Pristionchus trametes n. sp. (Diplogastridae) isolated from the mushroom Trametes orientalis in Kyoto, Japan

Abstract A new species of Pristionchus was isolated from fruiting bodies of the wood-decaying fungus Trametes orientalis collected from Kyoto, Japan. Attempts to culture it using bacteria, yeast, and freeze-killed wax moth larvae as food or substrate failed. The eurystomatous form of the species was not found in the collected material, and the species is typologically characterized by: its ‘small’ stoma with thin, membrane-like cheilostomatal plates, a small triangular right subventral tooth, thorn-like dorsal tooth, and small left subventral denticles; a short, blunt male tail spike; and a short, conical female tail. Although the posterior probability support was not high (66%), phylogenetic analysis of both small and large ribosomal RNA gene subunits suggests that the species is closely related to P. elegans and P. bucculentus. The new species can be distinguished from those two by its diagnostic characters comprising the stomatal morphology and male and female tail characters.

Most Pristionchus species are phoretic and necromenic associates of insects and other invertebrates, and are culturable using Escherichia coli strain OP50, a common food source of bacteriophagous nematodes (e.g., Ragsdale et al., 2015). However, one subgeneric clade of Pristionchus species associated with figs associates contains several species that are not currently culturable; these species exhibit highly divergent stomatal polymorphism, have specific associations with fig wasps, and inhabit fresh figs (Susoy et al., 2016). The clade may represent the phenotypic and genetic plasticity of the genus.
Despite dense taxon sampling of the genus (e.g., Herrmann et al., 2006;Kanzaki et al., 2021;Mayer et al., 2007), the species diversity of the genus is still far from saturated (Kanzaki et al., 2021;Rödelsperger et al., 2018), and further isolation of various species is necessary to improve the model system.
This study describes a species of Pristionchus recovered from fruiting bodies (mushrooms) of the wood-de caying fungus Trametes orientalis (Yasuda) based on its typological characters and ribosomal RNA sequences, which were used as species-specific molecular barcodes.

Nematode isolation and culturing attempts
No special permit was required for the material collection at the collection site, and no endangered or protected species was involved in the present study.
Several fruiting bodies of T. orientalis on a dead log were collected from Uji, Kyoto, Japan in July 2020. The wood substrate could not be identified because the bark was lost and the wood was partially decomposed. The fruiting bodies were enclosed in a plastic bag, transported to the laboratory, and examined for nematodes and insects.
Nematodes were collected from the inner surface of the plastic bag after removing the fruiting bodies by washing the bag with distilled water. The fruiting bodies were placed on distilled water agar (2.0% agar) to avoid dehydration and kept in the laboratory at room temperature (20-25°C).
The collected nematodes were briefly observed under light microscopy (Eclipse 80i, Nikon) in differential interference contrast mode to identify genera, and several individuals were transferred to E. coli OP50 growing on nematode growth medium (NGM). Ten gravid females were transferred to the medium in triplicate, and culture was attempted. In addition, five individual males and females were briefly observed to confirm their typological conspecificity; they were then picked up using a stainless steel insect pin (Insect pin #00, Shiga Kontyu) and transferred to nematode digestion buffer (NDB; Kikuchi et al., 2009;Tanaka et al., 2012) separately to obtain molecular profiles. The remaining nematodes were killed via heating at 55°C for 1 min and fixed in TAF (triethanolamine: formalin: distilled water = 2:7:91) as morphological material.
Because the first culture attempts were unsuccessful, nematodes were collected from the water agar under the fruiting bodies, and we attempted to culture them again using E. coli OP50 (the above procedure), yeast, freeze-killed Galleria mellonella (L.) larvae, and T. orientalis fruiting bodies.
Entomophilic yeast isolated from a carpenter bee (Xylocopa appendiculata circumvolans Smith) were streaked onto 1.0% malt extract agar (MEA: 1.0% malt extract, 2.0% agar), which was then inoculated with 10 gravid female nematodes. For culture with larvae, freeze-killed G. mellonella larvae were cut in half, placed on 2.0% water agar, and inoculated with nematodes. Next, the T. orientalis fruiting bodies from which the nematodes were originally isolated were cut into ca. 1.5 × 1.5 cm 2 pieces and placed on 2.0% water agar; the presence of nematodes was confirmed under a dissecting microscope (S8 Apo; Leica, Wetzlar, Germany). The culture media were kept in the laboratory and observed irregularly for 2 weeks. These attempts were repeated three to five times for each food source tested.

Morphological observation and micrography
Fixed nematodes were processed with glycerin using Seinhorst's method with some modifications (Minagawa and Mizukubo, 1994) and mounted in glycerin according to the methods of de Maeseneer and d'Herde (as described in Hooper, 1986). Mounted specimens were used for morphological observations and morphometric analyses, and kept as type specimens. All micrographs were obtained using a digital camera system (MC170 HD; Leica), and morphological drawings were made using a drawing tube connected to the microscope. To prepare the figures, the micrographs and drawings were edited using Photoshop 2019 (Adobe).

Molecular profiles and phylogeny
The nematodes transferred to NDB were digested at 55°C for 30 min, and the lysates served as template DNA for PCR. To confirm species identity, ca. 0.7 kb of the D2-D3 expansion segments of the large subunit of ribosomal RNA (D2-D3 LSU) was analyzed for all DNA samples following the method in Ye et al. (2007). We then sequenced a 4.2-kb segment of the ribosomal DNA region, including the near-full length sequence of the small subunit (SSU), internal transcribed spacer (ITS) region (ITS1, 5.8S rRNA, and ITS2), and D1 to D4 expansion segments of the large subunit (D1-D4 LSU), as well as partial (ca. 0.7 kb) segments of mitochondrial cytochrome oxidase subunit I (mtCOI), following the methods of Ekino et al. (2017), Kanzaki and Futai (2002), and Kanzaki et al. (2019Kanzaki et al. ( , 2021. The sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers LC633357 (rDNA) and LC633358 (mtCOI).
To determine the status of this previously unknown nematode within the genus, a Bayesian phylogenetic analysis was conducted based on concatenated SSU and D1-D4 LSU sequences. First, the compared sequences were aligned using the program MAFFT (Katoh et al., 2002;Kuraku et al., 2013; available at https://mafft.cbrc.jp/alignment/server/index.html) with the default settings. Base-substitution models for each gene were determined using the Akaike information criterion in MEGA X (Kumar et al., 2018). Bayesian analysis was performed using MrBayes 3.2 (Huelsenbeck and Ronquist, 2001;Ronquist et al., 2012): four chains were run for 4 × 10 6 generations, and the Markov chains were sampled at 100-generation intervals (Larget and Simon, 1999). After performing two independent runs, confirming convergence of the runs, and discarding the first 2 × 10 6 generations as 'burn in', the remaining topologies were used to generate a 50% majority-rule consensus tree. The sequences compared in the analysis are listed in Table 1.

Measurements
Summarized in Table 2.

Adult
Body cylindrical, stout. Cuticle thick, with fine annulation and clear longitudinal striations. Lateral field consisting of two lines, only weakly distinguishable from body striation with the presence of deirid. Head without apparent lips, and with six short and papilliform labial sensillae; four small, papilliform cephalic papillae present in males, as typical for diplogastrid nematodes. Amphidial apertures located on the lateral sector, slightly dorsally shifted, at level of margin of cheilo-and gymnostom. However, labial and cephalic papillae and amphidial aperture were not observed clearly in the most individuals examined. Stomatal dimorphism often found in the genus not observed, and all individual have 'small' (short and

Male
Ventrally arcuate, strongly ventrally curved at tail region when killed by heat. Testis single, ventrally located, anterior part reflexed to right side; spermatogonia arranged in three to five rows in reflexed part, then well-developed spermatocytes arranged as three to four rows in anterior half of the main branch, then mature ameboid spermatocytes arranged in single to two row(s) in proximal part of gonad. Vas deferens not clearly separated from other parts of gonad, formed by relatively large cells. Three (two subventral and one dorsal) cloacal gland cells observed at distal end of vas deferens and intestine. Spicules paired, separate; spicules smoothly curved in ventral view, adjacent to each other for distal third of their length, each smoothly tapering to pointed distal end. Spicule in lateral view smoothly ventrally arcuate, giving spicule about 100° curvature, oval manubrium present at anterior end, lamina/calomus complex (blade) clearly expanded slightly posterior to manubrium (ca. 1/5 of blade length from anterior), then smoothly tapering to pointed distal end. Gubernaculum conspicuous, about one-third of spicule length, broad anteriorly such that dorsal wall is slightly recurved with dorsal and ventral walls separate at 50 to 60° angle at posterior end; dorsal side of gubernaculum possessing single, membranous, anteriorly directed process and lateral pair of more sclerotized, anteriorly and obliquely ventrally directed processes. In lateral view, anterior half of gubernaculum with two serial curves separated by anteriorly and obliquely ventrally directed process, with anterior terminal curvature highly concave and almost closed, and with deep posterior curvature being one-third of gubernaculum length; posterior half forming tube-like process enveloping spicules. Cloacal opening (CO) forming simple slit. One small, ventral, single genital papilla (vs) on anterior cloacal lip; nine pairs of genital papillae (v1-v7, ad, pd) and a pair of phasmids (ph) present, with an arrangement <v1, (v2d, v3/v4), ad, (ph, V5-7, pd)> in nomenclature of Sudhaus and Fürst von Lieven, 2003), where v1 is located at ca 1.5 cloacal body diam. (CBD) anterior to CO; v2d to v4 are close to each other, gathering within less than half CBD; ad at ca. 1 CBD posterior to CO; and ph, v5-7 and pd close to each other around immediately anterior to the root of tail spike. Anterior five pairs of papillae (v1-4 and ad) almost equal in size, rather large and conspicuous, v7 and pd papillae obviously smaller than anterior five pairs, v5 and v6, sometimes difficult to observe with light microscope. Anterior two pairs of the ventral triplet papillae (v5 and v6) papilliform and borne from socket-like base, v7 simple or typical thorn-like in shape. Tip of v6 papillae split into two small papilla-like projections. Tail conical, with short, ca. 0.6 CBD in length, spike with bluntly pointed tip. Bursa or bursal flap absent.

Female
Relaxed or slightly ventrally arcuate when killed by heat. Gonad didelphic, amphidelphic; each gonadal system arranged from vulva/vagina as uterus, oviduct, and ovary. Anterior and posterior gonads are basically same in their structure, and only anterior gonad is described in detail here. Anterior gonad right of intestine, with uterus and oviduct extending ventrally and anteriorly on right of intestine and with a totally reflexed (= antidromous reflexion) ovary extending dorsally. Oocytes in ovary mostly arranged in three to four rows in distal two-thirds to three-fourth of ovary and in double or single row in rest of ovary, distal tip ovary reaching vulva to oviduct of opposite gonad branch depending on the developmental condition. Anterior end of oviduct (= junction tissue between ovary and oviduct) consists of rounded cells. Spermatheca not clearly distinct, but anterior part of oviduct immediately posterior to the junction consists of rounded cells works as spermatheca. Eggs in single to multiple-cell stage or even further  Relative position of phasmid to total tail (%) --57 ± 5.6 (48-66)

Molecular profile and phylogeny
Although the posterior probability support was low (66%), P. trametes n. sp. formed a clade with the two species of the elegans group: P. elegans P. buc culentus Kanzaki, Ragsdale, Herrmann, Röseler & (Fig. 5).

Differential diagnosis
Pristionchus trametes n. sp. is characterized by its short, narrow stoma with thin membrane-like cheilostomatal plates, small inverted 'V'-shaped dorsal tooth, triangular right subventral tooth with a blunt tip, and left subventral plate with three blunt denticules; male genital papillae with a v1, (v2d, v3/v4), ad, (ph, V5-7, pd) arrangement and short blunt male tail spike; and short conical female tail with a bluntly pointed or narrowly rounded terminus. Based on its membrane-like cheilostomatal plates, the new species is close in form to two elegans group species, P. elegans and P. bucculentus; these three species share this unique morphological character as well as a similar arrangement of genital papillae, i.e., the second pair of genital papillae is oriented laterally (v2d), and the second to fourth pairs (v2d, v3, and v4) are close to each other (Kanzaki et al., , 2013. Although phylogenetic support for a grouping of all three was low, these three species formed a sub clade within the genus in our phylogenetic analysis (Fig. 5).
Distinguishing P. trametes n. sp. from P. elegans are stomatal characters, although only a single (stenostomatous) form is known for the two species. Diagnostic characters of P. trametes n. sp., with respect to P. elegans, are the absence vs. presence of anterior serratae of the gymnostomatal tube and a triangular vs. flattened right subventral tooth. In terms of the relative length of anterior and posterior pharynx, the anterior pharynx of P. trametes n. sp. is slightly (1.1 times) vs. obviously (1.5 times) longer than the posterior pharynx. The position of male v1 paired papillae in P. trametes n. sp. is 1.5 vs. less than 1 CBD anterior to CO, the male tail tip has a short spike vs. a long and filiform appendage, and the female tail is short and conical vs. long and filiform . The new species is also distinguished from P. bucculentus by its stomatal shape, which shows only the stenostomatous vs. only the eurystomatous form, relative length of the anterior and posterior pharynx, with its anterior pharynx slightly (1.1 times) vs. obviously (1.5 times) longer than the posterior pharynx, the position of male v1 paired papillae (1.5 vs. ca. 1 CBD anterior to CO), a male tail tip with a short spike vs. a long and filiform appendage, and a short and conical vs. long and filiform female tail (Kanzaki et al., 2013).

Additional remarks on nematode isolation and culture attempts
In addition to the specimens initially collected from the plastic bag including the T. orientalis specimens, several aphelenochoidids, rhabditids, and plectids were recognized in the 2.0% water agar where the small pieces of T. orientalis were placed. No predation behavior of P. trametes n. sp. was seen during occasional observations.
Regardless of the methodology, none of the culture attempts was successful, i.e., no oviposition or propagation of P. trametes n. sp. was observed in the cultures. Further, several unidentified dipteran larvae and adult mushroom beetles (Coleoptera: Ciidae) infected the fruiting bodies placed on the 2.0% water agar, and some died on the medium. Although several mushroom beetles harbored other entomophilic rhabditids, P. trametes n. sp. was not observed on these dead bodies; therefore, an insect association of P. trametes n. sp. was not confirmed.

Discussion
This study describes a new species of Pristionchus. Because the isolates were not cultured successfully, several morphological characters were not observed sufficiently. For example, the labial and cephalic sensilla were not observed in many individuals. This could be because of the material condition, although males in a recently described species P. nudus Kanzaki, Herrmann, Weiler, Röseler, Theska, Berger, Rödelsperger & Sommer, 2021 had lost cephalic sensilla (Kanzaki et al., 2021). Similar degeneration might have occurred in P. trametes n. sp. Further detailed morphological observations of live specimens might elucidate these fine characters.
Pristionchus trametes n. sp. has several characteristic morphological traits that are uncommon in the genus. For example, the stomatal structure of the new species is unique. Stomatal dimorphism was not observed in this study; because of its "small" size, the species is tentatively considered monomorphic with a stenostomatous form. However, considering its teeth shape, the form seems intermediate between two forms, i.e., the anterior tip of the dorsal tooth curved slightly anteriorly, and the right subventral tooth is relatively large compared with the stoma size. In addition, the tail morphology (short, spiked in males and short, conical in females) is not found in Pristionchus spp. except for P. chinensis Kanzaki, Herrmann, Weiler, Röseler, Theska, Berger, Rödelsperger & Sommer, 2021, which belongs to the group that is sister to thre rest of the genus, excluding the clade of fig associates (Kanzaki et al., 2021).
In some respects, tail and stomatal morphology is similar to that of other diplogastrid genera. For example, the stomatal form, comprising a buccal cavity with two teeth, is similar to that of several Para sitodiplogaster Poinar, 1979 species in which the relatively narrow stoma has two teeth (e.g., Giblin-Davis et al., 2006;Wöhr et al., 2014), and several species of Allodiplogaster Paramonov and Sobolev in Skrjabin et al. (1954) have a short, spiked male tail and conical female tail (e.g., . Because Allodiplogaster is clearly distant from Pristionchus, representing a basal split within the family with respect to the latter genus (Susoy et al., 2015), these traits are likely to be independent conversions. The characteristic stomatal structure of the new species is likely related to the difficulty in culturing it. Comparative studies of their biological characters may give new insight into their functional morphology and feeding (nutritional) preferences.
Biologically, the new species was found in the frui ting bodies of a wood-decaying fungus, and its insect associations are unclear. Pristionchus bucculentus, a close relative of the new species, has been isolated from mushroom beetles in Japan, i.e., Episcapha gorhami Lewis from Hokkaido (Kanzaki et al., 2013) and Encaustes praenobilis Lewis from Aomori . These two species may share similar habitats, and an examination of mushroom substrates and related insects may reveal further diversity of mushroom-associated Pristionchus species.