On the identity of Eucephalobus oxyuroides (de Man, 1876) Steiner, 1936 (Rhabditida, Cephalobidae), with an updated taxonomy of the genus and notes about its phylogeny

Nematodes were obtained from soil samples using a modified Baermann (1917) funnel technique, killed by heating, fixed in a 4% formalin solution, transferred to pure glycerine following the technique of Siddiqi (1964), and mounted on glass slides for observation.

Observations were made using a Leitz Laborlux S (Leitz, Wetzlar, Germany) microscope and a Nikon Eclipse 80i (Nikon, Tokio, Japan) with differential interference contrast optics. Measurements were taken and drawings were made with the Leitz microscope using a drawing tube (camera lucida). Pictures were taken with the Nikon microscope and a Nikon Digital Sight DS-U1 camera. Micrographs of the same structure were combined using Adobe^® Photoshop^® CS. The terminology used for the morphology of stoma and spicules follows the proposals by De Ley et al. (1995) and Abolafia and Peña-Santiago (2017), respectively.

Specimens preserved in glycerine were selected for observation under SEM according to the methods of Abolafia (2015). They were hydrated 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).

For phylogenetic relationships, analyses were based on 18S and 28S rDNA gene sequences available in GenBank. The sequences were aligned using the ClustalW alignment tool implemented in the MEGA7 (Kumar et al., 2016). The ambiguously aligned parts and divergent regions were found using the online version of Gblocks 0.91b (Castresana, 2000) and were removed from the alignments using MEGA7. The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically selected using jModelTest 2.1.10 (Darriba et al., 2012). Phylogenetic trees were generated with the Bayesian inference method using MrBayes 3.2.6 (Ronquist et al., 2012). Drilocephalobus sp. (AY284679) and Teratolobus sp. (KJ652552) were chosen as outgroups for the 18S and 28S rDNA trees, respectively. Analysis under the GTR + I + G model was initiated with a random starting tree and run with the Markov chain Monte Carlo for 1 × 10⁶ generations. The tree was visualized and saved with FigTree 1.4.4 (Rambaut, 2018).

Measurements are provided in Table 1.

The Navarra population (northern Iberian Peninsula) was collected from soil of an oak (Quercus robur L.), near Pamplona. The two specimens were from Jaén (southern Iberian Peninsula) from soil of an oak (Quercus faginea Lam.) forest in Acebeas, Natural Park of Cazorla, Segura and Las Villas, province of Jaén.

Iberian specimens match well with the type material described by de Man (1876), but males show slightly larger general size (513-629 vs 447-514 µm long body) and slightly more anterior excretory pore (at 64-72 vs 59% of neck length from the anterior end). Unfortunately, details of post-vulval uterine sac, an important diagnostic character of the species, were not originally provided. Nevertheless, a few years later, de Man (1881) described additional material, including both females and males, and illustrated the female genital system with a long (about 2.5 times the body diameter) postvulval sac. These new specimens studied by de Man do not differ from the Iberian ones in any relevant morphological or morphometric trait, including the long post-vulval sac (2.5 vs 2.0-2.5 times the body diameter). Loof (1961) re-examined one female and one male of de Man’s material, but he was unable to provide any relevant morphological or morphometrical information about it, due to its bad condition according to this author. Thus, with due caution, it is herein assumed that Iberian and Dutch exemplars are conspecific.

Based on the material now examined, E. oxyuroides is characterized by its 0.49 to 0.70 mm long body, cuticle with 2 µm broad annuli, lip region continuous with the adjoining body and 6 µm wide, bearing short probolae, neck 132 to 158 µm long, spermatheca 23 to 55 µm long or 1.0 to 1.8 times the corresponding body diameter, uterus 48 to 80 µm long or 2.5 to 3.5 times as long as body diameter, post-vulval uterine sac 40 to 57 µm long or 2.0 to 2.5 times as long as the body diameter, vagina 7 to 9 µm long, V = 58 to 64, female tail conical elongate with very acute terminus (60-79 µm, c = 7.1-10.0, c′ =  4.9-5.8), male tail conical (36-49 µm, c = 10.9-14.3, c′ = 2.4-3.4) with an elongate acute mucro, and spicules 21 to 22 µm long.

The above description of E. oxyuroides allows analysis and discussion of the identity of previously recorded populations (Table 2).

Those populations or specimens examined by Örley (1880) from Hungary, Thorne (1937) from UK, and Boström (1984) from Sweden match well with the type and others described by de Man (1876, 1881, 1984), having in common a lip region with short labial probolae, excretory pore and nerve ring at metacorpus-isthmus junction, long post-vulval uterine sac ca. twice as long as the body diameter while lacking constrictions and septae (only provided in Boström, 1984), and male tail with elongate acute ventral curved mucro.

Mukhina (1981) described a population of Heterocephalobus nannus (Steiner, 1936a) Andrássy, 1967 from Primorsky Krai (Russia). Although only the male was illustrated, both the morphological and the morphometrics of this population fit well the diagnosis of E. oxyuroides. This it is herein regarded as belonging to this species.

Several records of the species significantly differ from the aforementioned in one or several relevant features. Thus:

Cobb (1893a, 1893b) described but did not illustrate Cephalobus similis from Australia, later regarded as identical with E. oxyuroides by Andrássy (1967). The female genital system of this species was considered as ‘probably double and symmetrical,’ a feature totally incompatible with the cephalobid pattern.

The genus Eucephalobus includes 13 valid species plus 2 species inquirendae vel incertae sedis (Fig. 5).

Figure 5:

Type species:

E. oxyuroides (de Man, 1876) Steiner, 1936a.

= Cephalobus oxyuroides de Man, 1876.

= Cephalobus (Eucephalobus) oxyuroides de Man, 1876 (Schneider, 1939).

= Cephalobus oxyuroides f. acaudata Micoletzky, 1921.

= Cephalobus similis Cobb, 1893a.

= Heterocephalobus nannus apud Mukhina, 1981 nec Steiner (1936a), n. syn.

Other species:

E. asteromucronatus Bostrom and Holovachov, 2011.

E. compsus (Steiner, 1935) Thorne, 1937.

= Neocephalobus compsus Steiner, 1935.

E. cornis (Thorne, 1925) Andrássy, 1967.

= Acrobeles (Acrobeloides) cornis Thorne, 1925.

=  Cephalobus cornis (Thorne, 1925) Thorne, 1937.

E. hooperi Marinari-Palmisano, 1967.

= Eucephalobus oxyuroides apud Kim et al. (2017), nec de Man (1876), n. syn.

E. iaculocaudatus Bostrom and Holovachov, 2011.

E. mucronatus (Kozłowska and Roguska-Wasilewska, 1963) Andrássy, 1967.

= Eucephalobus mucronatus Kozłowska and Roguska-Wasilewska, 1963.

= Cephalobus dubius var. apicata Maupas, 1900.

= Cephalobus persegnis var. apicata Maupas, 1900 (Micoletzky, 1921).

= Cephalobus striatus f. tubifera sf. typica Micoletzky, 1921.

= Cephalobus striatus f. microtubifera Micoletzky, 1921.

= Cephalobus persegnis apud Rühm, 1956 nec Bastian (1865).

= Eucephalobus arcticus Loof, 1971.

E. panaxi Mukhina, 1990.

E. paracornutus de Coninck, 1943.

E. plicicaudatus Mukhina, 1970.

E. strandicornutus (Allgén, 1934) Andrássy, 1967.

= Cephalobus strandicornutus Allgén, 1934.

= Cephalobus (Heterocephalobus) strandicornutus Allgén, 1934 (Brzeski, 1960).

= Heterocephalobus strandicornutus (Allgén, 1934) Brzeski, 1961.

E. striatus (Bastian, 1865) Thorne, 1937.

= Cephalobus striatus Bastian, 1865.

= Cephalobus (Eucephalobus) striatus Bastian, 1865 (Schneider, 1939).

= Cephalobus striatus f. atubifera Micoletzky, 1921.

= Cephalobus bursifer de Man, 1876.

E. tribei Swart and Heyns, 1997.

Species inquirendae vel incertae sedis:

E. heterocheilus (Steiner, 1935) Andrássy, 1984 (see Comments).

= Panagrolaimus heterocheilus Steiner, 1935.

= Heterocephalobus heterocheilus (Steiner, 1935) Andrássy, 1967.

E. setensis Kannan, 1960 (see Comments).

Species transferred to other genus:

E. nannus Steiner, 1936a to Panagrolaimus (see Comments).

= Tricephalobus nannus (Steiner, 1936a) Steiner, 1936b.

= Heterocephalobus nannus (Steiner, 1936a) Andrássy, 1967.

The identity of E. heterocheilus raises some doubt. It is characterized by having acute lips and cephaloboid stoma as well as pharynx, but the female reproductive system lacks a spermatheca and its ovary is long, reaching the posterior part of the intestine, features better agreeing with Panagrolaimus Fuchs, 1930. The male posterior end, with a fine mucro curved dorsad, also resembles that present in some Panagrolaimus species. It is possible that this species was originally described from material belonging to more than one species. It is hence regarded here as species inquirenda.

Andrássy (1984) considered E. setensis to be a species inquirenda. Its original description was based on only one Indian female, characterized by a combination of features (lips not acute, wide stomatal lumen, indistinct genital system with very short uterus and without spermatheca or post-vulval sac, and elongate tail covered by short setae-like filaments, probably artifact) that does not fit the Eucephalobus pattern. Thus, it is herein regarded as incertae sedis.

Some relevant morphological traits of E. nannus suggest that it does not belong to this genus. Its long gymnostom and very short stegostom, pharynx with short slightly robust metacorpus and elongate isthmus, and conoid female tail better fit the pattern of the genus Panagrolaimus resembling for instance P. verrucosus Fuchs, 1930. Thus, it is transferred to this genus as Panagrolaimus nannus (Steiner, 1936a) n. comb.

(1a) Female tail conoid elongate with acute tip 2.

(1b) Female tail short conoid, subcylindrical or clavate with rounded tip, with or without mucro 7.

(2a) Female tail conoid tapering sharply from its middle strandicornutus.

(2b) Female tail conoid tapering uniformly until the end 3.

(3a) Post-vulval uterine sac ca. twice longer than body diameter 4.

(3b) Post-vulval uterine sac as long as or shorter than body diameter 5.

(4a) Probolae short but visibly acute; male tail usually with mucro as long as gubernaculum (rarely shorter); spicules elongate with small rounded manubrium and long calamus oxyuroides.

(4b) Probolae very reduced, obscure; male tail with very short mucro; spicules robust with wide rounded manubrium and short calamus tribei.

(5a) Lip region as wide as adjacent part of body; post-vulval uterine sac slightly constricted at its middle length compsus.

(5b) Lip region narrower that the adjacent part of body; post-vulval uterine sac wider at its middle length 6.

(6a) Post-vulval uterine sac with tubular proximal part and swollen distal part; male tail with elongate fine mucro; spicules with calamus as long as wide hooperi.

(6b) Post-vulval uterine sac not well differentiated in tubular and globular part, this latter with solid lumen; male tail with short mucro, thicker at it base; spicules with calamus longer than wide panaxi.

(7a) Female tail with warty or irregular mucro 8.

(7b) Female tail with smooth seta-like mucro or lacking mucro 9.

(8a) Nerve ring and excretory pore more anterior, at metacorpus level; female tail subcylindrical with mucro placed centrally on tip asteromucronatus.

(8b) Nerve ring and excretory pore more posterior, at isthmus level; female tail conical with mucro placed ventrally on tip iaculocaudatus.

(9a) Female tail conoid with rounded terminus or slightly clavate 10.

(9b) Female tail subcylindrical 11.

(10a) Female tail slightly clavate, with or without mucro striatus.

(10b) Female tail conoid lacking mucro plicicaudatus.

(11a) Female and male tails lacking mucro cornis.

(11b) Female and male tails usually with mucro 12.

(12a) Tail with long and robust mucro placed ventrally on tip; spicules 23 to 25 µm long paracornutus.

(12b) Tail with short and thin mucro placed centrally on tip, sometimes absent; spicules 25 to 33 µm long mucronatus.

As derived from the molecular analyses based on the 18S and 28S rDNA fragments, the evolutionary relationships of Eucephalobus are presented in the trees of Figures 6 and 7, respectively. The phylogenetic tree generated by 28S sequences is robust, however, the 18S tree is poorly resolved having Bayesian probabilities with low values. The most relevant result of these analyses is that, in both cases, Eucephalobus sequences occupy a basal position, together with sequences of the genus Pseudacrobeles. Morphologically, these two genera are very similar taxa, especially when elongate-tailed species of Eucephalobus – for instance, E. oxyuroides– are compared to members of Pseudacrobeles, as they share simple (poorly developed) labial probolae, probably a plesiomorphic condition within Cephalobidae against the more complex (elongate and ramified) probolae that characterizes other lineages of the family and probably represents the corresponding apomorphic condition. Thus, present results propose a new approach to interpret the Cephalobidae phylogeny, in agreement with traditional postulates based on morphological data. Previous contributions, mainly based on molecular analyses, situated Eucephalobus species at intermediate (rather than basal) position within the subfamily Cephalobidae, closer to either the representatives of Zeldia (Nadler et al., 2006) or Cephalobus (Smythe and Nadler, 2006; Donn et al., 2012). On this subject, our results partially agree with those of van Megen et al. (2009), who also suggest a basal position of Eucephalobus, closer to Cephalobus and Heterocephalobus (synonym of Pseudacrobeles), although the phylogeny of the group was not very satisfactorily resolved by their analyses as Eucephalobus sequences appeared separated into two clades.

Figure 6:

Figure 7: