Descriptions of two new and one known species of Parkellus Jairajpuri, Tahseen and Choi, 2001 (Nematoda: Mononchidae) and their phylogenetic position among Mononchida

Abstract Two new species of Parkellus (Jairajpuri et al., 2001) from Vietnam and a population of Parkellus zschokkei (Ahmad and Jairajpuri, 2010; Menzel, 1913) from Ukraine are described, illustrated and their phylogenetic position among the Mononchida is presented. The molecular data (18S and 28S rDNA) are given for the three investigated species – first time for the genus Parkellus. Parkellus hagiangensis sp. nov. is characterized by a medium-sized buccal cavity, posterior position of the dorsal tooth located below the beginning of the pharynx, males having the ventromedian cuticular pores above and below the excretory pore, short spicules with conical proximal part; females with very faint pars refringens vaginae and small teardrop-shaped pieces, short pars distalis vaginae, the presence of small ventromedian vulval papillae. Parkellus tuyenquangensis sp. nov. is characterized by a medium-sized buccal cavity, posterior position of the dorsal tooth located above the beginning of the pharynx, males having the ventromedian cuticular pores above and below the excretory pore, medium-sized spicules with a cylindrical proximal part, very short lateral guiding pieces, females with very strongly sclerotized pars refringens vaginae, medium size teardrop-shaped pieces, short pars distalis vaginae thickened at the junction with pars refringens vaginae. The newly described species are morphologically most similar to P. parkus and P. zschokkei. An identification key to Parkellus species is presented.

Herein, the descriptions of two new species of predatory soil nematodes of the genus Parkellus are provided. Jairajpuri et al. (2001) established the monotypic genus Parkellus for the new species P. parkus collected in Korea from forest soil surrounding the roots of Euonymus oxyphyllus Miq. The main feature that distinguished the newly described genus Parkellus from the morphologically very similar Coomansus  is the location of dorsal tooth in the posterior half of the buccal cavity. Zullini and Peneva (2006) and Andrássy (2009) contested such a conclusion assuming that the occurrence of the above mentioned feature is not sufficient to make such a distinction. In 2010, Ahmad and Jairajpuri published a summary of the knowledge on the mononchid nematodes, in which they presented several changes in the taxonomy of this group of predatory nematodes. One of them was the transfer of nine species in which the dorsal tooth is located in the posterior half of the buccal cavity from genus Coomansus to the genus Parkellus. Andrássy (2011) did not support the separation of the genus Parkellus from the genus Coomansus because the distinguishing characters such as posteriorly situated dorsal tooth apex and a complex gubernaculum can be observed also in some species of Coomansus and regarded the genus Parkellus as a synonym of the genus Coomansus. However, Siddiqi (2015) believed that the size and position of the dorsal tooth on the dorsal metarhabdion are of fundamental importance in the classification and phylogeny of Mononchida, as is the occurrence of two subventral teeth and their relative position with respect to the dorsal tooth. He agreed with the separation of the genus Parkellus from the genus Coomansus. He has separation of several genera based on the position of dorsal tooth apex on dorsal metarhabdion: the new genus Supronchus from the genus Iotonchulus, Megaiotonchus from the genus Iotonchus (Iotonchidae), the new genus Pentonchella (Anatonchidae) (Siddiqi, 2015).
In the present study, two new species: Parkellus hagiangensis sp. nov. and Parkellus tuyenquangensis sp. nov. from Vietnam are described based on morphological, morphometric, and molecular data. Their phylogenetic position among the Mononchida is presented. An updated key to 12 Parkellus species is provided.

Material and methods
Nematode extraction, preservation, and morphological studies Soil samples were collected from a pristine forest in Vietnam and from an oak forest in Ukraine. Nematodes were extracted from soil samples using modified Baermann funnel technique (Southey, 1986).
They were heat killed, fixed in 4% formaldehyde (for morphological observations) or in a DESS mixture (Yoder et al., 2006) (for molecular analyses), transferred to anhydrous glycerol (Seinhorst, 1962), and mounted on glass slides for microscopic observation. Measurements were performed with a Nikon digital camera on a Nikon Eclipse Ni microscope at the Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST), Vietnam. Observations of morphological diagnostic features were performed in a Leica DM5000B light microscope using the Nomarsky differential interference contrast (DIC) technique. Illustrations were drawn and photographs were taken using a Leica DFC 500 camera on Leica DM5000B microscope at the Museum and Institute of Zoology Polish Academy of Sciences (PAS), Warsaw, Poland. Illustrations were edited by using Adobe Photoshop CC 2018. The spicule terminology is expressed according to Peña-Santiago et al. (2014).

Nematode lysis, amplification, and sequencing of rDNA fragments
After conducting the microscopic studies and preparing the photographic documentation, all nematodes subjected to molecular analyses were taken out from the temporary slides. Each retrieved nematode was transferred to a separate dish filled with PBS (phosphate buffered saline) solution. The dishes with nematodes were placed on a laboratory shaker. The nematodes were initially washed in PBS for 3 hr. Subsequently, the nematodes were transferred into dishes containing fresh PBS and were washed on a shaker overnight. In the final washing stage, the nematodes were transferred into dishes containing sterile milliQ water and were washed with it for 4 hr. After this procedure, the nematode individuals 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 Holterman et al. (2006), was added to every tube. The lysis occurred in a thermal cycler (Veriti 96-Well Thermal Cycler, Applied Biosystems, Foster City, CA, USA) at 65°C for 3 hr followed by a 5 min incubation at 100°C. The obtained single nematode lysate (crude DNA extract) was used either immediately as a DNA template for a PCR reaction or stored at − 20°C for future use.
Nearly full length 18S rDNA was amplified from the analyzed Parkellus individuals in two overlapping fragments, using the following primer combinations: 988F combined with 1912R and 1813F combined with 2646R (Holterman et al., 2006). The 28S rDNA sequence was also amplified in two parts using the 61F primer (Holterman et al., 2006) combined with the MCB1R (Dobosz et al., 2013) and the D2A primer combined with the D3B (Nunn, 1992).
All PCR reactions contained 12.5 μ l Color Perpetual OptiTaq PCR Master Mix (2x) (EURx, Gdansk, 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 the 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 40 cycles at 94°C for 30 sec, 54°C (58°C or 62°C in case of D2A-D3B primer combination) for 30 sec and 72°C for 60-70 sec with a final incubation for 7 min at 72°C. Amplicons were visualized under UV illumination after Simply Safe (EURx, Gdansk, Poland) gel staining and gel electrophoresis. Excess dNTPs and unincorporated primers were removed from the PCR product using the enzymatic mixture of Fast Polar-BAP -Thermosensitive Alkaline Phosphatase (EURx, Gdansk, Poland) and Exonuclease I -(EURx, Gdansk, Poland).
After sequencing the obtained Parkellus rDNA sequence fragments, as well as ribosomal DNA fragments of Coomansus parvus used in this study as a close reference species, were deposited in GenBank under the following accession numbers: MT799665-MT799669 (18S rDNA) and MT799670-MT799673 (28S rDNA).

Sequence analysis and phylogenetic studies
The newly obtained rDNA sequences were analyzed using the BioEdit program (Hall, 1999: v.7.2.5). The final 18S and 28S rDNA datasets for phylogenetic study included sequences from the three studied Parkellus, newly acquired Coomansus parvus and available sequences of Mononchida representa tives from GenBank. Representatives of Dorylaimida, Mermithida, and Trichinellida were used as an out group. The final multiple-sequence alignments con sisted of 1,688 overlapping characters in case of 18S rDNA and 1,047 characters in case of 28S rDNA. Genetic distances were estimated with the DNADist tool implemented in the BioEdit, using the default options (Kimura 2-parameter model of nucleotide substitution).
The Bayesian phylogenies were constructed with the program MrBayes v. 3.1 (Ronquist and Huelsenbeck, 2003). GTR substitution model with a proportion of invariable sites and gamma distribution for both 18S and 28S data sets was used. Two independent runs were performed with four Markov chains per run. The program was run for 2 × 10 6 generations in case of the 18S rDNA data set and for 1 × 10 6 generations in case of the 28S rDNA data set.
Sample frequency in both cases was 100 generations. The sampled trees from each run were combined in a single 50% majority-rule tree. Stabilization of the likelihood and parameters was checked with the program Tracer (Rambaut et al., 2014: v.1,6).

Etymology
The name of the species refers to its geographical origin from Ha Giang Province in Vietnam.

Description
Measurements see Table 1.

Adult
Relaxed specimens arcuate, more curved ventrally at posterior end. Body tapering slightly anterior to base of pharynx but more sharply toward posterior end.
Maximum body width at the level of vulval region. Cuticle smooth, 6.4 (5.3-7.5) μ m thick at the base of pharynx. Sublateral, subdorsal, and subventral body pores distinct along the entire body excluding tail. Lip region rounded, 3.4 (3.1-3.7) times as wide as high, slightly offset by a depression, its diameter almost the same as the adjacent body. Labial papillae small, conical, slightly protruding. Cephalic papillae comparatively more prominent, broadly rounded, slightly protruding beyond the body outline. Amphidial fovea cup-shaped, its aperture 5.4 (5.1-5.8) µm wide, located 20.8 (18.6-22.8) µm from the anterior body end. Buccal cavity spacious, oval-shaped, 1.8 (1.7-2.1) times as long as wide, with funnel-shaped base, its wall strongly sclerotized. Dorsal tooth medium-sized, located at the base of the vertical dorsal plate of the buccal cavity, below the beginning of the pharynx. Apex of dorsal tooth located 41.1 (38.1-44.6) µm or 71-76% of buccal cavity length from its anterior end, opposite to it lies a thin, subventral longitudinal ridge. Nerve ring encircling cylindrical, muscular pharynx at about 26.5 (23.8-29.2%) of its length. Excretory pore distinct, males have there are three well developed ventral pores, first one at 40.7-46.6 µm above and the next two at 24.7-29.7 and 74.3-82.6 µm behind the excretory pore. Pharyngo-intestinal junction nontuberculate. The intestine content nematodes, diatom shells, earthworm setae and detritus are visible. Rectum wide, arcuate, 0.8 times the anal body diameter long. Tail conical, ventrally bent, regularly tapering, with rounded tip. Hyaline part well developed, 17.0 (10.9-23.8) µm long. Caudal glands and terminal opening absent. Female: Genital system didelphic-amphidelphic, the sexual branches almost equally developed, anterior 225-238 µm long or 11-13% of body length, posterior 140-187 µm long or 8-9% of body length. Ovaries on alternate sides of the intestine, well developed, reflexed with numerous oocytes, not reaching the uterusoviduct junction, oviduct takes the form of a slender tube, which then expands to form a well-developed pars dilatata with distinct lumen, it is connected with the uterus by a muscular sphincter, uterus, relatively long, contains numerous sperm cells mainly in its distal part. Vagina extending inwards for 27.8-28.3 µm or 35-43% of body diameter, pars proximalis vaginae longer than wide, pitcher-shaped, surrounded by strong circular muscles, pars refringens vaginae with a very faint, small (6.7 x 4.7 µm), teardrop-shaped pieces, pars distalis vaginae short, vulva a transverse slit. Small, ventromedian vulval papillae, one or two on each side of the vulva are visible. Female tail with four pairs of sublateral caudal pores.     (Jairajpuri et al., 2001), P. zschokkei (Menzel, 1913), and P. tuyenquangensis sp. nov. From P. parkus, it is distinguished by having lower position of dorsal tooth apex (71-76% vs 61-65%), longer female tail (116-128 vs 85-90 µm) and males having the ventromedian cuticular pores above and below of the excretory pore. From P. zschokkei it differs by lower position of dorsal tooth apex (71-76% vs 47-60%), lower position of amphidial aperture (18.6-22.8 vs 12.0-16.0 µm) from anterior body end, males having the ventromedian cuticular pores above and below of the excretory pore and spicule curvature (114-119° vs 104-109°). From P. tuyenquangensis sp. nov. it is distinguished by having lower position of dorsal tooth apex (71.0-76.0% vs 60.0-70.0%), appearance of very faint and small pars refringens vaginae pieces vs strong sclerotized and medium size, structure of pars distalis vaginae (not thickened vs thickened at the junction with pars refringens vaginae), presence of small vulval papillae (vs absence), structure of proximal part of spicule (conical vs cylindrical).

Molecular characterization
Nearly full length (1,600 bp), identical 18S rDNA sequences were acquired from two individuals belonging to P. hagiangensis sp. nov. (MT799665) while identical, 1 kb long, 28S rDNA sequences were obtained from three individuals (MT799670). The results of the phylogenetic analyses using these molecular data are presented in Figures 9 and 10.

Type material
Holotype female, three paratype females, and three paratype males from Cham Chu and Na Hang population, in permanent mounts in glycerin, deposited in the nematode collection at the Department of Nematology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. Nine paratype females and nine paratype males from Cham Chu and Na Hang population deposited in the nematode collection of the Museum and Institute of Zoology, PAS, Warsaw, Poland. Two paratype females and three paratype males from Na Hang population deposited in State Museum of Natural History NASU, Lviv, Ukraine.

Etymology
The name of the species refers to its geographical origin from Tuyen Quang Province in Vietnam.

Description
Measurements, see Table 2.

Adult
Relaxed specimens arcuate, more curved ventrally at posterior end. Body tapering slightly anterior to base of the pharynx but more sharply toward the posterior end. Maximum body width at the level of vulva. Cuticle smooth, 7.1 (6.2-8.5) μ m thick at the base of the pharynx. Sublateral, subdorsal, and subventral body pores visible along the entire body excluding tail.
Lip region rounded, 3.4 (3.0-3.7) times as wide as high, slightly offset by a depression, its diameter slightly wider than an outline of the adjacent body. Labial papillae small, conical, slightly protruding. Cephalic papillae bigger, broadly rounded, slightly protruding beyond the body outline. Amphidial fovea cup-shaped, its aperture 5.2 (4.2-6.4) µm wide, located 19.5 (17.7-22.0) µm from the anterior body end. Buccal cavity spacious, oval-shaped, 2.0 (1.9-2.1) times as long as wide, with funnel-shaped base. Dorsal tooth small, located at the vertical dorsal plate of the buccal cavity, above the beginning of the pharynx. Apex of dorsal tooth located 35.7 (31.6-37.7) µm from anterior margin of the buccal cavity or 60-70% of the buccal cavity length from its anterior end, opposite to it lies a thin, subventral, longitudinal ridge.
Nerve ring encircling cylindrical, muscular pharynx at about 25.6 (22.0-28.6%) of its length. Excretory pore, ampulla and renette cells well visible. Males with three

Molecular characterization
For the P. tuyenquangensis sp. nov. we were able to obtain only the second part of the 18S rDNA gene (nearly 800 bp; MT799666) from one of the two molecularly-analyzed nematode individuals. Identical, 1 kb long, 28S rDNA sequences were successfully obtained from both of these nematodes (MT799671). The results of the phylogenetic analyses using these molecular data are presented in Figures 9 and 10. Parkellus zschokkei (Menzel, 1913) (Figs. 7 and 8 and Table 3 ). In total, 13 females and 11 males (population I) fixed with 4% TAF, mounted in glycerine, on permanent microscope slides and 16 females and 6 males (population I, II, III) fixed with DESS mixture and submitted for molecular study (see Material and methods).

Description
Measurements see Table 3.

Adult
Relaxed specimens arcuate, more curved ventrally at posterior end. Body tapering slightly anterior to base of pharynx but more sharply toward posterior end. Maximum body width at the level of vulva. Cuticle smooth, 4.3 (3.1-5.1) μ m thick at the base of pharynx, subcuticle with faint transverse striae. Sublateral, subdorsal, and subventral body pores distinct along the entire body excluding tail. Tail with four pairs of sublateral caudal pores.
Lip region rounded, 3.4 (3.0-4.0) times as wide as high, offset by a depression, wider than outline of the adjacent body. Labial and cephalic papillae conical, protruding beyond of the body contour, cephalic papillae comparatively more prominent. Amphidial fovea cup-shaped, its aperture relatively large, 7.3 (6.4-9.5) µm wide, located 13.5 (12.0-16.0) µm from the anterior body end. Buccal cavity spacious, ovalshape, 2.0 (1.9-2.2) times as long as wide, with funnel-shaped base. Dorsal tooth small, at the vertical dorsal plate of the buccal cavity, above the beginning of the pharynx. Apex of dorsal tooth located 28.1 (24.7-30.3) µm from anterior margin of buccal cavity or 47-60% its length from anterior end, opposite to it lies a thin, subventral longitudinal ridge. Nerve ring encircling cylindrical, muscular pharynx at about 29.4% (27.7-31.5%) of its length. Excretory pore and the ampulla well marked. Pharyngo-intestinal junction non-tuberculate. In the intestine content visible nematodes, diatom shells, earthworm setae, and detritus. Rectum wide and arcuate, 0.8 (0.7-1.0) times the anal body diameter long. Tail conical, ventrally bent, regularly tapering, with rounded tip. Hyaline part well developed, 12.9 (9.4-18.6) µm long. Caudal glands and the terminal opening absent. Female: Genital system didelphic-amphidelphic, the sexual branches almost equally developed, anterior 348 (256-423) µm long or 12.1% (9.3-14.6%) of body length, posterior 349 (297-423) µm long or 12.1% (10.5-16.3%) of body length. Ovaries on alternate sides of intestine, reflexed and well developed with numerous oocytes, not reaching the uterus-oviduct junction, oviduct takes the form of a slender tube, which then expands to form a well-developed pars dilatata with a distinct lumen, it is connected with the uterus by a muscular sphincter having a heavily sclerotized inner part, uterus, relatively long, contains numerous spermatozoids mainly in its distal part, sometimes sperm cells are also visible in sphincter and proximal part of pars dilatata. Vagina extending inwards for 37.4 (34.3-41.0) µm or 43.2% (37.3-54.4%) of the body diameter, pars proximalis vaginae longer than wide, pitcher-shaped, surrounded by strong circular muscles, pars refringens vaginae with strong sclerotized, teardrop-shaped pieces, 13.8 (11.2-15.6) × 4.8 (4.1-6.0) µm, pars distalis vaginae very short, thickened at the junction with pars refringens vaginae, Vulva as a transverse slit, sometimes the vulval lips extend beyond the body contour. Small, ventromedian vulval papillae, one to three on each side of vulva are visible. Male: Genital system diorchic, with opposed testes. Sperm cells elongate spindle shaped. Ejaculatory glands in tandem, generally distinct. Spicules total length along arc 1.3-1.4 times that at chord, 1.6 (1.4-1.8) times longer than body diameter at cloacal aperture; relatively thin, 8.4 (8.1-8.6) times longer than wide, ventrally curved, curvature 105.6°(104-109.5°), median piece marked between hump and lateral guiding pieces, occupying 26.3% (22.0-30.1%) of spicule total length. Dorsal contour arcuate, ventral side with moderately expressed hump and hollow, the former located at 30.2% (26.4-32.9%) of spicule total length from its anterior end, head narrow, on a dorsal side offset by a shallow depression, proximal part of spicules almost cylindrical, posterior end bifurcate, 4.9 (4.2-5.3) µm broad. Lateral guiding pieces with arched edges and a bifurcate terminus, this furcation  is symmetrical and well-marked. Gubernaculum well developed, 41.6 (36.2-46.3) µm long. Ventromedian supplements 21-24 in number, conical, regularly arranged, occupies 13.1% (11.8-13.9%) of body length. Above the supplements two cuticular pores are visible, the posterior most situated at 15.6 (13.8-16.6) µm from cloacal aperture, distance between the first and last supplement 360 (317-399) µm. Parallel to the supplements, on both sides of them, five to six subventral papillae of varying sizes are visible, largest one lies at the level of the anus. Almost at half of the tail length, on its ventral side a large medioventral caudal papilla is situated, below it lies one pair of subventral and two pairs of subdorsal papillae.

Molecular characterization
Both, the 18S and 28S rDNA -derived sequences were acquired from 11 independent nematode individuals belonging to P. zschokkei. Three of the 18S rDNA sequences differed from the other eight in 1 extra nucleotide. All the acquired 28 rDNA sequences were Table 3. Morphometrics of females and males of Parkellus zschokkei (Menzel, 1913) from Ukraine. Notes: L = body length; a = body length/maximum body width; b = body length/pharyngeal length; c = body length/tail length; c′ = tail length/body width at anus; V = (distance from anterior end to vulva/body length) x 100.

Key to species of Parkellus
Currently, 12 species of the genus Parkellus have been recorded. The following key is modified from Loof and Winiszewska-Ślipińska (1993) and Ahmad and Jairajpuri (2010    Three studied Parkellus species clustered together in a monophyletic group and were positioned within the M3 clade (following the nomenclature of Holterman et al., 2008), encompassing representatives of genera Clarkus, Coomansus, and Prionchulus (Figs. 9 and 10). This positioning was confirmed by the phylogenetic analyses based on both the 18S and 28S rDNA data.
The close relationships of both Vietnamese species are also in agreement with their morphology. Their common morphological features are the lower position of the amphidial aperture and the medioventral pores above and below the excretory pore that are present only in males. However, the main feature that distinguishes two species from each other is the position of the dorsal tooth in relation to the beginning of the pharynx, in the area where it surrounds the base of the buccal cavity. Other differences were described in detail in the diagnosis part, in the description of P. hagiangensis sp. nov.
Our research delivers so far the only molecular data that are available for the genus Parkellus as well as one of the first ones belonging to the genus Coomansus. Inspite of this, there is still not enough sequences available in the data bases representing Mononchida species diversity. Accordingly, only three out of twelve Parkellus and only two out of 25 Coomansus species have been characterized molecularly (Vu, 2021). That is why it is too early, to definitely resolve the ongoing discussion, whether Coomansus and Parkellus belong to the same or different genera. At this moment, the comparison of the available 28S rDNA sequence diversity (only the 260 bp region as only a fragment of such length was available for Clarkus), the DNA distance between Coomansus and Parkellus are at the same level (3-5%) as the one between Prionchulus and Clarkus. More over, the Coomansus gerlachei species, currently represented only by the sequences deriving from the work of Elshishka et al. (2015), which do not cluster well within the M3 clade, should be more closely studied. Its phylogenetic positioning outside the 'zschokkei-group' was already mentioned by Mladenov et al. (2019) in their conference abstract. In conclusion, the frequently-raised question regarding the synonimisation of Coomansus and Parkellus should be investigated in more detail in further research on Mononchida.

Conclusions
Two new species of the genus Parkellus from Vietnam, Parkellus hagiangensis sp. nov. and Parkellus tuyenquangensis sp. nov. are discovered based on the morphological and molecular characteristics. Their phylogenetic position in between Mononchida is fitted well within the M3 clade.
QTPL01.03/19-20 funded by the Vietnam Academy of Science and Technology (VAST) and the Polish Academy of Science (PAS) and 'The NEF Bio-ecological Nature Conservation Project in Mountainous Region of North Vietnam' funded by Nagao Environmental Foundation, Japan.