Morphological, Morphometrical and Molecular Characterization of Oscheius siddiqii Tabassum and Shahina, 2010 (Rhabditida, Rhabditidae) from India with Its Taxonomic Consequences for the Subgenus Oscheius Andrássy, 1976

Simple Summary Due to their potential entomopathogenicity, nematodes of the genus Oscheius have been in the spotlight of the scientific interest in recent years. Unfortunately, some of these species are poorly described and have inadequate or no molecular support. This fact complicates the systematics of the group, and a revision of these species is necessary to elucidate their taxonomic status. In the present study, we provide a detailed description based on the morphological, morphometrical, and molecular characteristics of Oscheius siddiqii from Uttar Pradesh, India, including the first scanning electron microscopy (SEM) studies of the species. Furthermore, based on morphological and molecular data, the status of some Oscheius species is discussed, and several synonymisations are proposed. Abstract An insect parasitic nematode belonging to the genus Oscheius was recovered from the agricultural soils from the Hapur district in western Uttar Pradesh, India. Morphological studies on this species exhibited its high resemblance with two Pakistani species: Oscheius siddiqii and O. niazii. No molecular data are available for these taxa but, morphologically, both species do not differ significantly from our strains and each other. Hence, these nematodes can be considered conspecific, and the correct name for this taxon is O. siddiqii, the first described species. The phylogenetic analyses of the ITS-, 18S-, and the 28S rDNA sequences showed that O. siddiqii is a sister taxon to the group formed by Oscheius microvilli, O. myriophilus, O. safricanus, and several unidentified Oscheius species. Additionally, our analyses show that based on molecular and morphological data, the species Oscheius rugaoensis and O. microvilli cannot be distinguished from O. chongmingensis and O. myriophilus, respectively, and are thus considered junior synonyms of these taxa. Furthermore, the available data are not sufficient to evaluate the status of Oscheius basothovii and O. safricanus, which are, in consequence, considered species inquirendae. These findings highlight the necessity of the proper morphological and molecular characterisation of the described Oscheius species.


Introduction
The genus Oscheius was proposed by Andrássy [1], with O. insectivorus (=Rhabditis insectivora Körner in Osche 1952) as type species. Sudhaus [2,3] distinguished two groups into the genus Oscheius, the Insectivorus-group, which includes species with a bursa leptoderan or pseudopeloderan and spicules with a crochet needle-shaped tip, and the Dolichura-group, which includes species with a peloderan bursa and spicules with a thin tubular tip. More recently, Abolafia and Peña-Santiago [4] categorised both the groups as subgenera, dividing them into Oscheius: Oscheius and Dolichorhabditis, respectively, according to the division proposed by Andrássy [5,6]. With respect to the subgenus Oscheius (or insectivorus-group), 23 valid species have been described at the time of writing [4,7] (Supplementary Table S1).
Unfortunately, some of these species are poorly described and have inadequate or no molecular support. This fact complicates the systematics of the group, and a revision of these species is necessary to elucidate their taxonomic status. In our recent study [7], we compared an Indian population of Oscheius sp. from India and six Oscheius species described from Pakistan (Oscheius citri, O. cobbi, O. cynodonti, O. esculentus, O. punctatus, and O. sacchari). The morphological comparison and molecular analysis of the only available genetic marker for the Pakistani species, the ITS rDNA sequence, showed that these nematodes do not differ from our Indian population and from each other; hence, they can be considered synonyms, and the correct name for this taxon is the first described species, O. citri. Such kinds of studies can significantly clarify group systematics and can improve our understanding of species diversity.
The molecular taxonomy of nematodes relies on phylogenetic reconstructions that are based on three nuclear ribosomal DNA genetic markers (conserved and variable regions of the 18S and 28S subunits and the more variable ITS region) [8]. In some nematode groups, the 18S and 28S genes lack the resolution that is required to distinguish between these closely-related lineages [9]. The quickly evolving internal transcribed spacer (ITS) region may display intrapopulation, or even individual variability [10,11], which could complicate its use in systematics [12]. Mitochondrial genes thus represent a viable option in nematode systematics [12]. Unfortunately, so far, no mitochondrial gene sequences are available for Oscheius nematodes, with the only exception being Oscheius onirici, which has several published sequences of the cytochrome oxidase I (COI) gene available in the NCBI GenBank database [13,14].
During the course of the nematological sampling survey in the Hapur district of western Uttar Pradesh, India, two Oscheius populations, labelled CS42 and CS43, were isolated from agricultural soil samples using the Galleria trap method [15] and were later morphologically identified as O. siddiqii Tabassum & Shahina, 2010.
Oscheius siddiqii was first described from soil samples taken from around the roots of rose (Rosa damascena L.) in Karachi, Sindh, Pakistan [16]. This species was previously described based on morphological and morphometric studies alone. In the present study, a detailed description based on the morphological, morphometrical, and molecular (small subunit, large subunit, and internal transcribed spacer (ITS1-5.8S-ITS2) rDNA gene sequences) characteristics of O. siddiqii from Uttar Pradesh, India, is provided, including the first scanning electron microscopy (SEM) studies of the species. We also provide the first COI sequences of O. siddiqii and several others Oscheius species. Furthermore, based on morphological and molecular data, the status of some Oscheius species is discussed.

Nematode Source
Twenty soil samples were collected in different agricultural fields of the Brijghat area of the Hapur district (28 • 47 N, 78 • 4 E and elevation of 219 m above sea level) in western Uttar Pradesh, India, in February 2018. The predominant climate in these areas is semi-arid and moderate to tropical monsoon (humid subtropical). Each sample had 1.5 kg of soil, which was made up of five soil subsamples collected at five sites within each field (one sample from each corner of the field and one from the centre of the field). Samples were collected at a 15-20 cm depth around the rhizosphere of crop plants [17]. These samples were tested for the presence of nematodes using the Galleria soil baiting technique [15]. Two soil samples taken from sugarcane fields (Saccharum officinarum L.) and cucumber (Cucumis sativus L.) cultivars were found to be positive for the presence of nematodes and were labelled as CS42 and CS43. The pH of the soil samples ranged from 9.6-10.0. The cadavers of Galleria mellonella L. (Lepidoptera, Pyralidae) from the soil baits were transferred to white traps after proper washing with double distilled water and sterilization with 0.1% NaOCl [12]. The nematodes that emerged from the white trap within 6-10 days were collected. The nematode cultures were kept in 250 mL flasks in the Bio-Oxygen Demand incubators at a temperature of 15 • C.
Slides and live specimens are also deposited at CCS University Meerut, University of Jaén, and the Institute of Entomology Czech Republic, respectively. Scanning electron microscopy and light microscopy images were taken at the University of Jaén, while the molecular studies were conducted at CCS University Meerut and at the Institute of Entomology, Czech Republic.

Morphological and Morphometrical Characterization
Prior to the light microscopic studies and morphometry analyses, the nematodes were propagated using the last instar larvae of G. mellonella by injecting 10 greater wax moth larvae with approximately 2000 surface-sterilized dauer larvae in sterilized Petri dishes using a 1 mL insulin syringe. The larvae died within 48-72 h [7]. Adult generations (females and males) and 3rd stage juveniles were isolated from the white trap within 7-10 days [18]. These were killed with hot water at 60 • C [19], fixed in TAF (7 mL formalin, 2 mL triethanolamine, 91 mL ddH 2 O) [20], dehydrated by means of Seinhorst's [21] method, and processed to glycerine [22]. The nematodes were mounted in a small drop of glycerine on permanent glass slides and were sealed with an extra amount of paraffin wax to prevent the flattening of the nematodes [23]. The observations and measurements of 136 specimens were taken using the Nikon DS-L1 image acquisition software mounted on a phase-contrast microscope (Nikon Eclipse 50i) in µm (Tables 1 and 2). A Nikon Eclipse 80i (Nikon, Tokyo, Japan) light microscope with differential interference contrast optics and a Nikon Digital Sight DS-U1 camera were used to photograph the best-preserved nematode specimens. Adobe ® Photoshop ® CS was used to process the micrographs. Nematode species were identified based on morphological and morphometric features using the keys described by Abolafia and Peña-Santiago [4]. The morphological and morphometrical traits used to characterise the species are given in Tables 1 and 2, and the morphometrical data were analysed in SPSS software to calculate different central tendency measures (Table 1).

Scanning Electron Microscopy
For the scanning electron microscopy, male and female specimens kept in glycerol were chosen for observation according to the protocol proposed by Abolafia [24]. The nematodes were hydrated in dH 2 O, dehydrated in a graded ethanol-acetone series, critical point dried with dry ice, mounted on SEM stubs with copper tape, and coated with gold in a sputter coater. Specimens were observed, and the microphotographs for ach sample were captured with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany). All of the light and scanning electron microscopy images were submitted to the X public database.
The PCR master mix included 7.25 µL of ddH 2 O, 1.25 µL of 10 × PCR buffer, 1 µL of dNTPs, 0.75 µL of each forward and reverse primers, 0.1 µL of DNA polymerase, and 1 µL of DNA-extract. The PCR profiles were used as follows for ITS: 1 cycle of 94 • C for 7 min followed by 35 cycles of 94 • C for 60 s, 50 • C for 60 s, 72 • C for 60 s, and a final extension at 72 • C for 7 min [26]; for 28S rDNA: 1 cycle of 94 • C for 7 min followed by 35 cycles of 94 • C for 60 s, 55 • C for 60 s, 72 • C for 60 s, and a final extension at 72 • C for 10 min [29]; for 18S rDNA: 1 cycle of 94 • C for 5 min followed by 37 cycles of 94 • C for 60 s, 55 • C for 90 s, 72 • C for 2 min, and a final extension at 72 • C for 10 min [30]. After PCR, 2 µL of PCR product was electrophoresed in a 1% TAE-buffered agarose gel stained with ethidium bromide (20 µL ETB per 100 mL of gel) for 45 min at 120 V [31]. For each locus, PCR products from five females were sequenced by Eurofins Genomics (Ebersberg, Germany) and PCR-product from a bulk of dauer juveniles was sequenced by Bioserve PVT Ltd. (Hyderabad, India). No variation was detected in any of the products for each locus except for the ITS region of the strain CS42, and one sequence for each fragment was deposited in GenBank under accession numbers MH837093 (ITS sequence, strain CS43, the amplicon length 939 bp), MH819729 (28S sequence, CS42, amplicon length 885 bp), MT835468 (18S sequence, strain CS42, amplicon length 1571 bp), and MH845237 (28S sequence, CS43, amplicon length 885 bp). Due to the presence of variability in the ITS region of the strain CS42, an additional 15 individuals were sequenced for this marker. The two sequence variants were deposited under the accession numbers MH837095 and MT835467 (ITS sequences, strain CS42, and amplicon lengths 1010 bp and 990 bp).

Sequence Alignment and Phylogenetic Analyses
The sequences were edited and compared with other sequences deposited in the GenBank NCBI database using the BLAST tool [32]. An alignment of our sequences with Biology 2021, 10, 1239 6 of 18 those of other Oscheius species was performed for each amplified locus (ITS, SSU and LSU) using the default ClustalW parameters in MEGA 7.0 [33] and was optimized manually in BioEdit [34]. The final alignment lengths were 904 bp (ITS), 476 bp (D2D3) and 1565 bp (SSU). Pairwise distances were computed using MEGA 7.0 [33] in order to compare the ITS rDNA locus of the selected Oscheius species from the "insectivorus" group.
The phylogenetic reconstruction was performed from the datasets using the Bayesian inference (BI). All of the characters were treated as being equally weighted. Heterorhabditis bacteriophora Poinar, 1976; H. zealandica Poinar, 1990; and H. downesi Stock, Griffin & Burnell, 2002; were used as outgroup taxa. Bayesian analyses were performed in MrBayes 3.2.7 [35]. The best fit model was identified as the GTR + G model test using the MrModeltest 2.0 program [36]. Metropolis-coupled Markov chains Monte Carlo (MCMCMC) generations were run for 1 × 10 7 cycles, and 1 tree was retained every 1000 generations. The Bayesian trees were visualized using the Mesquite program [37].

Analysis of the Cytochrome Oxidase I Gene
For the analysis, Ocheius siddiqii CS42 and five other Oscheius species or strains from our collection (Table 3) were used. The DNA was isolated from single females, and the cytochrome oxidase subunit I (COI) gene was amplified using the universal primers LCO-1490 (5 -GGTCAACAAATCATAAAGATATTGG-3 ) (forward) and HCO-2198 (5 -TAAACTTCAGGGTGACCAAAAAATCA-3 ) (reverse) [38] with the following PCR profile: 1 cycle of 94 • C for 2 min followed by 37 cycles of 94 • C for 30 s, 51 • C for 45 s, 72 • C for 2 min, and a final extension at 72 • C for 2 min. Sequences were deposited in GenBank under accession numbers OK142792-OK142797 (Table 3).

Description of Oscheius (Oscheius) siddiqii Tabassum & Shahina, 2010
Female (n = 20): Body larger than in males; 1.10-1.70 mm long; straight to slightly arcuate. Cuticle moderately transversely annulated. Lateral fields consisting of six distinct longitudinal incisures and other two sublateral poorly marked incisures. Lip region bearing six separate lips, with six inner long acute labial papillae and four outer short acute cephalic papillae in total; primary axils deeper than secondary axils. Amphidial apertures are porelike and ovoid. Stoma rhabditoid type; 4.1-5.9 times longer than wide and 3.4-6.2 times the lip region width: conspicuous cheilostom, poorly cuticularised rounded rhabdia; gymnostom and pro-mesostegostom cuticularised with straight tubular rhabdia; short metastegostom with well-developed glottoid apparatus; short telostegostom with rounded cuticularised rhabdia. Conspicuous deirids present at 81-93% of neck length. Pharynx differentiated into cylindrical procorpus with swollen and subcylindrical metacorpus that is about twice as long as it is wide; long isthmus that is narrower than the procorpus; ovoid basal bulb with conspicuous valvular apparatus. Nerve ring encircling the isthmus at 70-89% of the neck length. Excretory pore at basal bulb or posterior that is located at 100-110% of the neck length. Conspicuous hemizonid just above excretory pore at the level of the basal bulb. Intestine with the cardiac region with thinner walls. Didelphic and amphidelphic reproductive system; well-developed ovaries that are dorsally reflexed and that extend beyond the vulva; anterior and posterior ovaries on the same side of the intestine, with the anterior one larger than posterior one; oviducts with spermathecae are ovoid and filled with sperm and connected to dilated ovaries; long uteri that are divided in two parts are only observed in young females and comprise one distal tubular part and other proximal swollen part with thinner walls; uterine eggs in different embryonation stages compacted in uteri ranging in number from 6-24 and measuring 26- Didelphic and amphidelphic reproductive system; well-developed ovaries that are dorsally reflexed and that extend beyond the vulva; anterior and posterior ovaries on the same side of the intestine, with the anterior one larger than posterior one; oviducts with spermathecae are ovoid and filled with sperm and connected to dilated ovaries; long uteri that are divided in two parts are only observed in young females and comprise one distal tubular part and other proximal swollen part with thinner walls; uterine eggs in different embryonation stages compacted in uteri ranging in number from 6-24 and measuring 26-57 × 27-32 µm; vagina straight or slightly arcuate, ventral with lateral epiptygmata. Distinct rectum distinct that is 1.9-2.8 times anal body width, with three unicellular glands at its junction with the intestine. Large anus that is directed posteriorly. Straight, conoid tail with acute terminus. Distinct, pore like, phasmids located at 50-80% of tail length.
Biology 2021, 10, x FOR PEER REVIEW 9 of 19 57 × 27-32 µm; vagina straight or slightly arcuate, ventral with lateral epiptygmata. Distinct rectum distinct that is 1.9-2.8 times anal body width, with three unicellular glands at its junction with the intestine. Large anus that is directed posteriorly. Straight, conoid tail with acute terminus. Distinct, pore like, phasmids located at 50-80% of tail length.  vesicle. Ejaculatory glands not present. Tail with conoid anterior part that is flanked by the bursa, and short and thin posterior filiform section that is located outside of the bursa and that is sometimes inconspicuous under LM. Bursa leptoderan with nine pairs of genital papillae (1 + 1 + 1/3 + 3 + ph): three pairs pre-cloacal (GP1-GP3) and six pairs post-cloacal, with three pairs at mid tail length (GP4-GP6) and three pairs (GP7-GP9) at the terminal length; GP1, GP2, and GP3 are spaced apart; space between GP1 and GP2 is greater than the space between GP2 and GP3. Phasmids are easily observed and are located posterior to GP9 at 55-65% of the tail length. Long spicules that are broad and arcuate as well as larger than the gubernaculum and a manubrium that is ventrally bent and conoid to rounded; short calamus and lamina that are slightly ventrally curved with dorsal hump that is variable in size; long ventral velum and hooked tip. Gubernaculum with manubrium-corpus that is almost straight; well-developed crura with acute tip that is at 37-49% of the spicule length.
Biology 2021, 10, x FOR PEER REVIEW 10 of 19 orchic reproductive system with testis ventrally reflexed anteriorly on the left side of intestine. Vas deferens are broad and light tubes filled with sperm and lack demarcation of seminal vesicle. Ejaculatory glands not present. Tail with conoid anterior part that is flanked by the bursa, and short and thin posterior filiform section that is located outside of the bursa and that is sometimes inconspicuous under LM. Bursa leptoderan with nine pairs of genital papillae (1 + 1 + 1/3 + 3 + ph): three pairs pre-cloacal (GP1-GP3) and six pairs post-cloacal, with three pairs at mid tail length (GP4-GP6) and three pairs (GP7-GP9) at the terminal length; GP1, GP2, and GP3 are spaced apart; space between GP1 and GP2 is greater than the space between GP2 and GP3. Phasmids are easily observed and are located posterior to GP9 at 55-65% of the tail length. Long spicules that are broad and arcuate as well as larger than the gubernaculum and a manubrium that is ventrally bent and conoid to rounded; short calamus and lamina that are slightly ventrally curved with dorsal hump that is variable in size; long ventral velum and hooked tip. Gubernaculum with manubrium-corpus that is almost straight; well-developed crura with acute tip that is at 37-49% of the spicule length.  Third stage juvenile (J3) (n = 20): Robust body that is 0.50-0.60 mm long with straight or slightly curved at posterior end. Cuticle is almost smooth, with a lip region similar to adult specimens. Narrow stoma that 2.1-3.0 times as narrow as the lip region is wide. Pharynx clearly visible and differentiated into the three rhabditoid parts. Nerve ring surrounding the isthmus that is about 60-74% of the neck length. Excretory pore at or just posterior to basal bulb. Obscure deirid. Reduced cardia reduced that is surrounded by intestinal tissue. Rectum longer than anal body width. Anus prominent. Tail conoid; spicate with fine hair-like terminus without mucro with terminal hyaline part that is 42-66% of the tail length.
Third stage juvenile (J3) (n = 20): Robust body that is 0.50-0.60 mm long with straight or slightly curved at posterior end. Cuticle is almost smooth, with a lip region similar to adult specimens. Narrow stoma that 2.1-3.0 times as narrow as the lip region is wide. Pharynx clearly visible and differentiated into the three rhabditoid parts. Nerve ring surrounding the isthmus that is about 60-74% of the neck length. Excretory pore at or just posterior to basal bulb. Obscure deirid. Reduced cardia reduced that is surrounded by intestinal tissue. Rectum longer than anal body width. Anus prominent. Tail conoid; spicate with fine hair-like terminus without mucro with terminal hyaline part that is 42-66% of the tail length.

Molecular Characterization
Oscheius siddiqii is characterized by sequences of the ITS region, the SSU and D2D3 region of LSU of the rDNA and mitochondrial cytochrome oxidase I gene. Two variants of the ITS sequence were observed in the strain CS42, which had either C (40%) or T (60%) base at a position, which corresponded to position 335 bp of the sequence MT835467.   The analysis of the cytochrome oxidase I gene showed that the sequence of O. siddiqii differs from other available species from the "insectivorus" group by 3-9%, with O. chongmingensis being the most similar (97.3%).

Remarks
Unfortunately, some of the currently recognized Oscheius species have inadequate or no molecular support. This is because at present, since predominantly molecular identification of nematodes is used, the lack of molecular data for nematode species complicates the systematics of the group.
Generally, the species with no molecular data not only complicate systematic and phylogenetic studies but also present a problem for biodiversity studies, and it is thus desirable to match these older taxa with molecular data. This, however, presents a challenge, especially in the case of nematodes, where a number of cryptic species have been reported [39][40][41], and therefore, assigning the molecular data just based on morphological similarities poses an inherent risk of overlooking cryptic species. Nevertheless, such a situation can be possibly mended in the future if the original species is re-isolated. Alternatively, there will be more taxa than the actual evolutionary lineages, and according to Carstens et al. [42], in most contexts, it is better to fail to delimit species than it is to falsely delimit entities that do not represent actual evolutionary lineages. Moreover, Oscheius species, for which both morphology and molecular data are available, show interspecific differences in both datasets, and so far, no cryptic species have been reported in this group.
The material examined now from India agrees well with O. siddiqii and O. niazii Tabassum & Shahina, 2010 (the correct name of the latter should be O. niazensis because it refers to a village), the two very similar species that were both described from the same district in Pakistan. Regrettably, we were not able to gain the access to paratypes of these Pakistani taxa in order to study their morphology and to corroborate their identity despite numerous attempts. The descriptions of the type populations of both Pakistani species, despite the poor quality of their illustrations and the existence of some inaccuracies in the drawings, show negligible morphological and morphometrical differences (see Supplementary Tables S2 and S3), and the measurements mainly overlap, with O. niazensis having somewhat larger ranges. Therefore, with no significant morphological difference between both nematodes, we consider them to be two populations of a single species. Alternatively, the Pakistani and Indian populations could represent two morphologically identical species; however, we consider this possibility to be unlikely. Comparisons of the Indian populations examined now with both Pakistani species have shown that they resemble each other in terms of all of the important traits, such as stoma morphology (especially in O. niazensis), the arrangement of the genital papillae, with the GP2 and GP3 being spaced closer together, and the spicules, with the ventral bent tip, with no other important differences being detected. Unfortunately, the Pakistani species lack molecular data in order to adequately compare them to the Indian populations. Nevertheless, the morphological and morphometrical data show that the Indian populations and both Pakistani populations are the same species. According to the International Code of Zoological Nomenclature (ICZN), O. siddiqii, being the first described species in the paper published by Tabassum & Shahina [16], is the valid name for the taxon and the type population, whereas O. niazensis should be considered as its junior synonym.
For the first time, we provide the COI sequences for five Oscheius species, and these data will form the basis for further molecular systematic studies of the group. The sequence diversity among species was lower than it was in the ITS sequences, which is in agreement with the observations made with, e.g., heterorhabditid entomopathogenic nematodes [43] or populations of the parasitic nematode Camallanus cotti [44]. Nevertheless, the COI sequences show both interspecific and intraspecific variation and thus provide further resolution among Oscheius species.

Diagnosis (Based on Indian Populations)
Oscheius siddiqii is characterized by its body size, which ranges from 1.10-1.70 mm in females and from 0.92-1.44 mm in males; a cuticle with delicate transverse striations; lateral fields consisting of eight prominent lines; a lip region bearing six separate lips; a tubular stoma that is 13-20 µm in length; a pharynx with a more robust and subcylindrical metacorpus; a nerve ring at the isthmus-bulb junction; an excretory pore situated at the level of the basal pharyngeal bulb or slightly posterior; a didelphic-amphidelphic female reproductive system ; an equatorial vulva (V = 45-57) with a lateral epiptygmata; a rectum that is 1.9-2.8 times the anal body width; a female conical tail with an acute tip (123-180 µm long, c = 7.8-15.4, c' = 3.5-7.5) and a male conoid tail (36-49 µm long, c = 20.0-33.0, c' = 1.4-2.5) with small filiform part, spicules 39-55 µm long with a crochetlike tip; and a gubernaculum that is 21-26 µm long. A bursa leptoderan with nine pairs of genital papillae (GP) (1 + 1 + 1/3 + 3 + ph) and well developed phasmids are located posteriorly to GP9.

Phylogenetic Relationships of the Species of the Subgenus Oscheius
The reliable reconstruction of phylogenetic relationships within the subgenus Oscheius is hampered by the fact that for some established species, not all standardly used genetic markers are available. This not only complicates phylogenetic reconstructions of the relationship within the group but also presents a challenge for distinguishing the newly described species from the existing ones. In our analyses, the position of the majority of Oscheius species was consistent in all trees, and the majority of species were clearly separated from other species, with four prominent exceptions:  Andrássy, 1976 (EU195968) with 100% support, and both sequences are almost identical, with only one bp deletion difference. The D2D3 region is known to provide little resolution, and identical D2D3 sequences were observed in some closely related Meloidogyne species [45]. However, considering the fact that intraspecific differences in Oscheius species with more available D2D3 sequences (O. Myriophilus, O. chongmingensis) are significantly higher and range up to 13 bp, we assume that most likely, the sequence MN381940 actually belongs to O. insectivorus.

Oscheius basothovii and O. safricanus
Based on the analyses of the ITS and SSU sequences, Oscheius safricanus seems to be very closely related to O. myriophilus, and in the LSU tree, it falls within O. myriophilus strains. Unfortunately, it is not possible to compare the morphology because the line illustration provided by Serepa-Dlamini & Gray [46] is slightly modified from that of the description of Oscheius rugaoensis provided by Zhang et al. [47], and surprisingly, the LM micrographs, at least in the male (see Figure 3B), apparently correspond with some species of the genus Steinernema. According to the morphology of the spicules.
Interestingly, the ITS tree shows that Oscheius basothovii is even closer to O. myriophilus, with which it forms a monophyletic clade that is sister to O. safricanus. Unfortunately, no LSU and SSU sequences have been given in the description of O. basothovii [48], so it is not possible to assess the status of this species. Morphologically, the very poor quality of the illustrations provided for O. basothovii by Lephoto & Gray [48] makes it impossible to correctly evaluate its morphology and to compare it with O. myriophilus.
Both species, O. basothovii and O. safricanus, could be considered as species inquirendae.

Oscheius microvilli
The original description of O. microvilli only provides the ITS and SSU sequences under accession numbers KT825914 and KT825913, respectively [49]. In both of our phylogenetic reconstructions, O microvilli falls into the clade of other O. myriophilus strains with high support.
Furthermore, the differences between the ITS sequence attributed to O. microvilli and the respective sequences of other O. myriophilus are negligible and are within the standard intraspecific range (Table 5). In the case of SSU, the analysis revealed more than 60 differences ( Table 4). The fact that the conservative SSU harbors several times more differences than the variable ITS suggests that the SSU sequence might be inadequately edited. A closer look at the alignment shows that all of the differences occur at the beginning (1-41 bp) and at the end (1572-1615 bp) of the sequence and in positions 1209-1332 bp. In all of the other positions, the sequence is identical to the sequences of the O. myriophilus strains. The corresponding positions of the alignment are quite conservative, and these variable positions in the sequence KT825913 are probably sequencing errors. To conclude, the available molecular data attributed to O. microvilli are not sufficient to separate this taxon from O. myriophilus. Morphological comparisons of these two species do not display significant differences, with the exception of males, but the male morphology described in the description of O. microvilli agrees with Caenorhabditis sinica according the bursa morphology among other morphological characteristics and morphometry (see [4]). Therefore, O. microvilli is proposed as a junior synonym of O. myriophilus.

Oscheius rugaoensis
The original description of O. rugaoensis provided the ITS and SSU sequences [47], and in both the ITS and SSU trees, this species is a member of a monophyletic clade together with several strains of O. chongmingensis, suggesting that these nematodes are in fact conspecific. Moreover, distance analyses of the ITS and SSU sequences of O. rugaoensis and O. chongmingensis only show an insignificant number of differences (Tables 4 and 5). The NCBI GenBank database contains one D2D3 sequence attributed to O. rugaoensis (KT884891) that is, however, almost identical to the sequence attributed to O. necromenus (Sudhaus & Schulte, 1989), and in the LSU tree, their position strongly differs from the position of O. rugaoensis in both ITS and SSU trees. This sequence thus probably belongs to O. necromenus. In general, the available molecular data fail to support O. rugaoensis as a separate species from O. chongmingensis. Morphologically, O. rugaoensis shows a resemblance to O. chongmingensis, mainly in the morphology of the stoma, the position of the excretory pore and nerve ring, the morphology of both the female and male tails and spicules, the presence of pseudopeloderan type bursa, and the presence of small bristle-like sensilla posterior to the cloacal opening. Morphometrically, the also show close similitude to each other, such as in the size of the females; similar ratios (a, b, c, and c'); similar anal body width; similar SW%, GS% and V%; and other morphometric measurements (Supplementary Tables S2 and S3). Therefore, O. rugaoensis is proposed as a junior synonym of O. chongmingensis, while the population from Japan [50] attributed to O. rugaoensis should be considered conspecific with the population of O. necromenus that is described from Iran [51].

Conclusions
Potentially entomopathogenic nematodes of the genus Oscheius have been the subject of increased scientific interest in recent years. However, the research is complicated by unclear taxonomy, with some of the species being poorly described and having inadequate or no molecular support. In our study, we provide a morphological and molecular characterization of Oscheius siddiqii and propose O. niazensis as its junior synonym.
Using the available molecular data and newly sequenced COI gene of five Oscheius, we revised other species belonging to the genus Oscheius and discuss species delimitation. Our results show that a majority of Oscheius with the available molecular support are well characterized by the combination of traditional rDNA genes (ITS, LSU and SSU) but that the COI sequence provides further resolution among Oscheius species. Based on morphological and molecular data, we propose O. microvilii and O. rugaoensis as junior synonyms of O. myriophilus and O. chongmingensis, respectively. Furthermore, O. basothovii and O. safricanus should be considered as species inquirendae. Our results highlight the importance of the proper molecular characterisation of newly described Oscheius species. Therefore, any future species description should provide all three standardly used rDNA markers (ITS, SSU, and LSU) in combination with at least one mitochondrial COI gene.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/biology10121239/s1, Table S1: Diagnosis of the genus and list of species, Table S2: Comparative morphometrics of the females of the species of the subgenus Oscheius. All measurements in µm except for the indexes, Table S3: Comparative morphometrics of the males of the species of the subgenus Oscheius. All measurements in µm except for the indexes.