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

Abstract The identity of Eucephalobus oxyuroides is discussed after studying two Iberian populations that fit the original description of the species. A new characterization is consequently provided as follows: 0.49 to 0.70 mm long body, lip region continuous with the adjoining body and bearing short probolae prongs, neck 132 to 158 µm long, spermatheca 23 to 47 µm long or 1.0 to 1.8 times the corresponding body diameter, post-vulval uterine sac 40 to 54 µm long or 2.0 to 2.5 times as long as the body diameter, 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. Previous records of the species are revised. An emended diagnosis of the genus is proposed, and its taxonomy is updated with a list of species, key to their identification and illustrations. The evolutionary relationships of Eucephalobus, as derived from the analyses of 18S and 28S rDNA fragments, reveals that it occupies a basal position within the subfamily Cephalobidae.

Two E. oxyuroides populations from the Iberian Peninsula were collected in the course of a nematological survey conducted in natural areas. Their study revealed that they are very similar and tentatively regarded here as conspecific with the original material described by de Man. This contribution pursues their characterization, discusses the identity of previously reported populations, and updates the taxonomy of the genus.

Nematode extraction and processing
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.

Light microscopy
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.

Scanning electron microscopy (SEM)
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).

Phylogenetic analyses
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 × 10 6 generations. The tree was visualized and saved with FigTree 1.4.4 (Rambaut, 2018).
Material examined from Navarra (Spain): 11 females and 9 males, in good condition.

Measurements
Measurements are provided in Table 1.

Description Adult
They are moderately slender to slender (a = 25-33) nematodes of small size, 0.51 to 0.70 mm long. Upon fixation, habitus is nearly straight or somewhat curved ventrad. Cuticle is 2 µm thick, bearing transverse striation, and annuli is 2 µm wide. Lateral field is 3 to 4 µm broad, occupying 13 to 22% of mid-body diameter. Lip region is continuous with the adjacent body. Lips are conoid with protruding, conical labial and cephalic sensilla; primary and secondary axils are similar to each other. Labial probolae are three, biacute, bearing short acute prongs, each one fused to its more proximal lip. A fine incisure occurs between each labial probola and the adjacent lip pair. Amphids are very small, oval, located at the base of lateral lips. Stoma is cephaloboid, 2.2 to 2.3 times the lip region diameter long: cheilostom with strongly refringent, barshaped rhabdia, posteriorly thicker; gymnostom very reduced, with small rhabdia; stegostom consists of poorly refringent rhabdia, and metastegostom bearing dorsal acute rhabdion (dorsal tooth). Pharynx is also   cephaloboid: pharyngeal corpus is subcylindrical, 2.6 to 3.8 times the isthmus length, with procorpus and metacorpus not clearly separate; isthmus is comparatively thin, slightly tapering to its junction with basal bulb; the latter is ovoid with both valvular apparatus and posterior haustrulum well developed.  Intestine is without distinct specializations, but with slightly thinner walls at cardiac part.
Ovary is 116 to 160 µm long, usually with a flexure at its distal portion. Oviduct is very short, slightly areolate; spermatheca is 23 to 47 µm long, 1.0 to 1.8 times the corresponding body diameter long. Uterus is 48 to 80 µm long, 2.5 to 3.5 times as long as body diameter, tubular, and sometimes containing uterine eggs (53-62 × 21-23 µm). Post-vulval uterine sac is swollen, 40 to 54 µm long, and 2.0 to 2.5 times as long as the body diameter. Vagina is 7 to 9 µm long, extending inwards over 35 to 42% of the body diameter. Vulva is slightly protruding. Rectum is short, 1.1 to 1.7 times the anal body width; three large gland-like cells are distinguishable around the intestine-rectum junction.
Tail is conical-elongate with acute terminus. Phasmids are located at 19 to 23 µm or 28 to 33% of tail length from anus, and lateral fields terminating at phasmids.

Male
Reproductive system is monorchic, with testis reflexed ventrad anteriorly. Spicules are paired and symmetrical: manubrium is rounded, well developed and ventrad bent, short conoid calamus, and slightly curved ventrad lamina with acute tip in lateral view. Gubernaculum is well developed, slightly curved, about one-half of the spicule length, with thin corpus. Three small gland-like cells are distinguishable around the beginning of the cloaca. Tail is conical, slightly curved ventrad, ending in an elongate acute mucro. Genital papillae has seven pairs, two pre-cloacal and five post-cloacal: two pairs subventral at the middle of tail length, two ventral subterminal, and one lateral subterminal. One mid-ventral adcloacal papilla is present. Phasmids are at 21 to 24 µm or 46 to 55% of tail length from the cloacal aperture, close to middle genital papillae.

Other material examined
Two females examined from Acebeas (province of Jaén), with similar morphology and morphometry (Table 1) to the Navarra population, but one female is slightly smaller (493 µm long).

Distribution
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.

Discussion
Proposal of a new concept of E. oxyuroides 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.

Material fitting type material
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 (1876de Man ( , 1881de Man ( , 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.

Material not fitting type material
Several records of the species significantly differ from the aforementioned in one or several relevant features. Thus: 1. 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. 2. Rahm (1928Rahm ( , 1929 Andrássy (1964), who provided Demanian indices and illustrations of the tail in both sexes, suggest that it is not conspecific with the type due to a shorter post-vulval uterine sac (1.4 times the body diameter) and female tail (c′ = 3.6), male tail with very short mucro, and spicules lacking manubrium bent ventrad. It might not be a member of Eucephalobus. 4. The Hungarian females studied by Andrássy (1967) possess high labial probola prongs, excretory pore located at anterior part of isthmus, and post-vulval sac shorter than body diameter. They resemble E. compsus (Steiner, 1935) Thorne, 1937. 5. Monreal and Campoy (1982) recorded E. oxyuroides from northern Spain, provided Demanian indices and illustrated the tail of both sexes. The male tail lacks a distinct mucro, thus this material does not belong to this species. The lip region was not described, but these Iberian specimens might be members of the genus Pseudacrobeles Steiner, 1938. 6. The Brazilian population studied by Rashid et al. (1984) is distinguishable by its short post-vulval sac (about one body diameter), male with setalike or wart-like mucro, and spicules lacking a ventrad bent manubrium. As suggested by De Ley et al. (1993), it resembles Pseudacrobeles variabilis. 7. Gerber (1991) described (but did not illustrate) six females and one male from Austria characterized by having post-vulval sac 1.0 to 1.5 times the body diameter. This feature is not compatible with E. oxyuroides. 8. Boström (1993) studied one female and one male from Turkey characterized by having excretory pore apparently located at metacorpus-isthmus junction, post-vulval sac 1.2 times the body diameter and divided in two sections, and male tail bearing a short mucro. Although the author did not provide illustrations, available information suggests that this material resembles E. hooperi Marinari-Palmisano, 1967 or E. compsus. 9. The four Iberian females recorded by Abolafia and Peña-Santiago (2002) have their excretory pore and nerve ring situated at level of metacorpus, post-vulval sac more or less swollen and slightly divided in two sections, with septae. These specimens better fit E. compsus. 10. The very short post-vulval sac (one-half of body diameters) of the Bulgarian specimens examined by Iliev et al. (2003) is not compatible with E. oxyuroides. 11. Kim et al. (2017) described E. oxyuroides from South Korea, but this population resembles E. hooperi much more, due to the morphology of post-vulval sac (divided into two regions, proximally tubular and distally swollen) and tail in both sexes.

Diagnosis (emended)
Cephalobidae, Cephalobinae: they are small nematodes, 0.36 to 1.00 mm long, cuticle bearing transverse striation, lateral field with three longitudinal incisures, lip region continuous with the adjacent body and consisting of conoid lips, primary and secondary axils showing similar morphology, biacute labial probolae bearing prongs of variable length, each one fused at its more proximal lip, oval or rounded amphids located at the base of lateral lips, stoma and pharynx are cephaloboid, nerve ring is surrounding the metacorpus or the isthmus, excretory pore is at level of metacorpus or isthmus, female reproductive system is monodelphic-prodelphic, cephaloboid, ovary is usually with a flexure at its distal portion, oviduct is very short, spermatheca is well developed, post-vulval uterine sac is 0.5 to 2.5 times the body diameter, rectum is slightly longer than the anal body diameter, female tail is conical-elongate, conoid, or subcylindrical with seta-like, conoid or ragged mucro, phasmids are at anterior part of tail, male reproductive system is monorchic with testis reflexed anteriorly, spicules are slightly ventral curved, gubernaculum is almost straight, male tail is conical with seta-like, conoid or ragged mucro, genital papillae has seven pairs, two pre-cloacal and five post-cloacal, and phasmids are at mid-length of tail.

Comments on some species
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.
(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.
(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.
(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.

Notes about the phylogeny of Eucephalobus
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  or Cephalobus (Smythe and 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.