Longidorus polyae Lazarova, Elshishka, Radoslavov & Peneva

Lazarova, Stela S., Elshishka, Milka, Radoslavov, Georgi, Lozanova, Lydmila, Hristov, Peter, Mladenov, Alexander, Zheng, Jingwu, Fanelli, Elena, Francesca De Luca, & Peneva, Vlada K., 2019, Molecular and morphological characterisation of Longidoruspolyae sp. n. and L. pisi Edward, Misra & Singh, 1964 (Dorylaimida, Longidoridae) from Bulgaria, ZooKeys 830, pp. 75-98 : 77-82

Longidorus polyae Lazarova, Elshishka, Radoslavov & Peneva sp. n.

Description.

(for measurements see Table 1, Figs 1-7)

Female. Body assuming a spiral shape. Lip region narrow, 5-6 μm high, continuous with body profile, anteriorly flattened. Labial papillae, especially second circle, prominent, changing slightly the body contour. Cuticle 4-5 μm thick at postlabial region, 4-5 μm along the body, and 9-10 μm on tail posterior to anus. Guide ring 4-6 μm wide. Body pores conspicuous, 1 lateral pore anterior to or at the level of guide ring, 2 or 3 along odontostyle, 1 or 2 along odontophore, 3-5 in narrow part of the pharynx and 2-4 in bulb region as well as none dorsal and 5-6 ventral in pharyngeal region; numerous lateral pores observed along the rest of body. Amphidial fovea prominent, deeply bilobed, lobes long, slightly asymmetrical, amphidial aperture assumed to be a minute pore, hardly visible under light microscope. Odontostyle very slender, 1.5-2 μm wide at base. Two nerve rings observed, the first at some distance behind the odontophore and at 246.3 ± 11.1 (230-254) μm from anterior end, the second, more prominent, behind the first one at 354.8 ± 48.1 (327-440) μm from anterior end. Pharyngo-intestinal valve broadly rounded. Normal arrangement of pharyngeal glands: nuclei of the dorsal and ventrosublateral glands situated at 22.7-28.5 % and 54.3-58.5 % (n = 5) of the distance from anterior end of the bulb. Dorsal gland nuclei 2-2.5 μm in diameter, subventral gland nuclei 3-4 μm in diameter. In odontophore area of one female a small rudimentary odontostyle tip (vestigium) observed pointing forward. Peculiar crystalloid bodies of various sizes and shapes (mostly rod-like) found in the intestine of all females. Prerectum 465-497.5 μm long and rectum 33-42 μm or 0.6-0.8 of body diameter at anus. Tail bluntly conoid, rounded to hemispherical. Two pairs of lateral caudal pores. Vagina extending to ca. half the corresponding body width. Pars distalis vaginae 20-26 μm long; pars proximalis vaginae 21-27 μm long, thick walled. Uterus bipartite, moderately long, anterior uterus 230-406, posterior uterus 235-394 μm long, respectively; well-developed sphincter between uterus and pars dilatata oviductus, pars dilatata, and uteri containing numerous sperm cells; ovary small.

Male. Rarer than females. Habitus as in females, posterior part more strongly coiled ventrally. Shape of lip region similar to that in females. Cuticle 4.0-4.5 μm thick at postlabial region, 4 μm along the body and 5 μm on tail posterior to cloaca. One lateral pore anterior to or at the level of guide ring, 2 along odontostyle, 1 or 2 along odontophore, 3 or 4 in narrow part of the pharynx and 2 or 3 in pharyngeal bulb region, no dorsal and 6 ventral pores; numerous lateral pores present along the rest of the body. Two nerve rings observed, the first just behind the odontophore at a distance of 240.7 ± 12.0 (229-253) μm from anterior end, the second, more prominent behind the first one at 324 ± 12.7 (307.5-333) μm from anterior end. A small rudimentary odontostyle tip (vestigium) pointing forward observed in all males; in odontophore area (in 2 specimens) and in the slender part of pharynx (in 2 specimens). Pharyngo-intestinal valve broadly rounded. Tail short, bluntly conoid, dorsally convex, ventrally first straight then slightly concave. Three pairs of lateral pores on tail. One adclocal pair preceded by a row of 10 or 11 ventromedian supplements. Spicules slender, curved ventrally, lateral guiding piece sigmoid, 20-22 μm long. Spermatozoids oval (6-8 μm long).

Juveniles. Four juvenile stages can be differentiated based on the body, odontostyle, and replacement odontostyle length (Figs 5-7, Table 1). Habitus more or less an open C-shape, tail of the first stage juvenile digitate, with 9-11 μm long ventral peg, whereas in the subsequent developmental stages, bluntly rounded, c’ decreasing.

Type locality and plant association.

Balgarene village, Pre-Balkan zone of the Balkan Mountains, north-central Bulgaria (43°2'48.08"N; 24°46'24.53"E), 386 m a.s.l., private orchard, rhizosphere of Pyrus communis L.

Type material.

The holotype (PNT 42), 20 paratype females, 2 males, and 92 juveniles (overall PNT 43-101) from all stages are deposited in the nematode collection of the Institute of Biodiversity and Ecosystem Research, BAS, Sofia, Bulgaria; 1 female, 1 male and 6 juveniles in the USDA Nematode Collection, Beltsville, Maryland, USA; 1 female, 1 male and 6 juveniles in the Wageningen Nematode Collection (WANECO), Wageningen, the Netherlands; 1 female and 8 juveniles in the Nematode Collection of the Institute of Sustainable Plant Protection, Bari, Italy.

Molecular characterisation.

The NCBI BLASTn search of D2-D3 expansion segments of the 28S rRNA gene sequence showed highest similarity (93-94%) to several Longidorus species ( L. attenuatus Hooper, 1961; L. dunensis Brinkmam, Loof & Barbez, 1987; L. athesinus Lamberti, Coiro & Agostinelli, 1991; L. persicus Esmaeili, Heydari, Archidona-Yuste, Castillo & Palomares-Rius, 2017; L. euonymus Mali & Hooper, 1974; Longidorus sp. 1, and Longidorus sp. 3). The highest identity (93.0-93.1%) was calculated with populations identified as L. attenuatus and L. dunensis (accessions KT755457 and AY593057, respectively). However, in the 28S rDNA phylogenetic analyses L. polyae sp. n. grouped in a clade with four Longidorus spp. ( L. athesinus , Italy; Longidorus sp. 1, Longidorus sp. 2, and Longidorus sp. 6 from USA) with intermediate to high PP support (0.7-1.0) depending of the applied MSA algorithm (Fig. 9). The phylogenetic position of the new species based of 18S rRNA gene remained unresolved (Fig. 10) and was changing when different MSA algorithms and outgroups were tested. The pairwise sequence comparisons revealed highest identity (99.2%) with 18S rDNA sequences of L. attenuatus (AY687994), L. elongatus (de Man, 1876) Micoletzky, 1922 (EU503141), L. piceicola Liskova, Robbins & Brown, 1997 (AY687993), and L. uroshis Krnjaić, Lamberti, Krnjaić, Agostinelli & Radicci, 2000 (EF538760) (or 1564 identical residues of 1577 MSA length).

Diagnosis and relationships.

Longodorus polyae sp. n. is a comparatively large bisexual species average 7.98 (6.81-9.12 mm) with the odontostyle over 100 μm (114.0-127.5 μm) long; the lip region narrow (14.0-15.5 μm), almost continuous with body profile, anteriorly flat; amphidial fovea long, pocket-shaped, deeply bilobed, and lobes slightly asymmetrical; normal arrangement of pharyngeal glands; and tail short, bluntly rounded to hemispherical, four juvenile stages present, with the tail of the first-stage juvenile digitate.

The alpha-numeric codes for L. polyae sp. n. to be applied to the polytomous identification key for Longidorus species by Chen et al. (1997) and partial polytomous key proposed by Peneva et al. (2013) are: A5, B2, C34, D3, E3, F45, G12, H1, I2, J1, K7 (Table 2).

The group of large Longidorus species (code F34) with a moderately long odontostyle (code A45), pocket-shaped amphidial fovea, bilobed, symmetrical (code E2) or asymmetrical (code E3), normal arrangement of pharyngeal glands nuclei, short rounded tail (code H1) and digitate tail or tail with mucro (code K7) (according to Peneva et al. 2013) consists of a few species, namely: L. arthensis ; L. pauli Lamberti, Molinari, De Luca, Agostinelli & Di Vito, 1999; L. kheirii Pedram, Niknam, Robbins, Ye & Karegar, 2008; L. silvae Roca, 1993; and L. uroshis (Table 2). The new species differs from these by the presence of peculiar inclusions in the intestine. Furthermore, it differs from:

L. pauli by females having differently shaped amphidial pouches (asymmetrically vs symmetrically bilobed); plumper body (a = 95.7-119.5 vs 120.3-143.5); more posterior guide ring position (37-42 vs 27-36 μm); longer spicules (71.0-74.5 vs 61-69 μm); guiding piece in male sigmoid vs straight; different shape of tail in second- and third-stage juveniles (bluntly rounded vs conical) ( Lamberti et al. 1999);

L. uroshis by females having a longer body (6.81-9.12 vs 5.6-7.6 mm); narrower lip region (average 14.6 (14.0-15.5) vs average 17 (15.0-20.5) μm), higher c (184.8-260.0 vs 120.4-162.0) and lower c’ values (0.7-0.8 vs 0.9-1.0) ( Krnjaić et al. 2000);

L. kheirii by females having a narrower lip region (14.0-15.5 vs 19.5-23.0 μm) and pharyngeal bulb (25-29 vs 39.5-48 μm), higher c values (184.8-260.0 vs 119.0-167.8), amphidial pouches deeply bilobed vs not to slightly bilobed; differently shaped tail of the first- and second-stage juvenile and different ovarium structure ( Pedram et al. 2008).

L. arthensis by females having longer body (6.81-9.12 vs 5.14-6.74 mm) and odontostyle (114.0-127.5 vs 102.0-111.0 μm); asymmetrically vs evenly bilobed amphidial pouches, lower c’ values (0.7-0.8 vs 1.0); males with longer spicules (71.0-74.5 vs 60.0-66.0 μm) ( Brown et al. 1994);

L. silvae by females having two vs one nerve rings, differently shaped tail of the first-stage juvenile (subcylindrical, rarely cylindrical part/mucro with ventral position vs cylindrical mucro with central position), mucro shorter (9-11 vs 20-27 μm), higher c values (average 211.3 (184.8-260.0) vs average 166.7 (132.0-189.0) and posteriorly located vulva (50.9 (49.2-52.6) vs 48.6 (44.9-50.7) ( Roca 1993). Males common vs absent ( Roca 1993) or rare ( Barsi and Lamberti 2004; Barsi et al. 2007).

Additionally, it can be differentiated from L. athesinus , a phylogenetically related species (Fig. 9), by females having longer body (6.81-9.12 vs 3.7-5.8 mm) and odontostyle (114.0-127.5 vs 83.5-94.0 μm); higher a value (95.7-119.5 vs 56.2-88.1); differently shaped tail in first-stage juvenile (digitate vs bluntly conoid) ( Lamberti et al. 1991);

Morphometrical data of the most similar species are presented in Table 3.

Etymology.

Named after the first author’s sister Mrs Polya Kadiyska, a school teacher of art and iconography at the "Nikola Obretenov" Primary school in Rousse, Bulgaria.