Calcaridorylaimus castaneae sp. n. (Nematoda, Dorylaimidae) from Bulgaria with an identification key to the species of the genus

Abstract An unknown species belonging to the genusCalcaridorylaimus Andrássy, 1986 was collected from the litter of broadleaf forests dominated by Castanea sativa Mill. and mixed with Quercus daleshampii Ten. and Fagus sylvatica L. on Belasitsa Mountain, south-western Bulgaria. Calcaridorylaimus castaneae sp. n. is characterised by its long body (1.4–2.1 mm), lip region practically not offset, vulva transverse, short odontostyle (14.5–16 μm) and tail (75.5–110.5 μm, c=14.7–23.6; c’=2.9–4.4) in females and 38–46 μm long spicules with small spur before their distant end in males. It is most similar to C. andrassyi Ahmad & Shaheen, 2004, but differs in having transverse vs pore-like vulva and shorter spicules (38–46 μm vs 52–57 μm). An identification key to the species of the genus Calcaridorylaimus is proposed. Phylogenetic analyses were performed on 18S and D2-D3 expansion domains of 28S rRNA genes by Neighbor-Joining, Maximum Likelihood and Bayesian Inference methods. The phylograms inferred from 18S sequences showed closest relationships of the new species with some species belonging to the genus Mesodorylaimus. However, insufficient molecular data for members of both genera do not allow the phylogenetic relationships of Calcaridorylaimus and the new species described herein to be elucidated.


Introduction
During an ecological study of chestnut forests on Belasitsa Mountain (2003Mountain ( -2005 an undescribed species belonging to the genus Calcaridorylaimus Andrássy, 1986 was recovered. The genus Calcaridorylaimus is represented by nine species worldwide: C. calcarifer Andrássy, 1986, C. promissus Andrássy, 1986, C. ruwenzorii (De Coninck, 1935 Andrássy, 1986, C. signatus (Loof, 1975) Andrássy, 1986, C. simillimus Andrássy, 1986, C. sirgeli Heyns & Meyer, 1995 Gagarin, 1997, C. andrassyi Ahmad &Shaheen, 2004 andC. beatus Andrássy, 2011. The genus is distributed mainly in the southern hemisphere: three species occur in Africa, two in South America and one each in Antarctic, Central America, and Europe, and C. promissus was recorded from Australia and Alaska, North America (Andrássy 1986(Andrássy , 2003 (Fig. 1). The most characteristic features of these species are the shapes and structures of the spicules which are provided with a small spur before the distal tip. The new species is described based on both morphological and molecular data.

Sample collection
The litter samples were collected in 2003 by the last author (VP) from three sites on Belasitsa Mountain representing different types of broadleaf forests dominated by Castanea sativa Mill. mixed with Quercus daleshampii Ten. and Fagus sylvatica L. (Forest Management Plan database, sub-compartments 104g, 140b and 146a). Subsequently, on 17.10.2012 new litter samples were collected by Dr Michaela Ilieva from one of these sites, sub-compartment 140b, in order to obtain fresh material for molecular studies. Nematodes were recovered from the litter using the Baermann funnel method. They were killed by heat (65 °C), fixed in TAF (Triethanolamineformalin, Courtney et al. 1955), and processed to anhydrous glycerine (Seinhorst 1959). Drawings were prepared using an Amplival 30-G048b and a drawing tube РА-6У42. 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 programme Digitrak 1.0f (Philip Smith, Scottish Crop Research Institute, Dundee, UK).

Sequence and phylogenetic analysis
The sequences of the new species have been deposited in GenBank with the accession numbers KF717497 and KF717498 for the 18S and the D2-D3 rRNA genes, respectively. A BLAST (Basic Local Alignment Search Tool) search at NCBI (National Center for Biotechnology Information) was performed using the obtained sequences as queries to confirm their nematode origin and to identify the most closely related nematode sequences. The sequences revealing a similarity up to 97% and 85% with nematodes from various Dorylaimida families were included in the phylogenetic analyses of 18S and D2-D3 regions, respectively (Griffiths et al. 2006;Holterman et al. 2006;Meldal et al. 2007;Lesaulnier et al. 2008;Pedram et al. 2010;Pedram et al. 2011;Álvarez-Ortega and Peña-Santiago 2012a;2012b;Donn et al. 2012;Álvarez-Ortega et al. 2013). The Multiple Sequence Alignments (MSA) of both datasets were performed using the Clustal Omega tool (Sievers et al. 2011) via the EBI webserver: http://www.ebi.ac.uk/Tools/msa/clustalw2/. Subsequently, the MSAs were manually optimised and trimmed using MEGA 5 (Tamura et al. 2011). Eudorylaimus sp. (family Qudsianematidae) was used as an outgroup taxon for both 18S and D2-D3 rDNA sequence datasets (accession numbers AY284800 and AY593037, respectively; Holterman et al. 2008). The phylogenetic reconstructions of three datasets D2-D3, complete and partial 18S rDNA were performed using Neighbor Joining (NJ), Maximum Likelihood (ML) and the Bayesian Inference (BI) algorithms and implemented in MEGA 5.0 and MrBayes v. 3.2.1 (Huelsenbeck and Ronquist 2001, Tamura et al. 2011, Ronquist et al. 2012). The NJ phylogenetic inferences were performed under the following settings: Maximum Composite Likelihood method for computing evolutionary distances; Gamma distributed rates among sites, estimated values set up to 0.3429 (D2-D3) and 0.05 (18S rDNA); 2000 bootstrap replications. A total of 755 and 1593 positions in the final datasets were used for both analyses, respectively. General Time Reversible model (GTR) plus Gamma distribution rates (G) and 1000 bootstrap replications were used as ML analyses settings for all datasets. The Bayesian MCMC tree searches were conducted using MrBayes 3.2.1. Each analysis was run for 10, 000, 000 generations with a sample frequency of 1000 generations. The first 25% of the chains discarded as burning and the remaining 75% trees kept to summarise the tree topology, branch lengths, and posterior probabilities (PP) of branch support. The evolutionary models for nucleotide substitutions were set up as for ML analyses. Convergence diagnostic values were calculated every 1000 generations with a predefined stop value equal to 0.01. A single strict consensus tree was visualised using FigTree v1.4.0 graphical viewer (http://tree.bio.ed.ac.uk/software/figtree/). Posterior probabilities values of ≥0.80 (BI) and bootstrap values of ≥70 (NJ and ML) were considered as credible support values for nodes.
Male. General morphology similar to that of female, body curved ventrally in J-shape when fixed. Genital system diorchic, testes opposed, well developed. Spicules dorylaimoid, with double contour on dorsal arm, 1.3-1.6 times the corresponding body diameter long; ventral arm smaller than dorsal. A spur present dorsally before the distal tip, distinctly visible in extruded spicules. Lateral guiding pieces 9-11 μm long or ca. 23 % spicule length. In addition to adcloacal pair seven to twelve (mostly nine or ten), regularly spaced ventromedian supplements present (9 supplements in 8 specimens; 10 suppl. -in 7, and 7, 8 and 12 each in one specimen). Prerectum 4.5-7.5 times the corresponding body diameter long, extending 0.7-1.8 body widths anterior to the supplement series. Tail dorsally conoid and broadly rounded. One subdorsal and one subterminal pair of caudal pores.
Type material. Holotype and 80 paratype females and 47 males deposited in the nematode collection of the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria. Other paratypes deposited as follows: four females and two males in the Nematode Collection of the Foodland Environment Research Agency, Sand Hutton, UK (former Rothamsted Nematode Collection); three females and three males in the USDA Nematode Collection, Beltsville, Maryland, USA; two females and two males in the Riverside Nematode Collection, University of California, Riverside, USA; four females, and four males in the Wageningen Nematode Collection (WANECO), Wageningen, the Netherlands; four females and three males in the Nematode Collection of the Zoology Museum of the Ghent University, Belgium.
Etymology. The scientific name is derived from the generic name of dominant tree species, the sweet chestnut tree (Castanea) in the forest where this nematode was found.  Heyns and Meyer 1995 Shape of vulva: L -longitudinal; P -pore like; T -transverse; when average values are present ranges in parentheses; * from the drawing; ** C. promissus from Alaska (Andrássy 2003) was not included in a subsequent paper by the same author (Andrássy 2011); since most of the characters deviate substantially from the original description, probably this population belongs to another species.  (Meldal et al. 2007) and two sequences of unidentified species from environmental samples from Scotland (AJ875133 and JN049666) (Griffiths et al. 2006, Donn et al. 2012. The phylogenetic analyses based on 18S rDNA and D2-D3 of 28S rDNA sequences from various dorylaimid species with the highest matches of the BLAST search (up to 97% and 85%, respectively) were aligned along with our sequence. The phylograms obtained by NJ, ML and BI methods showed similar topology and differed only in the positions of poorly supported clades. The BI trees (Figs 7 and 8) with posterior probabilities higher than 0.8 and NJ-ML trees with bootstrap values above 70% are presented (Figs 9 and 10). The new species has clustered in a well-supported  Thorne andSwanger, 1936) andM. cf. nigritulus (Schneider, 1937)) and two unidentified nematodes from environmental samples in the 18S rDNA phylogenetic reconstructions (Figs 7 and 9); and to more distantly related species from various dorylaimid genera and families (Prodorylaimus, Labronema, Nevadanema, Paractinolaimus) in the partial D2-D3 LSU reconstructions (Figs 8 and 10).

Key to species of Calcaridorylaimus
The genus Calcaridorylaimus was erected by Andrássy (1986) to accommodate a few species having different shapes and structures of spicules from those of Mesodorylaimus, with the males and females being practically indistinguishable. The phylograms inferred from   18S sequences showed the closest relationships of C. castaneae with some members of the latter genus; however, the insufficiency of molecular data complementary to detailed morphological studies of species belonging to both genera does not allow the elucidation of evolutionary relationships among them and the position of the new species herein described.