Morphological and molecular characterisation of Tripylina gorganensis from the Slovak Republic as a contribution to the redescription of the species

Abstract Specimens of Tripylina gorganensis, collected from a natural beech forest in Slovak Republic, are described and illustrated. These nematodes were initially identified as an undescribed species, morphologically similar to Tripylina gorganensis described from Iran. An important feature distinguishing both species was the presence of post-vulval uterine sac (PUS) in specimens from Slovak Republic, which, according to the original description (Asghari et al., 2012), was absent in Tripylina gorganensis. However, a careful re-examination of type specimens performed in this study revealed that T. gorganensis also has the post-vulval uterine sac. Consequently, the findings of the morphological and molecular studies performed on the Slovak population and observations on the type material contribute to the redescription of T. gorganensis.

The genus Tripylina Brzeski, 1963 is represented by free-living, predacious species that inhabit soil, litter, moss, and other semi-wet biotopes (Andrássy, 2007) and are often found in close proximity to various tree species (Asghari et al., 2012;Xu et al., 2013;Zhao, 2009). This genus currently includes 22 species described from different regions of the world (Table 1).
Starting with the work by Zhao (2009), half of the described species have been characterized molecularly by partial sequences of either one or two of the ribosomal DNA genes (18 S and/or 28 S rDNA). The rDNA-based phylogenetic analyses revealed the well-supported monophyletic status of Tripylina (Asghari et al., 2012;Cid del Prado-Vera et al., 2012, 2016Xu et al., 2013;Zhao, 2009). The Tripylina clade was shown to be more closely related to the Enoplida representatives like Pseudocella Filipjev, 1927, Deontostoma Filipjev, 1916 or Alaimus de Man, 1880 rather than Tripyla Bastian, 1865, Tripylella Brzeski & Winiszewska-Ślipińska, 1993or Tobrilus Andrássy, 1959 supporting Andrássy's (2007) view that Tripylina could be subsumed under Enoplida, rather than the Tripylidae de Man, 1876 associated with the Triplonchida (Cid del Prado-Vera et al., 2012, 2016Zhao, 2009Zhao, , 2011. The present work focuses on morphological and molecular characterisation of the Tripylina gorganensis found during an ecological study, on the impact of natural disturbance on soil nematode communities' structures in a natural beech forest (Fagus sylvatica L.) in the Slovak Republic. Re-examination of the paratypes of T. gorganensis has pointed out the necessity to redescribe this species. T. gorganensis happens to be another European species following the reports of T. arenicola, T. longa, and T. macroseta.

Material and methods
Soil sampling, nematode extraction, and processing Soil In total, ten representative soil samples composed from five subsamples were collected from the five permanent research plots (10 × 10 m) established in the undisturbed natural beech forest in Opátka (48°47'N, 21°04'E, at 776 m a.s.l.), a small village in Košice district, eastern Slovakia. Samples were collected twice, five in May 2016 and five in September 2018, and examined in two laboratories, first in Slovakia and later in Poland.
Each sample (collected in 2016) was homogenized by gentle hand mixing. A total of 100 g of soil was then soaked in 1 l of tap water for 30-60 min and processed by the Cobb sieving and decanting method (Cobb, 1918) followed by the modified Baermann technique (van Benzooijen, 2006). Subsequently, nematodes were extracted from the aqueous soil suspensions using a set of two cotton-propylene filters. Suspensions containing nematodes were collected after 24 h. The nematode suspensions were subsequently examined under a stereomicroscope (40 × and 60 × magnification). After the excess water was removed, five Tripylina individuals was set aside for molecular analyses and therefore placed in an Eppendorf tube containing water and subsequently sent to Poland. The remaining nematodes were used to morphological and morphometric study, fixed with a hot 99:1 solution of 4% formaldehyde and pure glycerin (Seinhorst, 1962) and then processed to anhydrous glycerin (Seinhorst, 1959). Permanent slides were made and Tripylina nematode individuals T. sheri Brzeski, 1963 USA (California) T. stramenti (Yeates, 1972)  were studied using a light microscope Eclipse 90i Nikon, Japan at 100, 200, 400, 600, and 1000 × magnification.
The soil samples collected in 2018 were extracted in Poland by the decantation and sieving method followed by the centrifugal flotation method (van Benzooijen, 2006). Extracted nematodes were heatkilled using tap water at 60°C. Some of the selected individuals (as well as a few individuals from 2016 that arrived alive in water from Slovakia) were fixed in TAF and subsequently submitted for morphological analysis. Photographs and measurements were taken from 16 females and 14 males with a Leica DFC 500 camera on a Leica DM 5000B microscope. The remaining nematodes were fixed in DESS and temporary slides were prepared. With photographic documentation collected, the examined specimens were washed with PBS and subsequently with sterile water and then submitted for molecular studies.
Nematode lysis, amplification, and sequencing of 18 S and 28 rDNA fragments Ten selected individual nematodes (five collected in 2016 and five in 2018) were transferred to separate 0.2 ml polymerase chain reaction (PCR) tubes containing 25 μ l sterile water. An equal volume of the lysis buffer, as described in , was added. The lysis occurred in a Thermal cycler (Veriti 96-Well Thermal Cycler, Applied Biosystems, Foster City, CA, USA) at 65°C for 3 h followed by a 5 min incubation at 100°C. The obtained single nematode lysate (crude DNA extract) was either used immediately as a DNA template for a PCR reaction or stored at −20°C.
Nearly full length 18 S rDNA (1,6 kb) was amplified in two overlapping fragments using the following primer combinations: 988 F combined with 1912R and 1813F combined with 2646 R (as in . The 28 S rDNA (1 kb) sequence was also amplified in two parts using the 61 F primer  combined with MCB1R (Dobosz et al., 2013) and the D2A primer combined with D3B (Nunn, 1992). Amplification of the 18 S and 28 S rDNA fragments was performed in reactions containing 12.5 μ l Color Perpetual Taq PCR Master Mix (2x) or Color Perpetual OptiTaq PCR Master Mix (2x) (EURx, Gdańsk, Poland), 1 μ l of the forward and reverse primer (5 μ M each), the 3 μ l DNA template and sterile Milli-Q water to 25 μ l of a total volume. All PCR reactions were performed in Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA) as follows: an initial denaturation step at 94°C for 3 min, followed by 35-40 cycles at 94°C for 30 s, 54°C (58°C in case of D2A-D3B primer combination) for 30 s and 72°C for 70 s with a final incubation for 5-7 min at 72°C. Amplicons were visualised under UV illumination after Midori Green (Nippon Genetics Europe, Duren, Germany) or Simply Safe (EURx, Gdańsk, Poland) gel staining and gel electrophoresis. Excess dNTPs and unincorporated primers were removed from the PCR product using the Clean-Up Purification Kit (A&A Biotechnology, Gdynia, Poland) or alternatively the enzymatic mixture of Fast Polar-BAP -Thermosensitive Bacterial Alkaline Phosphatase (EURx, Gdańsk, Poland) and Exonuclease I -(EURx, Gdańsk, Poland) was used.
Both the 18 S and 28 S rDNA fragments were successfully amplified and sequenced from all 10 processed nematode individuals. There were no sequence variations, neither in the 18 S rDNA, nor within the 28 S rDNA region. The obtained sequence fragments of 18 S rDNA and 28 S rDNA were deposited in GenBank under the following accession numbers: MK722523 and MK733311.

Phylogenetic analysis
The newly obtained sequences of Tripylina (18 S and 28 S rDNA fragments) were analyzed with publicly available (GenBank) rDNA sequences, using the BioEdit program (v. 7.2.5; Hall, 1999). The 18 S rDNA dataset included the Enoplida sequences from representatives of the genera: Tripylina, Trischistoma, Trefusia de Man, 1893, Bathylaimus Cobb, 1894, Deontostoma, Phanoderma Bastian, 1865, Pseudocella, Thoracostoma Marion, 1870, Rhabdolaimus de Man, 1880, Syringolaimus de Man, 1888, Campydora Cobb, 1920, Oxystomina Filipjev, 1918, and Ironus macramphis Schuurmans Stekhoven & Teunissen, 1938 as an outgroup. The species used in the analysis were selected based on the phylogenetic information available in van Megen et al. (2009) and Cid del Prado-Vera et al. (2016). The GenBank sequences that were not unique (did not contain any nucleotide difference as compared to other sequences in the alignment) were excluded from the alignment as uninformative data. For instance, the sequence JQ433064 (T. ixayocensis; Cid del Prado-Vera et al., 2012) was not included as this short 18 S rDNA fragment was identical (BLAST 100% identical) to T. arenicola (KJ636242, KJ636243), T. manurewa (FJ480408) and other sequences from undetermined Tripylina species. The 28 S rDNA data set was constructed using representatives of Tripylina and Trischistoma only. Additionally, a Deontostoma sp. sequence was chosen as an outgroup. More distantly related genera, represented already in the 18 S data set, were not included into the 28 S rDNA analysis as they were too variable to align properly.
The final multiple-sequence alignments comprised of 1,705 overlapping characters in case of 18 S rDNA and 676 characters in case of 28 S. Substitution models for both, 18 S and 28 S rDNA data sets were tested using FindModel -an online implementation of the MODELTEST program (Posada and Crandall, 1998).
The Bayesian 18 S rDNA and 28 S rDNA phylogenies were constructed with the program MrBayes v. 3.1 (Ronquist and Huelsenbeck, 2003). Four independent runs were performed with four Markov chains per run in each analysis. The program was run for 1.5 × 10 6 generations in case of the bigger 18 S rDNA data set and for 400,000 generations in case of the smaller 28 S rDNA data set. The first 150,000 generations were discarded as burn-in in case of the 18 S rDNA analysis and 20,000 generations in case of the 28 S rDNA one. Sample frequency in both cases was 200 generations. The sampled trees were combined in single 50% majority-rule 18 S and 28 S rDNA trees. Stabilization of the likelihood and parameters was checked with the program Tracer (v. 1.6; Rambaut et al., 2014).

Measurements
Measurements of the European population of the Tripylina gorganensis are given in Table 2.

Material examined
Iran. Soil and litter, at a depth of 0-15 cm under a hawthorn tree (Crataegus monogyna L.) in a forest, Naharkhoran region, Gorgan, Golestan Province, northern Iran. One paratype female and two paratype males deposited at the National Nematode Collection, New Zealand (NNCNZ).
Slovak Republic. Soil and litter, at a depth of 0-20 cm under a beech tree (Fagus sylvatica L.) in a natural forest in Opátka (N 48°47', E 21°04', 776 m a.s.l.), a small village in the Košice district, eastern Slovakia. The description is based on the morphology and morphometry of 16 females and 14 males.
Voucher specimens were deposited in the National Nematode Collection in New Zealand (NNCNZ), the Crown Research Institute Landcare Research New Zealand Ltd, New Zealand (2 females, 2 males) and in the nematode collection at the U.S.D.A, Beltsville, USA (one female, one male). The remaining specimens were deposited in the Institute of Parasitology, Slovak Academy of Sciences, Slovak Republic (4 females, 4 males) and in the nematode collection at the Museum and Institute of Zoology, PAS, Warsaw, Poland (9 females, 7 males).

Remark
Specimens collected from a natural beech forest in Slovak Republic were first identified as an undescribed species, morphologically similar to Tripylina gorganensis described from Iran (Asghari et al., 2012). An important feature distinguishing both species was the presence of post-vulval uterine sac (PUS) in specimens from Slovakia, which, according to the original description (Asghari et al., 2012), was absent in Tripylina gorganensis. A careful re-examination of type specimens revealed that T. gorganensis also has a post-vulval uterine sac. As a consequence a redescription of T. gorganensis was needed. Morphologically, the specimens from Slovak Republic agree well with the paratypes of T. gorganensis. The morphometrics of the beech forest nematodes agrees with most of the dimensions in the original description except for some minor differences: they have a longer tail (76-95 vs 49-64 µm in females, 76-99 vs 45-67 µm in males) and a lower value of the coefficient c (18-25 vs 27-36 in females and 15-22 vs 24-40 in males). We consider these differences may be a part of the intraspecific variation within this species.

Diagnosis and relationships
T. gorganensis is characterized by having a long body, dorsal wall thickened almost over the entire length of stoma, a large dorsal tooth directed posteriad, subventral denticles located anterior to dorsal tooth, three ventromedian setae and two pairs of lateral cervical setae in the cervical region, a relatively long postvulval uterine sac, two subdorsal caudal setae situated a short distance anterior and posterior to anus and male having five to six papillate ventromedian supplements.
Tripylina gorganensis together with T. bravoae, T. iandrassyi and T. longa belongs to a small group of Tripylina whose common, distinctive features are: stoma with dorsal wall thickened almost on its entire length, the thickening gradually expands from the beginning of the mouth to the dorsal tooth, a large dorsal tooth directed posteriorly and the presence of males. Their close relationship, except T. longa for which ribosomal sequences are not available, is supported by the molecular data (Fig. 4). Phylogenetic analyses confirm the basal positioning of Tripylina gorganensis (Asghari et al., 2012) within a well supported Tripylina clade (Figs 4 and 5). BLAST analysis revealed the sequence identity between T. gorganensis populations from Iran and Slovakia of more than 99% (4 bp difference) in case of 18 S rDNA and of 97-98% (16-20 SNPs) in case of 28 rDNA.
Key to species of the Tripylina longa group