See Table 2

Female. Body plump, assuming a C to open spiral shape. Lip region continuous, anteriorly rounded. Labial papillae prominent. Cuticle 8–10 μm thick at poslabial region, 5–7 μm along the body and 12–14 μm on tail posterior to anus. Guide ring 6–7 μm wide. One lateral pore anterior to guide ring, 2–4 along odontostyle, 1–2 along odontophore, 4–5 in narrow part of the oesophagus and 3–4 in bulb region as well as 3–5 dorsal and 7–10 ventral; numerous lateral body pores observed. Amphidial fovea pouch like, short, almost as wide as long, funnel shape with code E5 according to Chen et al. (1997) and type 4 according to Decraemer and Coomans (2007), amphidial aperture assumed to be a minute pore, hardly visible under light microscope; fusus (sensillium pouch) at 51.6±2.7 (49.5–56) μm, n=7 from anterior end. Odontostyle slender, 2 μm wide at base. Pharyngo-intestinal valve, variable in shape (broadly rounded to heart-shape) and size, slightly wider than long: 19±1.4 (17–20) × 15.4±3.1 (12–19) μm, n=5. Normal arrangement of pharyngeal glands: nuclei of the dorsal and subventral glands situated at 23.6–32.1 % (n=3) and 50.7–58.9 % (n=8) of the distance from anterior end of the bulb. Dorsal gland nuclei 2 μm diam., subventral gland nuclei 3–4 μm diam. Nerve ring surrounding odontophore base, at 222.9±11.3 (203–242.5) μm from anterior end, a second nerve ring situated at a short distance behind the first one. Lateral chord 25–29 μm wide. Vagina extending to ca. half corresponding body width. Pars distalis vaginae 23–27 μm long; pars proximalis vaginae 28–35 μm long, thick walled. Uteri very long, anterior uterus 481.0±105.1 (372.5–662.5), posterior uterus 473.2±114.2 (357.5–660) μm long, respectively; well developed sphincter between uterus and pars dilatata oviductus, pars dilatata and uteri usually containing numerous sperm cells. Prerectum 426.9±79.7 (310–595) μm long, rectum 45.5±1.6 (43–48) μm or about 0.7–0.8 of body diameter at anus. Tail bluntly conoid, rounded to hemispherical. Two pairs of lateral pores.

Male. Habitus as in females, posterior part more strongly coiled ventrad. Shape of lip region similar to that in females. Cuticle 5–8 μm thick at poslabial region, 7–9 at guiding ring level, 4–6 μm along the body and 9–13 μm on tail posterior to cloaca. One lateral pore anterior to guide ring, 2–3 along odontostyle, 1–2 along odontophore, 3–5 in narrow part of the oesophagus and 3–4 in pharyngeal bulb region as 4 dorsal and 7–10 ventral; numerous lateral body pores present. Fusus at 52.3±3.7 (47–57) μm, n=7 from anterior end. Nerve ring surrounding odontophore base, at 231.8±12.2 (217.5–259.5) μm from anterior end, a second nerve ring situated at a short distance behind the first one. Pharyngo-intestinal valve, variable in shape (broadly rounded to heart-shape) and size, almost as long as wide: 16.6±3.2 (13–23) × 18±3.1 (13–22) μm, n=6. Lateral chord 20–25 μm wide. Supplements 3–4 adanal pairs followed by 10–14 arranged irregularly in a single row. Spicules massive, slightly curved ventrally, lateral guiding piece 27–28 μm long. Spermatozoids round small (4–6 μm diam.). Tail short, bluntly conoid, dorsally convex, ventrally slightly concave, three pairs of lateral pores.

Juveniles. Morphometrics obtained from juvenile specimens, and of the relationship between the lengths of their functional and replacement odontostyles and body lengths, confirmed the presence of four juvenile stages (Figure 9). Habitus in the shape of more or less open C, tail of the first stage juvenile conoid elongated whereas in the subsequent developmental stages the tail is conoid (second stage) to bluntly conoid (third and fourth stage).

Bachevo village, Rila Mountains, co-ordinates 41°56'14.97"N, 23°25'15.02"E, 1032 m asl, riparian vegetation; soil around the roots of wild cherry (Prunus avium L.), Juniperus communis L., Urtica dioica L. and grasses.

Holotype and 1 paratype females, 2 males, and 23 juveniles deposited in the nematode collection of the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria. Other paratypes deposited as follows: two females, one male and 8 juveniles in the Nematode collection of the Foodand Environment Research Agency, Sand Hutton, UK (former Rothamsted Nematode Collection); one female, one male and 6 juveniles in the USDA Nematode Collection, Beltsville, Maryland, USA; one female, one male and 8 juveniles in the Riverside Nematode Collection, University of California, Riverside, USA; one female, one male and 5 juveniles in the Nematode Collection of the Institute of Plant Protection, Bari, Italy; one female, one male and 12 juveniles in the Wageningen Nematode Collection (WANECO), Wageningen, the Netherlands.

Longidorus cholevae sp. n. is a comparatively large bisexual species (6.1–8.1 mm) with odontostyle over 100 μm (106–129 μm) long, lip region wide (21.5–24 μm), continuous, anteriorly rounded, amphidial fovea pouch like, almost as wide as long, posteriorly situated guide ring, short, bluntly rounded to hemispherical tail and normal arrangement of pharyngeal glands.

The alpha-numeric codes for Longidorus cholevae sp. n. to be applied to the polytomic identification key for Longidorus species by Chen et al. (1997) are, A45, B4, C23, D1, E5, F34, G12, H1, I2. The group of comparatively large Longidorus species (code F34) with a long odontostyle (code A45), pouch like amphidial fovea, elongate funnel (E4) or short (funnel or stirrup shaped (E5), normal arrangement of pharyngeal glands and short rounded tail (code H1) consists of a few species: Longidorus poessneckensis, Longidorus caespiticola, Longidorus macrosoma, Longidorus helveticus, Longidorus carniolensis, Longidorus macroteromucronatus Altherr, 1974, Longidorus pseudoelongatus Altherr, 1976, Longidorus pius. It differs from all these species except for Longidorus caespiticola and Longidorus pseudoelongatus, by the more anteriorly situated guide ring (ave. 32.6 (30–37) vs ave. 40 (36–43) μm in Longidorus poessneckensis; 37–48 μm in Longidorus macrosoma; 37.5–48 μm in Longidorus helveticus; 42–47 μm in Longidorus carniolensis; 38 μm in Longidorus macroteromucronatus and ave. 38.7 (35–41) μm in Longidorus pius). Among the above group the new species appears most similar to Longidorus poessneckensis from which itdiffers by adult specimens having different shape of amphidial pouches (almost as long as wide vs visbly longer than wide), males abundant vs males rare and different tail shape in first stage juveniles (elongate conoid with narowly tapering terminus vs elongate conoid with blunly rounded terminus) (Sturhan and Loof 2001, Kumari et al. 2009, Lišková and Kumari 2010, Kornobis and Peneva 2011). Further, it can be differentiated from:

Longidorus caespiticola by females having wider (21.5–24 vs 16–18 μm) and differently shaped lip region (rounded vs smoothly rounded, almost conical), shorter tail (28.5–38 vs 39–47 μm) and longer spicules (96–120vs 88.5–93 μm), and tail in first stage juveniles (elongate conoid vs bluntly conoid) (Boag and Brown 1975);

Longidorus macrosoma by female specimens having a somewhat shorter body (L= ave. 6.8 mm (6.1–8.1) vs ave. 9.1 mm (6.8–12), diferently shaped lip region (rounded vs slightly concave) and differently shaped tail of the first stage juvenile (elongated conoid vs digitate) (Brown and Boag 1975);

Longidorus helveticus by females having different shape of amphidial fovea (almost rounded vs elongated), shorter odontostyle (ave. 120.1 (106–129) μm vs ave. 135. 4 (127–145.5) in the type population and reported range for other populations 127–142 μm, differently shaped tail in first stage juvenile (elongated conoid vs mucronated) and shorter hyaline portion of tail (J=10–14 vs J=17.5–33 μm) (Lamberti et al. 2001, Barsi and De Luca 2005, Širca and Urek 2009, Kumari and Subbotin 2012);

Longidorus carniolensis – by having a shorter odontostyle (106–129 vs 136–157 μm); males with shorter spicules (96–120 vs 122–145 μm); different tail shape in first stage juvenile (elongate conoid vs rounded, conoidal, c’=1.8–2.2 vs c’=1.2–1.5) (Širca et al. 2011);

Longidorus macroteromucronatus – by females having wider lip region (21.5–24 vs 17.5 μm (calculated from the drawing by Altherr (1974), shorter odontostyle (106–129 vs 133 μm) and higher c values (c=171.2–220.4 vs c=160);

Longidorus pius – by different d and d’ values (following Brown et al. 1994) (d=1.2–1.4 vs d=1.7–1.9; d’=1.4–1.6 vs d’=1.9–2.1), shorter odontostyle (ave. 120.1 (106–129 vs ave. 136.5 and 137.5 (128–147.5) μm), shorter tail (28.5–38 vs 37–46.5 μm), higher c value (c=171.2–220.4 vs c=114.6–166.5) in females; males abundant vs males rare, and different tail shape in first stage juveniles (elongate conoid vs subdigitate, J=10–14 vs J=15–20 μm) (Barsi and Lamberti 2001, Barsi and De Luca 2008). Although in the original description the code for amphidial fovea shape is D1, but in the photos it appears more like that in the new species;

Longidorus pseudoelongatus – by having a longer body (L=6.1–8.1 vs L=5.1–5.6 mm), differently shaped (continious vs separated by constriction) and wider lip region (21.5–24 vs 12 μm), higher c (c=171.2–220.4 vs c=100–150) and lower c’ values (c’=0.5–0.7 vs c’=0.93) (Altherr 1976).

Further, Longidorus cholevae sp. n. is similar in body and odontostyle lengths (codes F34 and A45), and shape of anterior region and tail (codes D1 and H1) with a group of several other species from which it differs in amphidial fovea shape (see Appendix 1: a partial polytomous key): Longidorus kheirii, Longidorus raskii Lamberi & Agostinelli, 1993, Longidorus arthensis Brown, Grunder, Hooper, Klingler & Kunz, 1994, Longidorus fasciatus Roca & Lamberti, 1981, Longidorus uroshis Krnjaić, Lamberti, Krnjaić, Agostinelli & Radicci, 2000, Longidorus silvae Roca, 1993, Longidorus iuglandis Roca, Lamberti & Agostinelli, 1984, Longidorus saginus Khan, Seshardi, Weischer & Mathen, 1971, Longidorus picenus Roca, Lamberti & Agostinelli, 1984, Longidorus baeticus). The new species can be distinguished from Longidorus raskii, Longidorus arthensis, Longidorus fasciatus, Longidorus uroshis, Longidorus silvae, Longidorus picenus and Longidorus baeticus by its wider lip region (21.5–24 μm vs 15–19 μm; 14–19 μm; 12–14 μm; 14–20.5; 14–17 μm; 14–16 μm; 19–22 μm; 12–14.5 μm); from Longidorus raskii, Longidorus uroshis and Longidorus saginus by having different odontostyle length (106–129 μm vs 76–103 μm; 120–152 μm and 135–155 μm); from Longidorus kheirii, Longidorus raskii, Longidorus arthensis and Longidorus uroshis by the shorter tail (28.5–38 vs 47–72 μm; 36–50 μm; 36–46.5 μm and 38–57 μm); from Longidorus kheirii, Longidorus silvae and Longidorus picenus by having more anteriorly situated guide ring (30–37 vs 36.5–45 μm, 36–48 μm and 37–42 μm). Additionally, it can be differentiated from:

Longidorus kheirii by females having differently shaped lip region (rounded vs slightly concave), higher c value (171.2–220.4 vs 119–167.8), smaller pharyngeal bulb (114.5–146.5 × 30–38 vs 149.5–193.5 × 39.5–48 μm), males abundant, functional vs rare and not functional, differently shaped tail of the first stage juvenile as well as different morphometrics concerning the main characters such as body and tail length, functional and replacement odontostyle length (Table 1; Table 2 in Pedram et al. 2008).

Longidorus raskii by females having different tail shape in first stage juveniles (elongate conoid vs bluntly conoid); (Lamberti et al. 2001, Krnjaić et al. 2002, Barsi and De Luca 2005);

Longidorus arthensis by females having lower c’ value (c’=0.5–0.7 vs c’=0.8–1.1 and 0.9–1.1); males with longer spicules (96–120 vs 60–66 μm); different tail shape in first stage juveniles (elongate conoid vs digitate) (Brown et al. 1994, Lamberti et al. 2001);

Longidorus fasciatus by females having a more plump body (a=61.1–83.3 vs a=121–143) (Roca and Lamberti 1981);

Longidorus uroshis by males with longer spicules (96–120 vs 59–72 and 64–78 μm) and different tail shape in first stage juveniles (elongate conoid vs digitated) (Krnjaić et al. 2000, Krnjaić et al. 2002, Sturhan and Lišková 2002);

Longidorus silvae by female specimens having differently shaped lip region (rounded vs sub-acute and flattened anteriorly) and tail of the first and second stage juvenile (elongated conoid vs mucronated; conoid vs bluntly rounded, respectively), and males abundant vs males rare (Roca 1993, Barsi and Lamberti 2004, Barsi et al. 2007).

Longidorus iuglandis by having longer uteri (357.5–662.5 vs 140–160 μm) and differently shaped tail in the first stage juvenile (elongate conoid vs bluntly rounded) (Roca et al. 1984);

Longidorus saginus by having a longer body (L=6.1–8.1 vs 4.8–6.4 mm); lower c’ value (c’’=0.5–07 vs c’=0.8); more posteriorly situated vulva (V=46.7–53.4 vs V=40–45) (Khan et al. 1971);

Longidorus picenus by having, differently shaped tail in the first stage juvenile (elongate conoid vs mucronated) (Roca et al. 1985);

Longidorus baeticus by males having longer spicules (96–120 vs 80–95 μm) and differently shaped tail in the first stage juvenile (elongate conoid vs bluntly rounded to cylindrical).

The species is named after Dr Boryana Choleva, Faculty of Biology, University of Sofia, retired, for her substantial contribution to the knowledge of the fam. Longidoridae in Bulgaria.

Phylogenetic relationships of Longidorus cholevae with other Longidorus species

The amplification of D2-D3 expansion domains of the 28S rDNA and the ITS containing region yielded single fragments of 800 bp and 1384 bp, respectively, based on sequencing. The ITS1 and ITS2 sizes were 579 bp and 338 bp, respectively that resulted in the shortest ITS recorded for Longidorus so far. Intra-individual and intra-population sequence variability in ITS and no variability in D2D3 domains have been observed.

A BLAST search for D2-D3 region showed a 80-93% degree of similarity among Longidorus spp. suggesting that Longidorus cholevae can be easily identified from other species by using this ribosomal region. The closest species were Longidorus poessneckensis (93% similarity), Longidorus caespiticola, Longidorus macrosoma and Longidorus helveticus (92% similarity). Pairwise BLAST comparisons of the ITS sequence of Longidorus cholevae with those of Longidorus spp. from the database displayed high nucleotide dissimilarity and considerable variation in length.

Our preliminary phylogenetic analyses based on all the D2-D3 Longidorus sequences deposited in NCBI revealed that the new species clusters into a well-supported group of Longidorus species having a European distribution: Longidorus caespiticola, Longidorus macrososma, Longidorus poessneckensis, Longidorus helveticus and Longidorus carniolensis (trees not presented). The monophyly of this group has been highly supported also in other studies, including SSU phylogenetic analyses (Robbins et al. 2009, Gutiérrez-Gutiérrez et al. 2013). All these are large species, very similar in their morphology having long odontostyles, elongated or short not bilobed pouch-like amphidial fovea, continuous head region, short bluntly conoid to almost hemisphaercial tail, mainly amphimictic (only with Longidorus macrosoma and Longidorus poessneckensis males are rare). Longidorus caespiticola and Longidorus macrososma occur mainly in western Europe including the British Isles, Longidorus poessneckensis was reported from central (Germany, Slovakia and Czech Republik) and northern Europe (Poland); the first two species were found in association with a wide range of crops and forest trees (Brown and Boag 1975, Boag and Brown 1975); Longidorus poessneckensis with preference toflood plains and hill deciduos forest habitat (Lišková and Kumari 2010) and Longidorus helveticus associated with deciduous forest and orchard threes in central Europe (Lamberti et al. 2001, Širca and Urek 2009, Kumari and Subbotin 2012). Longidorus carniolensis is known only from Slovenia (grapevine) and Longidorus cholevae sp. n. – only from Bulgaria (riparian vegetation). Probably, Longidorus pius, known so far only from Macedonia and having similar morphology, is part of this group, however, no sequences of D2-D3 region are available.

Further, for phylogenetic analysis Longidorus species from GenBank with the highest match of BLAST search were aligned along with Longidorus cholevae D2-D3 and partial 18S-ITS1 sequences and these alignments included sequences from various populations (Table 1). The trees obtained by NJ, ML and BI methods showed similar topology and differed in the position of poorly supported clades, and thus only the BI trees with posterior probabilities higher than 0.8 and bootstrap values above 70% (NJ and ML) are presented (Figs 10–11).

Figure 10.

Phylogenetic relationships of Longidorus cholevae sp. n. and its closest species for the D2-D3 rDNA. Bayesian Inference strict consensus tree acquired under GTR+G model. Numbers at the nodes indicating posterior probabilities higher that 0.8 and bootstrap values more that 70% for ML and NJ are presented.

Figure 11.

Phylogenetic relationships of Longidorus cholevae sp. n. and its closest species for the partial 18S-ITS1 rDNA regions. Bayesian Inference strict consensus tree acquired under K2+G model. Numbers at the nodes indicating posterior probabilities higher that 0.8 and bootstrap values more that 70% for ML and NJ are presented.

The phylogenetic tree of the D2-D3 region (Fig. 10) showed two well-supported clades: Clade I consists of three subclades: two highly supported subclades containing various populations of I1) Longidorus helveticus and I2) Longidorus macrosoma, and one subclade having lower values for ML bootstrap support (52%) and BI posterior probabilities (0.72) I3) that includes the new species Longidorus cholevae, two populations of Longidorus carniolensis from Slovenia and two populations of Longidorus poessneckensis from the Czech Republic and Slovakia. The second clade (II) consists of two well-supported subclades: II1) consisted of Longidorus caespiticola from Slovenia and Belgium and one Longidorus cf. caespiticola from Bulgaria and subclade II2) consisted of three populations of Longidorus caespiticola from Scotland, Belgium and Germany. It is possible that these populations represent two different species that requires further investigation.

The phylogenetic reconstructions of the partial 18S-ITS1 region revealed more unstable groups due to the shorter sequence length and higher sequence variability. Three of the Longidorus spp. belonging to the above mentioned group (Longidorus cf. caespiticola, Longidorus helveticus and Longidorus macrosoma)and two additional species(Longidorus pius and Longidorus raskii) originating from Macedonia and Switzerland have been separated from other ITS1 Longidorus sequences (the tree not presented) and further analysed (Fig. 11). Three clades were distinguished, two well supported clades consisting of: 1) Longidorus macrosoma, Longidorus helveticus and Longidorus pius and 2) Longidorus cf. caespiticola and Longidorus raskii, and one not well resolved 3) containing only Longidorus cholevae sp. n. The species forming these clades have similar tail shape in first stage juveniles: digitate in clade 1, bluntly conoidal in clade 2, elongate conoidal in clade 3.