Morphological and Molecular Characterization of Oscheius saproxylicus sp. n. (Rhabditida, Rhabditidae) From Decaying Wood in Spain, With New Insights into the Phylogeny of the Genus and a Revision of its Taxonomy

Abstract A new species of the genus Oscheius, O. saproxylicus sp. n., collected in decaying wood obtained from an orchard in Southern Iberian Peninsula, is reported. A detailed description, including morphometrics, LM and SEM images, and molecular (18S and 28S rDNA genes) information is provided. The female is characterized by a moderately long body, lateral field with three longitudinal ridges, midbody vulva, and conical tail with acute tip. It was distinguished from its closest relative, O. dolichura, by a shorter tail and longer rectum. The male was not found. Morphological and molecular data support its belonging to Dolichura-group. Molecular analyses show that both Insectivorus and Dolichura groups are related to each other, being proposed as subgenera of the genus Oscheius as Oscheius and Dolichorhabditis. Finally, an updated taxonomy of the genus is presented, with generic and subgeneric diagnoses, list of species and a key to their identification.

proposed the new genus Oscheius under Rhabditidae (Örley, 1880), Rhabditinae (Örley, 1880), with O. insectivorus ( = Rhabditis insectivora Körner in Osche, 1952) as its type and only species. Oscheius was distinguished from other Rhabtidinae by its unusually short buccal tube, about as long as wide, and the absence of median pharyngeal swelling. Later, the same author (1983,1984) transferred a second species, O. koerneri ( = Rhabditis koerneri Osche, 1952), to the genus and provided its diagnosis. Sudhaus and Hooper (1994) provided new ideas about the taxonomy and the phylogeny of several rhabditid species: (i) accepted Oscheius as a subgenus of Rhabditis, (ii) regarded it as a monophyletic taxon based on three synapomorphies (long female rectum, terminal duct of the excretory system forwards coiled and with heavily sclerotized wall, and several features of spicule shape), (iii) considered Dolichorhabditis (Andrássy, 1983) as a junior synonym of Oscheius, and (iv) distinguished two species groups within the subgenus. One of these groups, the Insectivorus-group, included seven species with leptoderan or pseudopeloderan bursa (male tail with a filiform part standing out behind the bursa, a plesiomorphic state), and spicules with crochet needle shaped tip (apomorphic state). The second group, the Dolichura-group, with five species previously classified under Dolichorhabditis and having peloderan bursa (lacking the filiform part, an apomorphic condition) and spicules with thin tubular tip (plesiomorphic condition). Andrássy (2005) reinstated the generic range for Oscheius, listed a total of eight species under it, and distinguished it from Dolichorhabditis, with ten valid species, by several differences in stomatal teeth, bursa and spicules. The separation of both genera has been accepted in several contributions (Abolafia and Peña-Santiago, 2010;Gorgadze, 2010), but Sudhaus (2011) and Tabassum et al. (2016) maintained Dolichorhabditis as junior synonym of Oscheius as well the two monophyletic species groups within the latter.
An Oscheius population was collected in the course of a nematological survey conducted in southern Iberian soils. Its study revealed it belonged to a non-described form. The aims of this contribution are to characterize this material, to provide new insights on the phylogeny of the group, and to update its taxonomy.

Materials and methods
Nematode extraction and processing Nematodes were collected from dead wood using a modified trays technique (Whitehead and Hemming, 1965), killed by heat, fixed in 4% formalin, transferred to pure glycerine following the Siddiqi's (1964) method, and mounted on permanent glass slides. Moist, dead wood was maintained as a culture to extract specimens every several months.

Light microscopy (LM)
Observations were made using a Leitz Laborlux S (Leitz, Wetzlar, Germany) and Nikon Eclipse 80i (Nikon, Tokio, Japan) microscopes. Measurements were taken with the Leitz microscope, which has a drawing tube (camera lucida) attached to it, and Demanian indices and other ratios calculated. Drawings were made using the Leitz microscope. Images were taken with the Nikon microscope that was provided with differential interference contrast (DIC) optics and Nikon Digital Sight DS-U1 camera. Micrographs were edited 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 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).

DNA Extraction, PCR and Sequencing
Nematode DNA was extracted from single fresh individuals using the proteinase K protocol and PCR assays as described Castillo et al. (2003) somewhat modified. Specimen was cut in small pieces using a sterilized dental needle on a clean slide with 18 ml of AE 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) (Roche, Basel, Switzerland), and stored to -80°C within 15 min (for several days). The microtubes were incubated at 65°C (1 hr), then at 95°C (15 min). The microtube was centrifuged to 13,000 r.p.m. (or 15,900×g) for 3 min. and 2 μ l of the supernatant extracted DNA was transferred to a microtube containing: 2.5 μ l ×10 PCR reaction buffer, 5 μ l Q-solution ×5, 0.5 μ l dNTPs mixture (10 mM each), 1 μ l of each primer (10 mM), 0.2 μ l Taq DNA Polymerase (Qiagen, Venlo, The Netherlands) and ddH2O to a final volume of 25 μ l. The primers used for amplification of the D2-D3 region of 28S rRNA gene were the D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and the D3B (5'-TCGGAAGGAACCAGCTACTA-3') primers (De Ley et al., 1999). PCR cycle conditions were as follows: one cycle of 94°C for 3 min., followed by 35 cycles of 94°C for 1 min. + annealing temperature of 55°C for 45 s + 72°C for 2 min., and finally one cycle of 72°C for 10 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 a electrophoresis system (Labnet Gel XL Ultra V-2, Progen Scientific, London, UK). The bands were stained with RedSafe (×20,000) previously added to the agarose gel solution. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Venlo, The Netherlands), quantified using a spectrophotometer (Synergy HT, BioTek, Winooski, USA) and used for direct sequencing in both directions using the primers referred to above. The sequencing reactions were performed at "Centro de Instrumentación Científico-Técnica (CICT)" of the University of Jaén (Spain) using an Applied Biosystems Hitachi 3500 Genetic Analyzer. The sequences obtained were submitted to the GenBank database.

Phylogenetic analyses
For phylogenetic relationships, analyses were based on 18S and 28S rDNA. The newly obtained sequences were manually edited using BioEdit 7.2.6 (Hall, 1999) and aligned with another 18S or 28S rRNA gene sequences available in GenBank using Muscle alignment tool implemented in the MEGA7 (Kumar et al., 2016). The ambiguously aligned parts and divergent regions were known using the online version of Gblocks 0.91b (Castresana, 2000) (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) 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 tree was generated with Bayesian inference method using MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001;Ronquist and Huelsenbeck, 2003). Myolaimus byersi (KU180665 for 18S and KU180676 for 28S) was chosen as outgroup according to previous results by Kanzaki et al. (2009). The analysis under GTR+I+G model was initiated with a random starting tree and run with the Markov Chain   The specific epithet refers to the habitat where the species was found, decaying wood.

Material examined
Fifty one females in generally acceptable state of preservation.

Measurements
See Table 1. Description.

Female
Moderately slender to slender (a = 24-37) nematodes of small size, body 0.67 to 0.99 mm long. Upon fixation, habitus straight or somewhat curved ventrad. Cuticle 1 μm thick, nearly smooth under LM, but bearing very fine transverse striation when observed with SEM. Lateral field with three longitudinal ridges (alae), 3 to 6 μm broad or occupying Notes: Demanian indices (de Man, 1880): a = body length/body diameter; b = body length/pharynx length; c = body length/tail length; c' = tail length/anal body diameter; V = (distance from anterior region to vulva/body length) × 100.
one-tenth to one-fifth (11-19%) of mid-body diameter, and extending to phasmids. Lip region continuous with the adjacent body: lips rounded, separated by deep, U-shaped axils with six rounded labial and four acute cephalic sensilla. Amphids conspicuous, oval, with swollen margin. Stoma typical rhabditoid, 1.8 to 2.1 times the lip region width long or 2.6 to 4.0 times longer than broad: cheilostom lacking refringent rhabdia; gymno-promesostegostom (buccal tube) barrel-shaped, with gymnostom slightly narrower at its anterior part, glottoid apparatus of metastegostom with minute denticles, two per valve, and telostegostom consisting of small rounded rhabdia.
Pharynx rhabditoid: subcylindrical corpus 1.3 to 2.2 times longer than isthmus, and with not swollen metacorpus; isthmus robust, visibly thinner than metacorpus; basal bulb ovoid, with well-developed valvular apparatus. Cardia conoid, surrounded by intestinal tissue. Nerve ring located at 103 to 132 μm or 65 to 75% of neck length from the anterior end, at level of about middle isthmus. Excretory pore at 97 to 152 μm or 63 to 89% of neck length from the anterior end, at level of the middle or posterior part of isthmus. Deirids hardly in front of excretory pore, at 112 to 146 μm or 63 to 86% of neck length, at level of about middle isthmus. Intestine lacking any distinct differentiation, but its wall becoming thinner at cardiac part, and its cells often associated/infected with microsporidia spores (cf. Nishikori et al., 2018). Three large gland-like cells are present around the intestine-rectum junction. Rectum very long, 3.0 to 4.2 times the anal body diam. Reproductive system didelphic-amphidelphic, the anterior branch in dextral position to intestine and the posterior one in sinistral position: ovaries 64 to 156 μm long, with a flexure at their middle; oviducts short, 32 to 68 μm long, barely discernible, distally differentiated in a more or less spherical spermatheca with small female sperm cells inside; uteri very variable in length, 32 to 146 μm long or 1.5 to 4.9 times the corresponding body diameter, tubular, often containing several eggs in different developmental stages; vagina 6 to 9 μm long, extending inwards to 22 to 33% of body diameter; vulva not protruding, displaying lateral epiptygma. Tail conical-elongate with fine acute terminus, 1.4 to 2.2 times the rectum long. Phasmids located at 20 to 30 μm or 25 to 39% of tail length from anus.

Male
Unknown.

Remarks
During the culture of nematodes in dead wood under wet conditions, hundreds of Oscheius saproxylicus sp. n. specimens were obtained from a moist, dead wood culture. One hundred females and numerous juveniles were observed, but males were not found. Females exhibited consistent morphology. Generation of males by starvation in culture plate (Carta and Osbrink, 2005) could not be done. The presence of very small cells at uteri, which could represent hermaphrodite sperm (LaMunyon and Ward, 1998;Woodruff et al., 2010;Ellis and Schärer, 2014;Ellis and Wei, 2015), and the absence of males could indicate the evidence of hermaphroditism in this species.

Molecular characterization
One 923

Type locality and habitat
Spain, Jaén province, Jaén town, Puente de la Sierra (GPS coordinates: 37°42'36.5"N and 3°45'33.2"W, elevation 439 m), in association with decaying wood from dead white poplar trees present at the boundaries of an orchard.

Type material
Fourty seven females (holotype and paratypes) deposited in Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Spain; four female paratypes deposited in nematode collection of the Swedish Museum of Natural History, Stockholm (Sweden).

Phylogeny and Systematics of Oscheius Sensu Lato
Evolutionary relationships of Oscheius species have been previously analyzed with either traditional (morphological) or a modern (molecular) perspective, but an integrative approach is lacking yet. As mentioned in the introductory section, Andrássy (1976Andrássy ( , 1984Andrássy ( , 2005, on the basis of morphological data, defended the separation of Osheius from other Rhabditinae genera, especially from Dolichorhabditis, whereas Sudhaus and Hooper (1994; see more recently Sudhaus, 2011), by means of morphological cladistic analyses, advocated the synonymy of Oscheius and Dolichorhabditis. However he recognized two monophyletic species groups, the Insectivorus-group, including Oscheius species sensu Andrássy (op. cit.), and the Dolichura-group, including Dolichorhabditis species sensu Andrássy (op. cit.). Available molecular analyses (Ye et al., 2010(Ye et al., , 2018Darby et al., 2011;Campos-Herrera et al., 2015;Torrini et al., 2015;Tabassum et al., 2016;Lima de Brida et al., 2017;Valizadeh et al., 2017) have repeatedly confirmed the monophyly of both Insectivorus-and Dolichura-groups, and most of them agree that these groups are sister groups. Nevertheless, two contributions (Darsouei et al., 2014;Zhou et al., 2017) do not support this idea.
The molecular analysis of 18S and 28S rDNA sequences of the new species herein described, whose results are presented in the trees of Figures 4 and 5, respectively, confirm the monophyly of both species groups as well as that they are sister groups. Thus, O. saproxylicus sp. n. sequences form a highly supported (100%) clade with several representatives of the Dolichura-group. The well-supported (100%) Insectivorus sister group joins it in a larger, highly supported (100%) clade in the 28S tree but moderately supported (65%) clade in the 18S tree. In its turn, the Dolichura-group/clade consists of three highly supported sub-clades that should be a matter of further analysis when more sequences become available. Internal relationships within the Insectivorus-group/clade cannot be elucidated yet.
Regarding the outer relationships of Oscheius species, the topology of the 28S tree shows that they could share a most recent common ancestor with representatives of the genus Metarhabditis (Tahseen et al., 2004). However, the 18S tree topology is inconsistent with that of the 28S relative to the more distantly positioned Metarhabditis in this deeper phylogenetic tree. This could be explained because small subunit rDNA sequences are better for elucidating higher relationships within organisms, phyla and classes, while large subunit sequences are useful for distinguishing among genera and species (Hillis and Dixon, 1991). Morphologically, both genera are superficially similar, differing in lip region (lips separated vs grouped in pairs), female rectum length (very long vs always short, as long as the anal body width), and bursa (three vs two well developed genital papillae at its posterior end. The long female rectum of Oscheius certainly is an apomorphic condition, and the paired lips and the presence of only two genital papillae in the bursa of Metarhabditis probably represent apomorphic states of their respective characters. Tabassum et al. (2016) transferred Metarhabditis species and its synonyms (Asif et al., 2013) to Oscheius, but the authors did not justify their action, which is not herein followed. The relationships with other genera of Rhabditidae remain more obscure as the branching of the tree is not definitely resolved.
Both morphological (Sudhaus and Hooper, 1994;Sudhaus, 2011) and molecular (among others Darby et al., 2011;Campos-Herrera et al., 2015;Torrini et al., 2015;Lima de Brida et al., 2017;Ye et al., 2018) evidences support the monophyly of Oscheius, with two well-defined monophyletic subgroups among its species. A reasonable translation of these ideas to classification results in the maintenance of Oscheius as valid genus, with Dolichorhabditis as its junior synonym, and the proposal of two subgenera: Oscheius for the Insectivorus-group of species and Dolichorhabditis for the Dolichura-group of species.

Updated Taxonomy of Oscheius
In the following diagnoses of the genus and its two subgenera, a list of their species and a key to their identification are presented. The diagnoses are mainly based on Sudhaus' (2011) ideas about the concept of the genus and the differences between the two species groups. In addition, the status of several species, in particular those described in recent years, is discussed. Subgenus Oscheius Andrássy, 1976 syn. Oscheius sensu Andrássy (1976Andrássy ( , 1984Andrássy ( , 2005. (Fig. 6).

Diagnosis
Stoma tubular with metastegostom bearing warts. Bursa leptoderan. Male tail conoid with tip out of the bursa, filiform, variable in length. Spicules distally hook-shaped, like a crochet needle.

Diagnosis
Stoma tubular or barrel-shaped with metastegostom bearing setose teeth. Bursa peloderan. Male tail tip not reaching beyond bursa end. Spicule tips shaped like a probe head.
cynodonti Much resembling O. rupraekramae, it can be separated from this in its smaller general size (female body 1.25-1.66 vs 1.80-2.40 mm long). Female tail in the former is mentioned to be comparatively longer (c' = 4.0-6.7 (3.1 in LM picture and 3.8 in drawing) vs 2.6-3.7), but c' is ca. 3.1 in LM picture and 3.8 in line drawing, thus raising a doubt about the consistency of this difference.

debilicauda
The identity of this species has raised some controversy as Andrássy (2005) considered it as a valid species whereas Sudhaus (2011) regarded it as junior synonym of O. dolichura. Both taxa are distinguishable by their female rectum length (slightly longer than vs ca. 1.5-2.0 times the anal body diam.), female tail length (c' = 3-4 vs c' = 4.7), morphology of spicules (manubrium rounded, swollen and strongly bent ventrad vs rounded but not swollen and slightly bent ventrad), and genital papillae arrangement (GP4-6 at level of vs posterior to cloacal). Thus, they are herein kept as valid and separate species. dolichura apud Tabassum and Shahina (2002), nec Schneider (1866) oxyuris The identity of this species maintains some doubts being regarded by Andrássy (1983)  sacchari Very similar to O. shamimi, both species are distinguishable by minor and questionable differences: slightly larger females (1.36-2.02 vs 0.76-1.52 mm), slightly posterior position of the excretory pore (at basal bulb level vs at isthmus level), and slightly shorter spicules (47-55 vs 53-67 μm) and gubernaculum (18-22 vs 22-28 μm), insufficient differences to maintain them as separate species.
tipulae Morphologically, this species and O. onirici are nearly indistinguishable, but molecular analyses show relevant differences. Thus, they represent a case of cryptic species within the subgenus Dolichorhabditis.
wohlgemuthi Tahseen and Nisa (2006) mentioned this species as Oscheius wohgelmuthi, but they did not justify the nomenclatorial change which was later officially promoted by Sudhaus (2011). On the other hand, the morphological pattern of this species, with swollen metacorpus (spheroid), moderate length rectum (1.2-1.6 times ABW) and male with bursa bearing nine genital papillae with GP1 and GP2 more widely spaced than GP2 and GP3 (1+2/3+3), agrees better with the members of the Brassicae-group (Sudhaus, 2011) of the genus Rhabditis (Dujardin, 1845).
zarinae Very similar to O. debilicauda, both species being distinguishable in a few minor (but apparently relevant) differences in their general size (body 0.75-1.56 vs 0.53-0.75 mm long), female tail shape (posterior half very thin, almost filiform vs thicker, not filiform), and spicule length (32-48 vs 29 μm).