Three new species, one new genus and subfamily of Dorylaimida (de Man, 1876) Pearse, 1942, and revisions on the families Tylencholaimellidae Jairajpuri, 1964 and Mydonomidae Thorne, 1964 (Nematoda: Dorylaimida)

Three new species of the order Dorylaimida (de Man, 1876) Pearse, 1942 were identified and described. Paratylencholaimus sanshaensis gen. nov. sp. nov. from Hainan is proposed as a new member of the family Tylencholaimellidae Jairajpuri, 1964. Paratylencholaimus gen. nov. is close to Phellonema Thorne, 1964 and Goferus Jairajpuri & Ahmad, 1992 but can be differentiated mainly by having basal part of odontophore rod-like and without knobs, and basal part of pharynx expanded gradually. Tylencholaimus zhongshanensis sp. nov. from Guangdong and Dorylaimoides shapotouensis sp. nov. from the Ningxia Hui Autonomous Region are also described herein. Phylogenetic analyses based on the 18S rDNA and the D2–D3 region of the 28S rDNA support that the three new species are valid. The classifications of the families Tylencholaimellidae and Mydonomidae Thorne, 1964 are revised mainly based on the analysis of the morphology of odontostyle and odontophore. After these revisions, Paratylencholaiminae subfam. nov. including Paratylencholaimus gen. nov. and Goferus is proposed. Athernema and Agmodorus of Tylencholaimellidae are transferred into Mydonomidae, and the subfamily Athernematinae of Tylencholaimellidae is dismissed. The main characteristics of the family Mydonomidae and Tylencholaimellidae are revised. Keys to the genera of Mydomonidae and Tylencholaimellidae are included.


INTRODUCTION
In the classification proposed by Andrássy (2009), which is based on the classification created by Peña Santiago (2006), the superfamily Tylencholaimoidea contains a wide range of genera and species and includes six families: Leptonchidae Thorne, 1935, Tylencholaimidae Filipjev, 1934, Mydonomidae Thorne, 1964, Tylencholaimellidae Jairajpuri, 1964, Aulolaimoididae Jairajpuri, 1964and Encholaimidae Golden & Murphy, 1967. Tylencholaimoidea can be differentiated from other superfamilies of Dorylaimida (de Man, 1876) Pearse, 1942 mainly by having tylencholaimoid or dorylaimoid cuticle, cap-like lip region, markedly short basal expansion of the pharynx, common occurrence of both pro-and opisthodelphy, and few male supplements. However, Andrássy (2009) stated that this classification of Tylencholaimoidea should be artificial as the species of this superfamily can 'hardly represent a homogeneous trend in the evolution of this group' . Peña Santiago (2014) again stressed that the superfamilies classification of the suborder Dorylaimina Pearse, 1936 is not supported by morphology or molecular analyses (Holterman et al., 2008) . Peña Santiago (2014) canceled the superfamilies of Dorylaimina and kept the families, and moved Encholaimidae under Nordiidae.
During nematode investigations in China, three new species of Dorylaimina were identified. One from Guangdong belongs to the genus Tylencholaimus de Man, 1876 (Tylencholaimidae), and the second one from the Ningxia Hui Autonomous Region is a new member of the genus Dorylaimoides Thorne & Swanger, 1936 (Mydonomidae). These species are herein described as Tylencholaimus zhongshanensis sp. nov. and Dorylaimoides shapotouensis sp. nov. The third one from Hainan is interesting. It equips with dorylaimoid cuticle that is different from Tylencholaimidae (equips with tylencholaimoid cuticle), but other characteristics are highly similar to those of Tylencholaimidae. And later, three more populations of this species from Guangdong were collected. With further examinations, this species was suggested to be a member of a new genus of the family Tylencholaimellidae, herein described as Paratylencholaimus sanshaensis gen. nov. sp. nov. Detailed descriptions based on microscopy and phylogenetic analysis based on the 18S rDNA and the D2-D3 region of the 28S rDNA of the three new species were presented. In addition, the classification of Tylencholaimellidae and Mydonomidae was discussed, one new subfamily of Tylencholaimellidae was proposed, and keys to the genera of the revised Tylencholaimellidae and Mydonomidae were provided.

Morphology and morphometrics
Soil samples were collected from the rhizosphere soil of some plants from Hainan, Guangdong and Ningxia, respectively. Nematode populations were extracted from samples using the modified Baermann funnel method (Whitehead & Hemming, 1965). Then, specimens were gently killed at 62 • C for 3 min, fixed in 4% FG fixative, dehydrated using the glycerol-ethanol method and then mounted on permanent slides for further examination (Xie, 2005). The specimens were observed, measured and photographed as described by Wu et al. (2017). Locations of the pharyngeal gland nuclei were measured as described previously (Andrássy, 1998). Measurements are given as mean (minimum-maximum) with SD indicated when n > 30. Nematodes were prepared for SEM observations as described by Abolafia & Peña Santiago (2005) and observed with a FEI XL-30-ESEM electron microscope at 10 KV.

Phylogenetic analysis
The sequences of the three new species were respectively compared with sequences in GenBank using BLAST. Sequences of species of Tylencholaimidae, Leptonchidae, Mydonomidae, Tylencholaimellidae, Mononchida Jairajpuri, 1969 andNygolaimina Ahmad &Jairajpuri, 1979 were aligned along with the sequences of the three new species. The sequence alignments were performed, and conservative regions were selected using MEGA v6. For the Bayesian inference (BI) analysis, the substitution saturation was tested by DAMBE. The best-fit models were selected by AIC (Akaike Information Criterion) in MrModeltest v2.3. Bayesian trees were constructed by using MrBayes v3.1.2 running the chain for 1,000,000 generations with a sample frequency of 1,000 generations, and setting the 'burnin' at 2500. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) were given for appropriate clades.

Nomenclatural acts
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:427B5E52-23B0-4474-B179-4FA9FC5E7C9C. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.   the body diameter at mid-body, lateral pores indistinct. Lip region cap shaped, offset from the body by a constriction, 2.3-2.8 times as wide as high or 30-38% of the body diameter at posterior end of the neck region. Lips amalgamated, labial and cephalic papillae distinct. Amphidial foveae cup shaped, opening at the level of the constriction, apertures narrow, 30% in average of the lip region width. Odontostyle slender, 0.7-0.9 times the lip region width long, its aperture one-fourth to one-third of its length. Odontophore rod-like with small basal knobs, 0.8-1.3 times as long as the odontostyle. Guiding ring single, indistinct. Nerve ring situated at 41-45% of the pharyngeal length. Anterior part of pharynx slender, basal expansion occupying 33-38% of the total pharyngeal length. Pharyngeal gland nuclei located as follows: D = 68-74%, AS1 = 41-45%, AS2 = 40-50%, PS1 = 66-80%, PS2 = 72-83%. Cardia conoid to rounded. Genital system prodelphic, postvulval sac completely absent. Ovary 30-124.5 µm long. Oviduct slender, 58-95 µm long. Junction of oviduct and uterus indistinct. Uterus simple and slender, 24-44 µm long. Sperm not observed in the genital system. Vulva transverse in ventral view. Vagina approximately 46.5-57% of the corresponding body width long, anteriorly directed. Pars proximalis vaginae with conoid walls, 5-8 µm long and 6-7.5 µm wide, pars refringens lacking, pars distalis vaginae 3-4 µm long. Prerectum 3.2-4.8 times and rectum 0.6-0.9 times anal body diameter long. Tail hemispheroid to elongate-hemispheroid, 1.1-1.4 times the anal body diameter long. Male. Unknown. All soil samples were processed, but no males were found.

Habitat and locality
Rhizosphere soil of Phalaenopsis sp. from Zhongshan, Guangdong, China.

Etymology
The new species is named after its type locality, Zhongshan City.

Diagnosis and relationships
Tylencholaimus zhongshanensis sp. nov. is characterized by having a body length of 473-605 µm; lip region offset and approximately one-third of the body diameter at posterior end of the neck region; amphid aperture 30% as wide as the lip region; odontostyle 5-6 µm, 0.7-0.9 times the lip region width long; odontophore rod-like with small basal knobs, 5-7 µm long, 0.8-1.3 times as long as the odontostyle; basal expansion of pharynx occupying 33-38% of the total pharyngeal length; female genital system prodelphic; postvulval sac completely absent; vulva transverse; prerectum 3.2-4.8 times anal body diameter long; tail

Notes.
All measurements are in m (except for 'L in mm) and shown in the form: mean (minimum-maximum). n, number of specimens observed; L, body length; a, L/ maximum width; b, L/ pharyngeal L; c, L/ tail length; c , tail length/ body diameter at anus; V, distance of vulva from anterior end × 100/L; G1, anterior uterine sac × 100/L; G2, posterior genital branch × 100/L. 16-21 µm, hemispheroid to elongate-hemispheroid, 1.1-1.4 times the anal body diameter long.

Molecular characterization and phylogenetic analysis
The sequences of 18S rDNA and D2-D3 region of 28S rDNA of Tylencholaimus zhongshanensis sp. nov. were obtained, and interindividual variabilities were both observed. Four sequences for 18S rDNA (1,747 bp; accession numbers: MG921272 to MG921275) and three sequences for the D2-D3 region of 28S rDNA (829 bp; accession numbers: MG921305 to MG921207) were deposited in GenBank. The BLAST search for the 18S rDNA showed the highest similarity (96%) to the sequences of T. helanensis (KU992903 and KU992904). For the D2-D3 region of 28S rDNA, both sequences showed the highest similarity (86%) to the sequences of T. helanensis (KU992905 and KU992906). In Bayesian trees for both the 18S rDNA and D2-D3 region of 28S rDNA (Figs. 4 and 5), the sequences of Tylencholaimus zhongshanensis sp. nov. formed a clade with 88% and 100% supports, respectively, and clustered together with other Tylencholaimus species.

Etymology
The new species is named after Shapotou Region, a successful soil restoration area in China.

Molecular characterization and phylogenetic analysis
Each sequence of 18S rDNA and D2-D3 region of 28S rDNA of Dorylaimoides shapotouensis sp. nov. (1,743 bp and 825 bp, respectively) was obtained and deposited in GenBank (accession numbers: KU662325 for the 18S rDNA and KU662324 for the D2-D3 region of 28S rDNA). The BLAST search for the 18S rDNA showed the highest similarity (99%) to the sequence of D. micoletzkyi (AY284830) and showed 8 nucleotide differences. The D2-D3 region of 28S rDNA showed the highest similarity (95%) to the sequences of D. micoletzkyi (AY593004) with 40 nucleotide and 4 gaps differences. In the 18S rDNA phylogenetic reconstructions (Fig. 4), the new species clustered together with other species of Dorylaimoides with 84% support. In the D2-D3 region of 28S rDNA phylogenetic reconstructions (Fig. 5), the new species is located in a 100% supported clade with D. micoletzkyi and D. limnophilus (an opisthodelphic species). Most measurements of Dorylaimoides shapotouensis sp. nov. overlap those of eight documentary populations of D. micoletzkyi (Table 2), but Dorylaimoides shapotouensis sp. nov. can be easily differentiated from D. micoletzkyi mainly by the pharynx morphology and the odontophore length. Peña Santiago & Peralta (1997a) and Peña Santiago & Peralta (1997b) published a series of papers on the genus Dorylaimoides, and comprehensively discussed the suitability of using the female genital system types and the tail to identify the species of Dorylaimoides and made a key to the species and groups based on the female genital system types (didelphic, opisthodelphic and pseudodidelphic). In the 28S rDNA Bayesian tree, Dorylaimoides shapotouensis sp. nov. showed a closer relationship with another didelphic species, D. micoletzkyi rather than with the opisthodelphic species D. limnophilus. However, the inner relationships of Dorylaimoides remain unclear in the 18S rDNA Bayesian tree. Thus, to clarify the evolutionary relationships among the three groups with different genital system types, more molecular data of Dorylaimoides are needed.

Diagnosis
Tylencholaimellidae. Paratylencholaiminae subfam. nov. Cuticle dorylaimoid with fine transverse striations. Lip region cap-shape, offset from the body. Amphidial fovea not sclerotized. Odontostyle straight, tubular with small aperture, without dorsal accessory pieces. Odontophore rod-like and basal part slightly expanded. Guiding ring simple. Pharynx slender in anterior part, the basal part expanded occupying one-third of the total pharyngeal length. Female genital system didelphic. Vulval lips not sclerotized. Tail short, rounded to conoid-round. Males unknown.

Etymology
The new genus is named as Paratylencholaimus (latin para-= similar), as its inner characteristics are similar to those of the genus Tylencholaimus, but it has a different type of cuticle which is dorylaimoid.

Etymology
The new species is named after the Sansha City, which is its type locality.

Type habitat and locality
Rhizosphere soil of Euphorbia sp. from Yongxing Island, Sansha City, Hainan, China.

Feeding type
One noticed phenomenon was observed in the Zhongshan population of Paratylencholaimus sanshaensis gen. nov. sp. nov. The anus of this population showed morphological diversity: nematodes with normal color intestines have a flat anal region, while the nematodes with a black mass in intestines have bulge anus. The culture medium of Phalaenopsis sp., which this population is collected from, consists of three levels: sphagnum on the top, coco coir in the middle and barks at the bottom. It can be suggested that the nematodes with a black mass in intestines may feed on the barks. This finding indicates that the food source can influence the morphology of nematodes, and the feeding type of Paratylencholaimus sanshaensis gen. nov. sp. nov. is omnivorous.

Discussion on new genus classification
In Dorylaimida, there are two cuticle types: (a) dorylaimoid: inner layer not loose and without radial elements; (b) tylencholaimoid: inner layer loose with irregular  (Peña Santiago, 2006;Peña Santiago, 2014), Paratylencholaimus gen. nov. should be placed under the subfamily Tylencholaimellinae due to its amphideal fovea that is not sclerotized. However, Paratylencolaimus gen. nov. and its close relative genus Goferus has odontophore without distinct basal knobs and basal expansion occupying greater than one-fifth of the total pharyngeal length. In contrast, the remainder genera of Tylencholaimellinae (Dorella Jairajpuri, 1964, Margollus Peña-Santiago, Peralta & Siddiqi, 1993, Tylencholaimellus Cobb in Cobb, 1915, Doryllium Cobb, 1920, Oostenbrinkella Jairajpuri, 1965 and Phellonema Thorne, 1964) except Agmodorus Thorne, 1964 (see further) have distinct basal knobs and basal expansion about or less than one-fifth of the total pharyngeal length. To adjust this, we propose to place the new genus and Goferus under a new subfamily, namely, Paratylencholaiminae subfam. nov.

Tylencholaimellidae
Athernematinae Ahmad & Jairajpuri, 1978Athernema Ahmad & Jairajpuri, 1978Tylencholaimellinae Jairajpuri, 1964 Agmodorus Thorne, 1964Dorella Jairajpuri, 1964Doryllium Cobb, 1920Goferus Jairajpuri & Ahmad, 1992Margollus Peña-Santiago, Peralta & Siddiqi, 1993Oostenbrinkella Jairajpuri, 1965 Phellonema Thorne, 1964Tylencholaimellus Cobb in Cobb, 1915 The odontostyle of Athernema and Agmodorus has not the typical tube shaped. We also found that these two genera with the arched odontophore, conoid to filiform tail and the opisthodelphic female genital system are more closely related to the family Mydonomidae Thorne, 1964 which is mainly characterized by having odontostyle asymmetry, odontophore straight or arched, basal expansion cylindrical and no longer than one-third of the pharynx length, female didelphic or opisthodelphic. Thus, we propose to transfer Athernema and Agmodorus into the family Mydonomidae Thorne, 1964 according to the morphology mentioned above, and under the subfamily Mydonominae Thorne, 1964 according to the body length less than three mm, and cancel the subfamily Athernematinae.
The main characteristics of the families Mydonomidae, Tylencholaimellidae and the subfamily Tylencholaimellinae should be revised as follow: Mydonomidae: cuticle dorylaimoid; lip region continuous or slightly offset, occasionally cap like; lips rounded, usually amalgamated; odontostyle short, asymmetry or not typical tubular, with distinct lumen; odontophore straight or arched; basal expansion cylindrical and no longer than one-third of the pharynx length, occasionally offset; female genital system didelphic-amphidelphic or mono-opisthodelphic; spicula dorylaimoid; ventromedial supplements spaced, 1-20; tail variable, short and rounded to elongate or filiform, similar or dissimilar in sexes.
Tylencholaimellidae: cuticle dorylaimoid; lip region cap-like, more or less offset, lips amalgamated; odontostyle short, tubular, occasionally with accessory stiffening piece; odontophore with or without basal knobs or flanges; basal expansion short pyriform, usually offset, occupying one-fifth to one-third of the pharynx length; female amphidelphic or opisthodelphic, exceptionally prodelphic; vulva transverse; spicula dorylaimoid; ventromedial supplements none to two, spaced; tail short and rounded to filiform, similar in sexes.