Eleven females and 21 juveniles, two females and one juvenile from Cernica forest, Ilfov County, Romania on slide numbers NE 35–37 stored at the reference collection of the National Phytosanitary Laboratory, Voluntari, Romania, 9 females and 20 juveniles - at the personal collection of the first author; nine females and 30 juveniles from Bran, Braşov County, Romania, stored in the nematode collection of IBER, Bulgaria, slide numbers N2-29/2/1-19.

Figures 1–7.

Measurements See Tables 1–3.

Females (Figs 1A, B1–4, G2–4, 5E, 6E, J, O, 7) based on the Larix population, Bran, Braşov County.

Habitus spiral shaped, more strongly coiled in posterior part of body. Cuticle 3–4 μm thick at guide ring region, ca 3 μm in mid-body, and 5–6 μm on tail posterior to anus. Lip region broadly rounded anteriorly, rounded laterally, almost continuous with rest of body. Amphideal fovea pocket-shaped, varying from not lobed to symmetrically bilobed at base (according to terminology proposed by Decraemer and Coomans 2007) extending to ca half the distance anterior end-guide ring. Left and right fovea of about equal size (12.7 (11–14) μm, n = 5), sensillar pouch (fusus) just posterior the guide ring, the distance from the fovea to fusus 24 (23–29 μm). Pharyngeal bulb occupying 25 (22–29) % of total pharynx length; dorsal nucleus located at 29.5 (27–32) % (n = 7) of bulb length; ventro-sublateral nuclei at 54 (48–57) % (n = 8) (left) and 54 (52–56.5) % (n = 8) (right); opening of the dorsal gland at 9 (7.5–11) % and opening of the ventro-sublateral glands at 84 (80.5–90.5) % of the distance from anterior end of pharyngeal bulb, respectively. In one female, a small vestigium (5 μm) observed in wall of slender pharynx. Two nerve rings observed, the first one at 207.2 ± 8.8 (193–218) μm from anterior end, surrounding about mid-odontophore; the second at 329 ± 11.6 (313–344) μm from anterior end, n = 6, (first at 235.7 ± 12.7 (215–255) and second at 329.3 ± 18.6 (290–343) μm from anterior end, n = 7, Cernica forest). Tail bluntly conical, dorsally convex, flat or shallowly concave ventrally. Two pairs of caudal pores. Reproductive system didelphic, two branches of about equal size. Vagina occupies ca 50 % of corresponding body width; pars distalis vaginae and pars proximalis vaginae 13–15 μm and 15–19 μm long, respectively. Uteri short, anterior uterus 96.3 ± 13.5 (80–120) μm long, posterior 91.0 ± 10.5 (76–107) μm. Uteri shorter in Cernica population – anterior uterus 80.9 ± 7.0 (70–90) μm long and posterior 78.3 ± 8.3 (70–95) μm long. Sphincter between uterus and pars dilatata oviductus well developed. Sperm observed in both uteri of one female.

In the population from Cernica forest two females with reserve odontostyles have been observed (Table 3).

Male. Not found.

Juveniles (Figs 1C–F, H–K; 6A–D, F–I, K–N, 7).

General morphology similar to adult females. Body habitus similar in all stages, open C- to J-shaped. Tail of all juvenile stages conical, but becoming more rounded and c’ decreasing in subsequent stages: tail of first stage juvenile elongate conoid with slightly digitate terminus, in the second stage – elongate conoid, in third – bluntly conoid, variable, with narrow to widely rounded terminus, in fourth – resembling that of female, bluntly conoid (Fig. 5). In several juveniles, the abnormalities in their development did not allow to assign them to a particular stage and the morphometrics are presented separately (Table 3). The lengths of functional and replacement odontostyles used to infer the developmental stages were in contradiction with other measurements such as L, a, b, c etc. which were in correspondence with a different stage, or the functional odontostyle was in the ranges of one stage while the replacement one was not in the ranges of the next stage; in one occasion the length of replacement odontostyle was less than that of the replacement one (Table 3).

The amplification of the ITS and the D2-D3 expansion domains of the 28S rRNA gene yielded fragments of 1646 and 756 bps, respectively, based on sequencing. The ITS sequences of L. piceicola from Romania were obtained for the first time in the present study. They showed 98 % similarity (962/984 identities, 9 gaps) when compared with the corresponding sequence of L. intermedius (KT308890) and 86 % with the ITS sequence of L. elongatus Hooper, 1961 (AJ549986, AJ549987). Intraspecific variation for the ITS sequences was low, with only two nucleotides difference and no indels.

D2-D3 rDNA sequences obtained from both Romanian populations were identical to each other and to the sequence of L. piceicola from Slovakia (AY601577, He et al. 2005). The phylogenetic relationships of L. piceicola with several related species is presented in Figure 8. Longidorus intermedius revealed sister relationships with L. piceicola and the sequences from both species formed a well-supported clade. In addition, five sequences of L. intermedius from Germany (AF480074, Rubtsova et al. 2001), Russia (KF242311 and KF242312, Subbotin et al. 2014), Spain (KT308868, Gutiérrez-Gutiérrez et al 2013 and JX445117, Archidona-Yuste et al. 2016), and the L. piceicola sequence were realigned separately and pairwise distances estimated. A total of 737 positions was included in the dataset. The between species dissimilarities (p-distances) were 0.3–0.9 % (or 2–6 bp differences). Similarly, the intraspecific p-distances of L. intermedius from the three European countries were 0.4-0.9 % (i.e. 3–6 bp).

The SNPs analysis comparing all D2-D3 sequences of L. piceicola and L. intermedius revealed three parsimony-informative sites (i.e. nucleotide positions with transitions 89T/C, 134T/C and 297A/G) when compared to the reference sequence of L. piceicola (AY601577) (Table 4). The most similar sequence to the L. piceicola sequence was that of L. intermedius from Germany, revealing the highest similarity and only two interspecies differentiating nucleotides at positions 89 and 134 compared to the reference sequence (Table 4).