The morphological and molecular identity of Longidorus piceicola Lišková, Robbins & Brown, 1997 from Romania (Nematoda, Dorylaimida)

Abstract Longidorus piceicola, a new geographical and host record from Romania, was described and illustrated on the basis of two populations originating from a coniferous and a deciduous forest. The main morphological characters of specimens from Romania correspond very well with the type material collected from the soil around Picea abies L. (Slovakia) except for the shorter body and tail. The D2-D3 fragment of 28S rDNA from both populations was amplified and sequenced, and the sequences were identical to L. piceicola sequence from Slovakia. The partial 18S-ITS1-5.8S-ITS2 rDNA regions from one of the populations were sequenced for the first time. The evolutionary relationships between L. piceicola and the closest species L. intermedius based on D2-D3 sequence divergence and single-nucleotide polymorphisms are discussed. Although having very low sequence dissimilarity (0.3–0.9 %) both species have distinct morphology and biology. Longidorus piceicola differs from L. intermedius in having a much longer odontostyle, body, distance anterior end - guide ring, a wider lip region, more ventromedian supplements (11 vs 5–7) in the male, and develops through four rather than three juvenile stages. Furthermore, L. piceicola occurs more frequently in association with conifers, while L. intermedius is found mainly in oak forests.


Introduction
Longidorus piceicola Lišková, Robbins & Brown, 1997 was originally described from Slovakia  in association with Picea abies L. Subsequently, it was recovered from different localities in Bosnia and Herzegovina, Serbia and Montenegro (Barsi and Lamberti 2001), and Poland (Kornobis and Peneva 2011) in forests dominated by coniferous trees. Here two new findings of this species in Romania are reported. The aims of this paper are to characterize morphologically and molecularly the populations recovered and to discuss the phylogenetic relationships with the most closely related species.

Sampling and processing
Specimens were collected from the rhizosphere of a Larix decidua Mill. forest near to Bran, Braşov County, Romania (45.3050N, 25.2156E), ca 760 m a.s.l. on 15.10.2013, and from the soil around roots of deciduous trees (Quercus sp., Tilia sp., and Fraxinus sp.), Cernica forest, Ilfov County (44.2637N, 26.16514E) and ca 60 m a.s.l. on 4.08.2014. Nematodes were isolated from soil samples by a decanting and sieving technique (Cobb 1918); specimens recovered were heat killed at 55 °C for two minutes, fixed in a 4 % formalin/1 % glycerol mixture, processed to anhydrous glycerol (Seinhorst 1959), and mounted on glass microscope slides. Drawings were prepared using an Olympus BX51 compound microscope with differential interference contrast (DIC). Photographs were taken using an Axio Imager.M2-Carl Zeiss compound microscope equipped with a digital camera (ProgRes C7) and specialised software (CapturePro Software 2.8). Measurements were made using an Olympus BX41 light microscope, a digitising tablet (CalComp Drawing Board III, GTCO CalCom Peripherals, Scottsdale, AZ, USA), and computer Digitrak 1.0f programme, (Philip Smith, Scottish Crop Research Institute, Dundee, UK) and a Leica DMLB microscope with a Leica DFC 295 camera and LAS V 4.2 software.

DNA extraction, amplification and sequencing
The genomic DNA extraction, amplification, and sequencing of single specimens of L. piceicola from both populations in Romania were carried out independently in two laboratories: one at the Institute for Sustainable Plant Protection, Bari, Italy and the other at the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria. Both protocols are presented separately below.
Institute for Sustainable Plant Protection (Bari Unit): specimens (Cernica locality) for molecular analysis were kept in DESS solution (Yoder et al. 2006) before extraction. Genomic DNA was extracted from six individual female nematodes as described by De Luca et al. (2004). The crude DNA isolated from each individual nematode was directly amplified. The partial 18S-ITS1-5.8S-ITS2 regions were amplified using the forward primer TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and the reverse primer AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3') ) and the D2-D3 expansion segments of 28S rDNA was amplified using the D2A (5'-ACAAGTACCGT-GAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') primers (De Ley et al. 1999). PCR cycling conditions used for amplification were: an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 50s, annealing at 55°C for 50s and extension at 72°C for 1 min and a final step at 72°C for 7 min. The size of amplification products was determined by comparison with the molecular weight marker ladder 100 (Fermentas, St. Leon-Rot, Germany) following electrophoresis of 10 ml on a 1 % agarose gel. PCR products of the ITS and D2-D3 regions were purified for cloning and sequencing using the protocol provided by the manufacturer (High Pure PCR elution kit, Roche, Germany). Purified ITS fragments were cloned in TA cloning vector (Invitrogen) and several clones were sequenced using an ABI Prism 377 sequencer (PE Applied Biosystem, Foster City, CA).
Institute of Biodiversity and Ecosystem Research: Genomic DNA was extracted from two single female worms L. piceicola from Bran locality using a standard nematode digestion protocol (Holterman et al. 2006). The D2-D3 expansion segments of the 28S rRNA gene were amplified using the same primers D2A and D3B (De Ley et al. 1999). Each PCR reaction was performed under the following conditions: initial denaturation 94°C for 5 min; 40 cycles (denaturation 94°C for 30 secs; primer annealing 50°C for 30 secs; extension 72°C for 1 min), and final extension 72°C for 10 min. For further details, see Nedelchev et al. (2014). The amplified products were sequenced by Eurofins MWG Operon, Germany.

Sequence and phylogenetic analysis
The sequences of the L. piceicola have been deposited in GenBank with the following accession numbers: KY086070 and LT669801 for D2-D3 expansion domains of 28S rRNA gene; LT669802 and LT669803 for the ITS region. The D2-D3 and ITS sequences were compared with those of other nematode species available at the GenBank sequence database using BLASTN similarity search tool revealing similar results for both regions. The closest D2-D3 sequences to L. piceicola were aligned using ClustalX 2.1 (Larkin et al. 2007). The estimates of evolutionary divergence between the sequences of L. piceicola and L. intermedius Kozlowska & Seinhorst, 1979 (numbers of base differences and p-distances) and Single Nucleotide Polymorphism (SNP) varia-tions (six transitions and four transversions) were performed with MEGA7 (Kumar et al. 2016). Furthermore, sequences revealing the highest similarity to L. piceicola were used for phylogenetic analyses; however only a midpoint rooted tree based on a reduced number of sequences (26) comprising several related species was presented here. The multiple sequence alignments used for phylogeny reconstructions were carried out using GUIDANCE2 Server (http://guidance.tau.ac.il) with the default settings (Sela et al. 2015). Bayesian Inference (BI) algorithm implemented in MrBayes 3.2.5 was used for phylogenetic relationships reconstruction (Huelsenbeck and Ronquist 2001;Ronquist et al. 2012). For further details, see Lazarova et al. (2016).

Longidorus piceicola Lišková, Robbins & Brown, 1997
Material examined. 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.
In the population from Cernica forest two females with reserve odontostyles have been observed (Table 3).
Male. Not found.
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).    Sequences and phylogenetic analyses. 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  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).

Discussion
Morphologically, the specimens of L. piceicola from Romania are similar to the typepopulation from Slovakia , except for the slightly longer body (av. 5.88 vs 5.19 mm) and shorter tail (av. 34.5 vs 42 μm, av. c = 172 vs c = 125) in the population from Cernica forest. Barsi and Lamberti (2001) (Ferris et al. 2012) whereas atypical development in juveniles has not been recorded previously to such a great extent (ca 30 % of all juveniles studied from L. decidua forest were atypical). Ferris et al. (2012) hypothesized that "anatomical aberrations possibly are results from accidents in transcription of the genetic code or mutations which may or may not be mechanistically limiting to reproduction and therefore may or may not be maintained in the genome through either apomixis or amphimixis".
Longidorus piceicola was previously recovered in association with P. abies, Abies alba L., Fagus sylvatica L., Carpinus betulus L. and Vitis vinifera L. in Slovakia, West Balkans and Poland , Barsi and Lamberti 2001, Kornobis and Peneva 2011, Skwiercz et al. 2015, and our findings in coniferous forest dominated by larch and mixed deciduous forest (Fraxinus, Quercus and Tilia) in Romania extend the geographical and plant association ranges further southeast.