Description of Longidorus cholevae sp. n. (Nematoda, Dorylaimida) from a riparian habitat in the Rila Mountains, Bulgaria

Abstract A description is provided of Longidorus cholevae sp. n., a bisexual species associated with wild cherry (Prunus avium L.) from the Rila Mountains, Bulgaria. The position of L. cholevae sp. n. among other species of the genus was elucidated by using morphological and molecular data. Phylogenetic analyses were performed of D2-D3 expansion domains of the 28S rRNA and the partial ITS1 containing regions by Neighbor-Joining, Maximum Likelihood and Bayesian Inference methods. The species is characterised by a female body length of 6.1–8.1 mm; long odontostyle (106–129 μm); lip region wide (21.5–24 μm) rounded and continuous with the body profile; amphidial pouches short and wide, funnel-shaped; a posteriorly situated guide ring (30–37 μm); normal arrangement of pharyngeal glands, and short bluntly rounded to hemispherical tail. Four juvenile stages indentified, first stage with elongate conoid tail. Males with 2–4 adanal pairs and a row of 11–13 single ventromedian supplements, spicules 96–120 μm long. Based both on morphological and molecular data the new species appearred to be the most similar witha group of species distributed in Europe sharing common charcters such as amphidial fovea, lip region and tail shapes, and having similar odontostyle and body length: L. poessneckensis, L. caespiticola, L. macrososma, L. helveticus, L. carniolensis and L. pius. An updated list of Longidorus species and a partial polytomous keys to the Longidorus species with long odontostyle (code A45) and short tail (code H1) are provided.

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).
A partial polytomous keys was prepared for the identification of Longidorus species with long odontostyle (A45) and short tail (H1). This key, based on that by Chen et al. (1997), but incorporating newly described species after 1997 and the addition of some new characters: J -number of juvenile stages -J1 -4 stages; J2 -3 stages; K -shape of tail in J1 -using the same codes as for female tail and introducing K7 -tail digitate or with mucro.

DNA extraction and amplification
Specimens for molecular analysis were kept in DESS solution (Yoder et al. 2006). Genomic DNA was extracted from fifteen individual 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 18S-Ext (5'-TGATTACGTCCCTGCCTTT-3') and the reverse primer 26S-Ext (5'-TTTCACTCGCCGTTACTAAGG-3') (Vrain et al. 1992) and the D2-D3 expansion segments of 28S rDNA was amplified using the D2A (5'-ACAAGTAC-CGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') primers (Castillo et al. 2003). 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.

Sequencing and phylogenetic analysis
PCR products of the ITS region from two individual nematodes 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). Similarly, the D2-D3 regions of rDNA from two individual nematodes were purified and used for direct sequencing.
The sequences of the new species have been deposited in GenBank with the accession numbers: FR775757 -FR775760 for the ITS clones; and FR775761, FR775762 for the D2-D3 regions. Additionally, another four sequences (ITS and D2-D3) belonging to a population identified as Longidorus cf. caespiticola Hooper, 1961 were produced and deposited using the same methodology (see Table 1 for accession numbers and locality). The morphometrics of this population and detailed discussion will be presented in another publication.
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).
Type material. 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.
Further, L. 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 2: a partial polytomous key): L. kheirii, L. raskii Lamberi & Agostinelli, 1993, L. arthensis    Etymology. 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 intrapopulation 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 L. cholevae can be easily identified from other species by using this ribosomal region. The closest species were L. poessneckensis (93% similarity), L. caespiticola, L. macrosoma and L. helveticus (92% similarity). Pairwise BLAST comparisons of the ITS sequence of L. 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: L. caespiticola, L. macrososma, L. poessneckensis, L. helveticus and L. 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 L. macrosoma and L. poessneckensis males are rare). Longidorus caespiticola and L. macrososma occur mainly in western Europe including the British Isles, L. 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; L. poessneckensis with preference to flood plains and hill deciduos forest habitat (Lišková and Kumari 2010) and L. helveticus associated with deciduous forest and orchard threes in central Europe , Širca and Urek 2009, Kumari and Subbotin 2012. Longidorus carniolensis is known only from Slovenia (grapevine) and L. cholevae sp. n. -only from Bulgaria (riparian vegetation). Probably, L. 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 L. cholevae D2-D3 and partial 18S-ITS1 sequences and these alignments included sequences from various populations (Table1).
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).
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) L. helveticus and I2) L. macrosoma, and one subclade having lower values for ML bootstrap support (52%) and BI posterior probabilities (0.72) I3) that includes the new species L. cholevae, two populations of L. carniolensis from 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.
Slovenia and two populations of L. poessneckensis from the Czech Republic and Slovakia. The second clade (II) consists of two well-supported subclades: II1) consisted of L. caespiticola from Slovenia and Belgium and one L. cf. caespiticola from Bulgaria and subclade II2) consisted of three populations of L. caespiticola from Scotland, Belgium and Germany. It is possible that these populations represent two different species that requires further investigation. 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 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 (L. cf. caespiticola, L. helveticus and L. macrosoma) and two additional species (L. pius and L. 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, L. helveticus and L. pius and 2) Longidorus cf. caespiticola and L. raskii, and one not well resolved 3) containing only L. 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.