Redescription and phylogenetic analysis of the type species of the genus Panagrellus Thorne, 1938 (Rhabditida, Panagrolaimidae), P. pycnus Thorne, 1938, including the first SEM study

Abstract The identity of Panagrellus pycnus, the type species of the genus Panagrellus, is discussed after studying specimens from a cultured population collected in Italy that fits the original material of the species. A new characterization is consequently provided as follows: body 0.93–1.32 mm long, lip region continuous with the adjoining body, stoma with gymnostom very reduced, pharynx with not swollen metacorpus, neck 161–203 µm long, excretory pore at level of the metacorpus, post-vulval uterine sac 99–162 µm long or 2.6–3.8 times as long as the body diameter divided in a short tubular proximal part and a long swollen distal part, vulva post-equatorial (V = 63–69), female tail conical elongate with acute terminus (133–170 µm, c = 6.8–8.1, c’ = 4.9–7.0), male tail conical elongate with acute terminus (104–137 µm, c = 7.8–10.9, c’ = 3.6–5.1), and spicules 70–81 µm long having angular hook-like and very curved ventrad lamina ending in a spatulate tip with a refringent forked axis. The evolutionary relationships of this species and the genus Panagrellus, as derived from the analyses of 18S and 28S rDNA fragments, are discussed. Additionally, the phylogenetic relationships among the members of the infraorder Panagrolaimomorpha is studied, being the genus Tarantobelus transferred to the family Panagrolaimidae and the new subfamily Tarantobelinae n. subfam. is proposed to accommodate it.

Panagrellus Thorne, 1938 is an infrequent genus belonging to the family Panagrolaimidae Thorne, 1937 (proposed as subfamily by Thorne (1937) and erected to family by Paramonov (1956)), and includes 15 species (Abolafia et al., 2016;Ivanova et al., 2018). This genus is characterized by having usually ventral curved spicules with hooked manubrium and bifurcate lamina tip. Its type species, P. pycnus, was described and succinctly illustrated by Thorne (1938) on the base of male and female specimens collected in slime secreted by wounds from the trunk of Great Plains cottonwood tree in Utah, USA. Later, Goodey (1943) transferred this species to the new erected genus Turbator Goodey, 1943 (currently junior synonym of Panagrellus together to Tylorhabdus Sukul, 1971). Andrássy (1958Andrássy ( , 2005 and Varga (1958) reported this species from organic matter undergoing fermentation in Hungary. Hechler (1971a) revised the Thorne's material and redescribed P. pycnus providing some line illustrations of the stoma and of the male and female posterior ends. This species is distinguished from its congeners by having spicules with angular hook-shaped manubrium and lamina very thin lacking dorsal hump and bearing spatulate terminus having forked refringent axis.
In the present paper, a cultured population of P. pycnus collected in Italy and used as food for carnivorous tardigrades is studied providing new LM and SEM illustrations and molecular data.

Nematode extraction and culture
The nematodes were initially obtained from rotting pears on the ground in Borgata Città, Bologna, Italy (44°34′42.7″N 11°10′26.6″E; 27th October 2019; leg. Matteo Vecchi). Other than nematodes, the fruits were heavily colonized by Drosophila spp. and Niti dulidae beetles. Fruit pulp was spread on Yeast-Sucrose Agar (Yeast extract 1%, Sucrose 2%, Agar 1%) plates and after 1 week of incubation at 21°C a single gravid female was handpicked with a loop to start an isofemale line. Nematodes were mass cultured at 21°C in 0.3 L plastic containers on a substrate composed of 19 g of wholegrain wheat breakfast cereal (Weetabix ® ), 3 g of dry brewer's yeast and 60 mL of distilled water.

Nematode processing
The specimens were killed by heating, fixed in a 70% ethanol solution, transferred to pure glycerin following the Siddiqi's (1964) technique, and mounted on glass microscope slides with the glycerine-paraffin method (de Maeseneer and d'Herde, 1963) somewhat modified using hot liquid paraffin.

Light microscopy (LM)
Observations were made and measurements were taken using a Nikon Eclipse 80i (Nikon, Tokyo, Japan) microscope with a drawing tube (camera lucida) attached to it. Demanian indices and other ratios were calculated according to de Man (1881). Pictures were taken with a Nikon microscope equipped with differential interference contrast (DIC) optics and an associated Nikon Digital Sight DS-U1 camera.
Micrographs were combined using Adobe ® Photoshop ® CS. The terminology used for the mo rphology of stoma and spicules follows De Ley et al. (1995) and Abolafia and Peña-Santiago (2017), respectively.

Scanning electron microscopy (SEM)
Specimens preserved in glycerine were selected and prepared for observation under SEM according to Abolafia (2015). They were cleaned in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).

Molecular analyses DNA extraction, PCR, and sequencing
Nematode DNA was extracted from single individuals using a modified DNA extraction and PCR assays described by Castillo et al. (2003) and Archidona-Yuste et al. (2016). The specimens were cut in small pieces using a sterilized dental needle on a clean slide with 18 ml of TE buffer (10 mM Tris-Cl + 0.5 mM EDTA; pH 9.0), transferred to a microtube and adding 2 μ l proteinase K (700 μ g/ml −1 ) (Roche, Basel, Switzerland), and stored to -80°C within 15 min (for several days) until processing. The microtubes were incubated at 65°C (1 hr), then at 95°C (15 min). For DNA amplification, 3 μ l of the extracted DNA was transferred to a microtube containing: 0.6 μ l of each primer (10 mM), 3 μ l Master Mix Taq DNA Polymerase (5x Hot FirePol Blend Master Mix) and ddH2O to a final volume of 20 μ l. The primers used for amplification of the region of 18S rRNA gene were the forward primer SSU F_04 (5′-GCTTGTCTCCAAAGATTAAGCC-3′) and the reverse primer SSU R_26 (5′-CATTCTTGG CAAATGCTTTCG-3′) (Blaxter et al., 1998). The primers used for amplification of the D2-D3 region of 28S rRNA gene were the forward primer D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and the reverse primer D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (De Ley et al., 1999;Nunn, 1992). PCR cycle conditions were as follows: one cycle of 94°C for 15 min, followed by 35 cycles of 94°C for 45 sec + annealing temperature of 55°C for 45 sec + 72°C for 45 sec, and finally one cycle of 72°C for 5 min. After DNA amplification, 5 μ l of product was loaded on a 1% agarose gel in 0.5% Tris-acetate-EDTA (40 mM Tris, 20 mM glacial acetic acid and 2 mM EDTA; pH = 8) to verify the amplification using an electrophoresis system (Labnet Gel XL Ultra V-2, Progen Scientific, London, UK). The bands were stained with 1.25 µl RedSafe (20,000x) previously added to the agarose gel solution (25 ml). The sequencing reactions of the PCR products were performed at Sistemas Genómicos (Paterna, Valencia, Spain) according the Sanger et al. (1977) method. The DNA sequences obtained for P. pycnus (MZ656001 for the 18S rDNA and MZ656000 for the 28S rDNA) and Tarantobelus arachnicida Abolafia and Peña-Santiago, 2018 (MZ655998-MZ655999 for the 18S rDNA and MZ656002-MZ656003 for the 28S rDNA) were submitted to the GenBank database.

Measurements
Measurements are provided in Table 1.

Description Adult
Moderately slender to slender nematodes of small size, 0.93-1.32 mm long. Upon fixation, habitus nearly straight in females or somewhat curved ventral, J-shaped, in males. Cuticle 1-2 µm thick, bearing minute transverse striations, with annuli 1-2 µm wide. Lateral fields occupying 10-20% of mid-body diameter, with four longitudinal incisures or three alae. Lip region continuous with the adjacent body: lips six, separate, slightly rounded, with protruding, rounded labial and cephalic sensilla; primary and secondary axils with similar morphology, primary ones slightly deeper. Oral opening large, with smooth margin. Amphids very small, oval, located at middle length of lateral lips. Stoma panagrolaimoid, 0.7-1.1 times the lip region diameter long: cheilostom large, slightly wider than long, with strongly refringent, barshaped rhabdia, posteriorly thicker; gymnostom very reduced, with small rhabdia; stegostom funnelshaped with poorly refringent rhabdia, metastegostom bearing dorsal acute rhabdia (dorsal tooth). Pharynx also panagrolaimoid: pharyngeal corpus robust, subcylindrical, 2.2-4.0 times the isthmus length, with procorpus and metacorpus not well discernible; isthmus comparatively thin; basal bulb ovoid, with both valvular apparatus and posterior haustrulum well developed. Cardia small, surrounded by intestinal tissue. Nerve ring at 70-85% of neck length from the anterior end, surrounding the anterior part of isthmus. Excretory pore at 52-75% of neck length, at level of the posterior part of metacorpus. Hemizonid located at level of isthmus. Deirids 78-90% of neck length, at level of isthmus-bulb junction. Intestine without distinct specializations, but with slightly thinner walls at cardiac part; intestinal lumen with rest of diatom frustules.

Female
Reproductive system monodelphic-prodelphic. Ovary very long, lacking flexure at post-vulval region, having oocytes arranged in several rows at its distal part and then in only one rows at its proximal part. Oviduct short, slightly longer than the body diameter developing a scarcely discernible spermatheca at its proximal part. Uterus very long, 6.8-10.9 times as long as body diameter, tubular, frequently including uterine eggs (20-33 × 28-54 µm) inside in different stages of development. Post-vulval uterine sac well developed, 2.6-3.8 times as long as the body diameter, with very thin walls, frequently poorly discernible, with proximal part short, tubular, and distal part large,   swollen. Vagina extending inwards to 31-43% of body diameter, sigmoid. Vulva slightly protruding. Rectum short, 0.9-1.3 times the anal body width; three small gland-like cells are distinguishable around the intestine-rectum junction. Tail conical-elongate with acute terminus. Phasmids located at 34-40% of tail length from anus.

Male
Reproductive system monorchic, with testis reflexed ventrad anteriorly. Spicules paired and symmetrical: manubrium ventrally bent, angular hook-shaped, reduced calamus, and very curved ventrad lamina lacking dorsal hump, with well-developed ventral velum, narrower at its middle length, and spatulate tip in lateral view having a refringent forked axis. Gubernaculum well developed, slightly curved, about 0.4 times of the spicules length, with thin corpus. Three small gland-like cells are distinguishable around the beginning of the cloaca. Tail conical, slightly curved ventrad, conoid anterior to phasmid and them filiform. Genital papillae seven pairs, tree pre-cloacal (GP1 and GP2 subventral, GP3 lateral) and four postcloacal: two pairs subventral (GP5, GP6) and one subdorsal (GP4) at the middle of tail length and one subdorsal (GP7) at beginning of the filiform part. One mid-ventral adcloacal papilla (MP) present. Phasmids at 32 to 50% of tail length from the cloacal aperture, close to genital papillae GP7.

Remarks
The material examined in this study agrees well with the type population of P. pycnus described by Thorne (1938), especially in the morphology of the spicules although, unfortunately, Thorne (op. cit.) did not provide their measurements. Apparently, according to the drawing illustrations, the author did not observe the post-vulval uterine sac which is now know to be very large but (in the specimens examined in this study) its walls are very thin and sometimes not very well discernible. Also, the vagina structure was not illustrated with precision. Later, Hechler (1971a) described lectotype specimens of this species and observed the presence of post-vulval uterine sac but it was not described or illustrated. The main characters to identify this species, the spicules morphology (with manubrium having dorsal angular side and ventral hook-like side; Fig. 4) and morphometry (56-61 vs 54-70 µm, measured as a straight line, or chord, connecting the spicule manubrium with the lamina tip) agree perfectly with P. pycnus, while the gubernaculum is slightly longer (29-32 vs 25-27 µm). Unfortunately, most of the measurements were not provided in the previous records of the species (Table 2).
On the other hand, the most similar species to P. pycnus is P. leperisini Massey, 1974, with which it could be confused by having similar spicules morphology. However, P. pycnus presents larger body (0.80-1.4 mm in Thorne's description, 0.90-1.41 in Hechler's description and 0.93-1.32 mm in the present paper vs 0.74-0.97 mm), lip region wider (wider than the adjacent part of body vs narrower) and longer spicules (70-81 vs 60 µm).

Molecular characterization and phylogenetic position
One 928 bp 18S rDNA sequence (GenBank accession number MZ656001) and one 766 bp 28S rDNA sequence (GenBank accession number MZ656000) were obtained for P. pycnus. After the molecular analysis, P. pycnus exhibits a high level of rDNA similarity with other Panagrellus species (Fig. 6).

Discussion
On the identity of Panagrellus pycnus and other related species The morphology and morphometry of the material examined now agree with the type population of Panagrellus pycnus described by Thorne (1938) and redescribed by Hechler (1971a, b). The main characters to distinguish this material from other species are the size of the spicules (70-81 µm length and reaching the GP1) and the presence of a manubrium with angular dorsal side and hooklike ventral side. Two species, P. ceylonensis and P. leperisini, are similar to P. pycnus, but the former species differs in size (70-81 µm reaching the GP1 vs 81-89 µm length reaching the GP1 in P. ceylonensis and 56-62 µm length not reaching the GP1 in P. leperisini) and morphology (lamina ventrally more curved at first third vs homogeneously curved but ventrally convex at its mid-length in P. ceylonensis and slightly ventrally more curved anterior and posteriorly in P. leperisini).
Other species have very similar spicules and could be confused with P. pycnus. For example, some populations of P. pycnus could have been confused with P. redivivus. Thus, de Man (1914, as Anguillula silusiae), Goodey (1943), Hechler (1970, 1971a, Zullini (1982 and Ferris (2009) described several populations of Panagrellus agreeing with P. redivivus, showing specimens with spicules that do not reach the pre-cloacal GP1 and possessing a dorsal hump with a slight ventral bend to the calamus. However, the material examined by Corrêa de Carvalho and Álvares Corrêa (1953) and Rühm (1956) does not agree well with R. redivivus as observed Hechler (1971a); thus, the material described by Corrêa de Carvalho and Álvares Corrêa (op. cit.) shows spicules reaching the GP1 (vs not reaching the GP1 in P. redivivus, see de Man, 1914 as P. silusiae (Aubertot, 1925;Goodey, 1922;Hechler, 1971a)     angular hook-like manubrium; on the other hand, the material described by Rühm (op. cit.) is very similar to P. ceylonensis agreeing in measurements, excretory pore at basal bulb level and spicules slightly sigmoid with narrow hooked manubrium. On the other hand, Stock and Nadler (2006) characterized morphological and molecularly three species of the genus Panagrellus. However, there are some discrepancies about their identity. With respect to the morphology of the illustrated spicules, the spicule named as 'P. ceylonensis' (see Fig. 3B) is more similar to P. redivivus and P. silusioides by having ventral bent manubrium and wide velum; the spicule named as 'P. redivivus' (see Fig. 3D) agrees with P. ceylonensis (although it is unusually short, 65 µm long) and P. parasiticus (junior synonym of P. redivivus) by having spirally curved manubrium and narrow velum; and the spicule named as 'P. dubius' (see Fig. 3E), agreeing with this species by having long bifurcated terminus but also it is similar to P. leperisini by the size and by having angular hooklike manubrium. However, other morphological and morphometrical characters are necessary to correctly identify these species.

On the phylogenetic position of Panagrellus pycnus in the genus Panagrellus
The material examined now of Panagrellus pycnus is phylogenetically related with other sequenced species of the genus Panagrellus. The species included in this genus are characterized by having very short gymnostom with minute rhabdia, post-vulval uterine sac differentiated in a short tubular proximal part and a large swollen distal part, and female and male tails conoid-elongate. An important character of the spicules is the ventral curvature and hook-shaped manubrium. There are two primary clades in the rDNA phylogenetic tree of Panagrellus (Fig. 6). One includes the species P. levitatus and P. redivivoides and is characterized by the more robust spicules with rounded manubrium, like other genera of the family Panagrolaimidae. The second clade includes P. ceylonensis, P. dubius, P. pycnus and P. redivivus and is characterized by thinner and elongate spicules, increasing the curvature of the manubrium from slightly ventrally curved to hook-like.
Conversely, the subfamily Panagrolaiminae is shown as polyphyletic. Four genera belonging to this sub family, Panagrobelus Thorne, 1939, Panagrolaimus Fuchs, 1930Procephalobus Steiner, 1934 and Pro panagrolaimus Andrássy, 2005 appear not directly related, unlike what was proposed by Andrássy (1984Andrássy ( , 2005. However, additional morphological and molecular studies are necessary to confirm the relationships between these genera and, after that, it could be suitable to reestablish the subfamily Procephalobinae Paramonov, 1956 to include Procephalobus and Propanagrolaimus, among others.
Nevertheless, the genus Macrolaimus Maupas, 1900 currently belonging to the family Chambersiellidae Thorne, 1937subfamily Macrolaiminae Sanwal, 1971, appears related with Panagrolaimus. Unfortunately, not enough species have been sequenced to clarify its phylogenetic position. To this respect, Kitagami et al.
(2019) sequenced a species identified as Panagrolaimus sp. (LC382049), however, it agrees with some species of the genus Macrolaimus according the stoma morphology being showed in the present phylogenetic tree belonging to this last genus (see Supplementary  Fig. S1 in Kitagami et al., op. cit.).
On the other hand, the genera Plectonchus Fuchs, 1930 andTarantobelus Abolafia andPeña-Santiago, 2018 proposed as belonging to the family Brevibuccidae Paramonov, 1956 by Andrássy (1976Andrássy ( , 1984Andrássy ( , 2005 and Abolafia and Peña-Santiago (2018), respectively, appear not closely related with this family in this new study. According to this, both genera are transferred to the family Panagrolaimidae. Regarding the genus Plectonchus (with post-equatorial vulva and without post-vulval uterine sac) appears related to the panagrolaimid genus Panagrobelus (with equatorial vulva and very short post-vulval uterine sac); however with only limited 18S rDNA sequence in support of the genus, there is a clear need to obtain detailed morphological and molecular data, without which the genus should be considered incertae sedis until to obtain a detailed morphological-molecular study of its species. Secondly, the genus Tarantobelus appears forming part of a well-supported, separate clade which is proposed now as a new subfamily, Tarantobelinae n. subfam., clearly differentiated from other subfamilies, especially by having short and robust pharyngeal isthmus and vulva post-equatorial. This new subfamily is composed by the only genus Tarantobelus.
Diagnosis: small body with cuticle nearly smooth, lateral field inconspicuous, lip region with six separated lips, each bearing a small cuticular flap topping it, panagrolaimoid stoma with well-developed gym-nostom having broad and refringent rhabdia, panagrolaimoid pharynx with isthmus robust and slightly longer than basal bulb, excretory pore situated at level of isthmus, female reproductive system monodelphicprodelphic, length of post-vulval sac less than the corresponding body diameter, vulva post-equatorial and distinctly protruding, female tail conical with acute tip, male tail conical with a long and thin mucro, spicules curved ventrad with rounded manubrium, and thick gubernaculum.