First record and description of juvenile stages of Longidorus artemisiae Rubtsova, Chizhov & Subbotin, 1999 (Nematoda: Longidoridae) in Poland and new data on L. juglandicola Lišková, Robbins & Brown, 1997 based on topotype specimens from Slovakia

This paper presents the first geographical record of the needle nematode Longidorus artemisiae Rubtsova, Chizhov & Subbotin, 1999 outside Russia. This species was found in Poland near the city of Skierniewice in association with nettle (Urtica dioica L.). Morphometric and morphological data are provided, including the first description of juveniles of this species. Nematodes of the Polish population differ from the type-population in Russia in possessing a thicker body (lower ‘a’ index) in both sexes; males having a longer body and longer spicules; different sex ratio (1:2 in Polish population vs 1:1 in the type-population) and somewhat less expanded lips. Molecularly, the Polish population was characterised by sequencing D2-D3 28S rDNA and ITS1 markers. Additionally, new data on these two markers are provided for another species, Longidorus juglandicola Lišková, Robbins & Brown, 1997, obtained from topotype specimens from Slovakia. Surprisingly, despite the high morphological similarity of these two species, analysis of their phylogenetic position did not show close phylogenetic relation and several other species (less similar in general morphology) appeared more closely related to both L. artemisiae and L. juglandicola.


Introduction
The genus Longidorus Micoletzky, 1927 consists of obligatory plant ectoparasites, many of which are of economic importance as plant pests. This importance is further augmented by the fact, that eight species of the genus are known as vectors of viruses of the genus Nepovirus (Taylor & Brown, 1997). Additionally, Longidorus is rich in species (158 nominal species according to Peneva et al., 2013), which are discriminated mainly on the basis of morphology and morphometrics. However, this approach is complicated by high levels of intraspecific variability in morphometrics and minor interspecific differences leading to substantial overlap among species and increased potential for misidentification (Gutiérrez-Gutiérrez et al., 2013).
To date, 15 species of the genus Longidorus have previously been reported from Poland (Kornobis & Peneva, 2011;Kornobis 2012). During a survey of the occurrence of the nematodes of the family Longidoridae in Poland, a previously non-recorded species Longidorus artemisiae Rubtsova, Chizhov & Subbotin, 1999 was found. This species was described from Russia on the basis of morphology and morphometrics of adult specimens. Subsequently, Rubtsova et al. (2005) obtained a partial sequence of the D2 domain of 28S rRNA gene from paratype specimens and Subbotin et al. (2014) obtained sequences for D2-D3 domains of 28S rRNA gene and partial sequences of the 18S rRNA gene from other Russian populations and assessed the phylogenetic position of this species. Here we present first record of L. artemisiae in Poland. This population was, however, characterised by large intra-specific differences compared to the population from Russia. This paper provides details on the morphology and morphometrics of the Polish population of L. artemisaie including the description of juveniles and sequences of D2-D3 domain of 28S rRNA gene and ITS1.
Additionally, differences between the type-population from Russia and the population from Poland made the latter somewhat similar to another species, Longidorus juglandicola Lišková, Robbins & Brown, 1997. Longidorus juglandicola was described from Slovakia (Lišková et al., 1997) from the rhizosphere of the walnut (Juglans regia L.) on the basis of morphology and morphometrics of adult and juvenile specimens. Thereafter records of L. juglandicola distribution come only from several localities in Serbia (Barsi & Lamberti, 2002;Krnjaic et al., 2002). As no data on molecular markers of L. juglandicola were available, sequences from specimens of the type-population of L. juglandicola collected at the type-locality Sorozka (Slovakia) in 2015 are presented together with data on phylogenetic relationships of both species.

Materials and methods
A total of 925 soil samples were taken during a survey of the occurrence of longidorid nematodes in Poland. In Slovakia, a soil sample was taken from the typelocality of L. juglandicola (Sorozka, a hill in eastern Slovakia, see Lišková et al., 1997) in May 2015. The soil sample was taken from the rhizosphere of the same walnut tree (Juglans regia L.) in the typelocality. Nematodes were extracted using decant and sieving method (Brown & Boag, 1988), fixation for molecular study in DESS (Yoder et al., 2006); the remaining specimens were heat-killed and fixed in TAF (Courtney et al., 1955). For study of morphology and morphometrics of L. artemisiae, specimens were transferred to glycerol as described by Seinhorst (1959). Microscopic slides were made using paraffin ring method and fiber glass to support the coverslip. Observations, measurements and photographs were made using Leica DM5000 microscope. From DESS fixed specimens temporary mounts were made with specimens of both species and photographs illustrating morphology were made (not presented) to retain possibility of checking the morphology of the specimens if necessary. All measurements are in micrometres and are given as the range followed by the means in parentheses.
Subsequently, temporary mounts were dismantled and genomic DNA was isolated from individual specimens using a QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's a protocol. DNA concentration was measured using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). Amplification and sequencing of the ITS1 rDNA region was performed using primers rDNA2 (Vrain et al., 1992) and rDNA58S (Cherry et al., 1997); amplification of D2-D3 28S rDNA using primers D2A and D3B (Nunn, 1992). PCR conditions were as follows: 95°C for 5 min, 35 cycles at 94°C for 45 s, 59°C for 1 min, 72°C for 1 min 30 s and a final extension at 72°C for 10 min (ITS1) and 94°C for 4 min, 37 cycles at 94°C for 30 s, 55°C for 40 s, 72°C for 1 min and a final extension at 72°C for 8 min for 28S rDNA. For D2-D3 28S rDNA PCR amplicons were directly sequenced, for ITS1 PCR amplicons were cloned into pCR TM 4-TOPO Ò vector (TOPO Ò TA Cloning Ò Kit for Sequencing, Invitrogen) and used to transform into One Shot Ò TOP10 competent cells for further sequencing. Sequencing was performed by Genomed (Warsaw, Poland).
[Based on 13 specimens; see also metrical data in Table 1 and Fig. 1A-G). Body C-shaped to open spiral-shaped, more coiled posteriorly. Cuticle with fine, with transverse striations. Cuticle thickness: 3.4-4 at guide ring, 3-4 along most body width, and 7 and 9-10 on ventral and dorsal part of tail. Lips anteriorly flattened to slightly convex, laterally rounded and slightly expanded, set-off from rest of body by almost indistinct to clear constriction. (1.08 ± 0.0) a Ltgo, body length-oesophagus length-tail length; Note some values from the work of Rubtsova et al. (1999) are rounded Amphidial fovea pouch-like, protruding to about half of anterior end-guide ring distance, posterior end not bi-lobed. Nerve-ring located at base of odontophore to less than a corresponding body width posterior to it. Pharyngeal bulb occupying about 1/3 of pharynx length. Dorsal and ventro-sublateral gland nuclei 2-2.5 and 3-3.5 wide, respectively. Vagina occupying 46-61 (57.5)% of the corresponding body width, pars distalis and proximalis vaginae 13-15 (4.6) and 15-20 (17.1) long, respectively. Genital tract morphology typical of the genus with sperm cells present in most of examined specimens. Tail bluntly-conoid, ventrally usually flat, sometimes slightly concave or convex; pair of pores present at each side of tail. Male.
[Based on 12 specimens; see also metrical data in Table 1 and Fig. 1H-L). Less frequent than females, sex ratio 1:2. General morphology similar to females, with differences in posterior body part and genital tract. Posterior body part more coiled than in females, tail ventrally concave. Adanal supplements 1-3 pairs, followed by 9-12 single adanal supplements. Juveniles.
[Based on 23 specimens; see also metrical data in Table 2 and Fig. 2A-H). Four developmental stages present. Body from J-shaped to C-shaped in J1, C-shaped in J2, J3 and J4. Shape of lip region in all stages similar to adults, only in J1 lips are not expanded to slightly expanded and not set-off to almost indistinctly set off. Tail shape conoid with a rounded tip in J1 becoming more bluntly rounded in subsequent stages.
Phylogenetic position of L. artemisiae and L. juglandicola The newly-generated sequences of D2-D3 28S rDNA and sequences containing partial 18S, whole ITS1 region and partial 5.8S are listed together with their GenBank accession numbers in Table 3 Table 3) were used for phylogenetic reconstruction.
In the ITS1 region-containing sequences, four sequence variants were recovered for L. artemisiae and two for L. juglandicola. These sequences were trimmed to include ITS1 only and used for both BLAST search and phylogeny reconstruction. BLAST search of sequences did not show exact matches with sequences of neither L. artemisiae nor L. juglandicola from this study. Sequences of L. artemisiae revealed 80-84% similarity with the sequences for L. elongatus (De Man, 1876) Thorne &Swanger, 1936 andL. intermedius Kozłowska &Seinhorst, 1979 (accession  AF511417, AJ549986-AJ549987, GU199044 and KT308890). Other sequences were characterised by very low query coverage, resulting in low total scores of BLAST searches. None of sequences available in the GenBank database was significantly similar to sequences obtained in this study for the type-population of L. juglandicola from Slovakia. To reconstruct the phylogeny, ten first results from BLAST searches were included to the alignment (some sequences repeated in both searches). The final list of ITS1 sequences used for phylogeny reconstruction is presented in Table 4. The final alignment of the D2-D3 28S rDNA sequences contained 689 positions and the alignment of ITS1 sequences comprised 896 positions. The phylogenetic trees are presented on Fig. 3 A and B. Some relationships were unresolved, however results from both D2-D3 28S rDNA and ITS1 sequences enable two conclusions. First, L. artemisiae as well as L. juglandicola formed their own clades, well distinguishable from the other species in this analysis. Secondly, these two species are not closely related, as many other species appear more closely related to each of them.

Discussion
Morphology and morphometrics of the population of L. artemisiae from Poland studied here revealed several differences in comparison with the original description by Rubtsova et al. (1999). These include a more slender body in the specimens from the typepopulation ('a' index value of 109-155 (133 ± 1.9) and 113-162 (133 ± 1.7) vs 86.6-114.3 (100.8 ± 7.6) and 104.6-133.6 (118.8 ± 9.91) in females and males, respectively. Low 'a' index can sometimes be a result of inappropriate preparation of microscopic slides, where specimen is flattened by coverslip. Here microscopic slides were made with care and using glass fibre (see 'Materials and methods') Nevertheless, we have prepared additional slides which additional specimens. On these slides pieces of broken coverslip (130-170 lm thick according to the manufacturer) were used instead of glass fibres. Measuring body width of several specimens from such slides did not reveal any important differences compared to values given in Table 1 (data not shown). Other differences worth mentioning include: longer body of Table 3 List of species used in the analyses and GenBank accession numbers for D2-D3 28S rDNA sequences Species Accession number Reference L. artemisiae Rubtsova, Chizhov & Subbotin, 1999KF242313-6 Subbotin et al. (2014 male specimens from Poland, 6,201 ± 393.5 vs 5,600 Funding Analysis of morphology in this work was carried on the equipment acquired as part of the Innovative Economy Operational Programme conducted by the Museum and Institute of Zoology, Polish Academy of Sciences. Project No. WND-POIG.01.03.01-00-133/09 co-financed by the European Union from the European Regional Development Fund (0.5). The technical support for the realization of this work was also provided within the frame of the project 'Centrum of Excellence for Parasitology' No. 26220120022 supported by the operating program 'Research and Development' funded by the European Fund for Regional Development (0.4) and project VEGA 2/0013/16 (0.1).

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Conflict of interest The authors declare that they have no conflict of interest.
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