15 specimens of all developmental stages per 200 cm3 of soil sample.

Type-material.

Holotype female and 2 female, 5 male and 8 juvenile (3 J1, 1 J2, 2 J3, 2 J4) paratypes deposited in the Nematode Collection of Agricultural Institute of Slovenia, Ljubljana, Slovenia; two female, two male and 10 juvenile (3 J1, 2 J2, 2 J3, 2 J4) paratypes - in the Wageningen Nematode Collection (WaNeCo), Wageningen, the Netherlands; one female, one male and 6 juvenile (3 J1, 1 J2, 1 J3, 1 J4) paratypes - in the Nematode Collection of The Food and Environment Research Agency, Sand Hutton, UK (former Rothamsted Nematode Collection); one female, three male and 6 juvenile (3 J1, 2 J3, 1 J4) paratypes - in the Nematode Collection of the Zoology Museum, Ghent University, Belgium; one female, three male and two juvenile (1 J3, 1 J4) paratypes - in the Nematode Collection of the Institute of Plant Protection, Bari, Italy; two female, one male and 6 juvenile (3 J1, 1 J2, 1 J3, 1 J4) paratypes - in the Nematode Collection of the University of California at Riverside, USA; one female, one male and two juvenile (1 J3, 1 J4) paratypes - in the USDA Nematode Collection, Beltsville, Maryland, USA; 4 female, 5 male and 8 juvenile (3 J1, 1 J2, 2 J3, 2 J4) paratypes - in the Nematode Collection of the Institute of Biodiversity and Ecosystem Research, BAS, Sofia.

Etymology.

The species epithet carniolensis was derived from Carniola which is the Latin name of the Kranjska province, a historical region that comprised parts of what is now Slovenia.

rDNA sequence analysis.

Cluster analyses of the D2-D3 expansion regions of the 28S rDNA nuclear gene sequences of Longidorus carniolensis sp. n. and closely related species (Table 1) were performed and a phylogenetic tree was constructed (Fig. 12). The sequences of both populations of Longidorus carniolensis sp. n.from Drašiči and Krmačina were identical. They formed a distinct clade within a cluster of the closely related sequences of Longidorus poessneckensis , Longidorus helveticus , Longidorus macrosoma , Longidorus caespiticola and Longidorus latocephalus . The closest sequence to Longidorus carniolensis sp. n.was the sequence of Longidorus poessneckensis (Acc. No EF538750) with 91.9% of similarity.