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

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 Nitidulidae 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 whole-grain wheat breakfast cereal (Weetabix®), 3 g of dry brewer’s yeast and 60 mL of distilled water.

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.

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 morphology of stoma and spicules follows De Ley et al. (1995) and Abolafia and Peña-Santiago (2017), respectively.

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).

Measurements are provided in Table 1.

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).

Panagrellus pycnus is characterized by having 0.93–1.32 mm long body, lip region continuous with the adjoining body, lips separated six having rounded sensilla, amphids small, stoma with gymnostom very reduced, pharynx with not swollen metacorpus, neck 161–203 µm long, excretory pore at level of the metacorpus, female reproductive system monodelphic-prodelphic with 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), spicules 70–81 µm long having angular hook-shaped and very curved ventrad lamina ending in a spatulate tip with a refringent forked axis, and gubernaculum 29–32 µm long.

The Panagrellus species are easily differentiated by the size (Table 3) and morphology (Fig. 5) of the spicules (Abolafia et al., 2016). Three main groups are distinguished according the morphology of the spicules. The first group, (Fig. 5B–D) with more simple spicules, includes three species [P. dorsobidentatus (Rühm, 1956) Baker, 1962, P. ludwigi (de Man, 1910) Goodey, 1945 and P. ventrodentatus (Heindl-Mengert, 1956) Baker, 1962] having robust spicules with irregular truncated manubrium, probably a plesiomorphic character. The second group (Fig. 5E–I), with five species [P. japonicus (Yokoo and Ota, 1961) Andrássy, 1984 P. levitatus P. nepenthicola (Menzel, 1922) Goodey, 1945, P. redivivoides (Goodey, 1943) Goodey, 1945 and, probably, P. ulmi Abolafia, Alizadeh and Khakver, 2016], presents ventrally curved spicules with rounded manubrium, straight or slightly ventral bent, and lamina with variable bifurcated tip. The third group (Fig. 5J–P), composed by seven species [P. ceylonensis, P. dubius, P. filiformis (Sukul, 1971) Andrássy, 1984 P. leperisini, P. pycnus, P. redivivus and P. silusioides Tsalolikhin, 1965], have ventrally curved spicules with ventral bent manubrium and lamina with well-developed bifurcated tip, probably an apomorphic character. Between this last group, the curvature degree of the manubrium is a diagnostic character. Thus, spicules with spirally curved manubrium appear in P. ceylonensis (with scarcely sigmoid lamina and short bifurcated terminus), P. dubius (with very curved lamina and long bifurcated terminus) and P. filiformis (with curved lamina while the terminus was not well illustrated). Manubrium poorly ventrally bent appears in P. redivivus and P. silusioides, having scarcely to very bent manubrium respectively, and slightly developed dorsal hump at lamina, while hook-shaped manubrium appears in some P. ceylonensis (narrow manubrium and poorly curved lamina), P. leperisini and P. pycnus (both species with wide manubrium and ‘C’-like lamina), more ventrally curved in P. ceylonensis, while P. pycnus is more anteriorly curved than P. leperisini.

Figure 5:

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).

Figure 6:

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 hook-like 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) having almost 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 hook-like manubrium. However, other morphological and morphometrical characters are necessary to correctly identify these species.

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.

Panagrellus is recovered as paraphyletic (Fig. 6) with Baujardia Bert, Tandingan De Ley, Van Driessche, Segers and De Ley, 2003 located inside the genus. Indeed, Baujardia is very similar to Panagrellus, only differentiated from it by the morphology of the stoma, slightly longer in Baujardia. Unfortunately, only a sequence based on 18S rDNA was obtained of Baujardia mirabilis Bert, Tandingan De Ley, Van Driessche, Segers and De Ley, 2003, the only species of the genus, leading to the possible consideration of the synonymy of Baujardia.

With respect to the subfamily Panagrellinae Andrássy, 1976 containing the genera Baujardia and Panagrellus, is shown as monophyletic. This subfamily was synonymized with Baujardinae Andrássy, 2005 by Abolafia et al. (2016) and the present molecular analysis agree with these authors.

Other subfamilies of the family Panagrolaimidae as Medibullinae Siddiqi, 1993 [tranferred to Panagrolaimidae by Abolafia and Peña-Santiago (2018), including Shahnema Siddiqi, 2014] Tricephalobinae Andrássy, 1976 (including Halicephalobus Timm, 1956 and Turbatricinae Goodey, 1943 (including Turbatrix Peters, 1927) are shown as monophyletic.

Conversely, the subfamily Panagrolaiminae is shown as polyphyletic. Four genera belonging to this subfamily, Panagrobelus Thorne, 1939, Panagrolaimus Fuchs, 1930 Procephalobus Steiner, 1934 and Propanagrolaimus Andrássy, 2005 appear not directly related, unlike what was proposed by Andrássy (1984, 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, 1937 subfamily 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 and Tarantobelus Abolafia and Peña-Santiago, 2018 proposed as belonging to the family Brevibuccidae Paramonov, 1956 by Andrássy (1976, 1984, 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.