Two new Species of Pristionchus (Nematoda: Diplogastridae) include the Gonochoristic Sister Species of P. fissidentatus

One strain of P. paulseni sp. n. was isolated from an adult of Lucanus dybowski taiwanus (Miwa, 1937) (Coleoptera: Lucanidae) collected at Pilu sacred tree, Taroko National Park, Taiwan. The strain of P. yamagatae sp. n. was isolated from an adult of Holotrichia sp. (Hope, 1837) (Coleoptera: Scarabaeidae, Melolonthinae) collected at Yuza, Yamagata, Japan. Both strains have been kept in laboratory culture on NGM agar plates seeded with Escherichia coli strain OP50 under the culture code and freezing voucher numbers RS5918 (P. paulseni sp. n.) and RS5964 (P. yamagatae sp. n.).

Light microscopic observations for drawings and morphometrics were conducted using live nematode material, which was handpicked from culture plates (Kanzaki et al., 2013).

A species phylogeny of the complete Pristionchus genus was reconstructed as described in Rödelsperger et al. (2018). In the following, we give a brief summary about the generation of the Pristionchus phylogeny (please see Rödelsperger et al., (2018) for full details). For all cultivable Pristionchus species, worms were grown on NGM plates seeded with E. coli OP50 at 20°C and total RNA was isolated from 2–3 mixed-stage plates per species using standard Trizol extraction following the manufacturers’ instructions (Zymo Research, CA, USA). RNA-seq libraries were prepared using TruSeq RNA library preparation kit v2 (Illumina, Inc., CA, USA), according to the manufacturer’s instructions, from 1 µg of total RNA in each sample and sequenced on the Illumina HiSeq3000 platform, yielding a median of 14m paired reads (2 × 150 bp) per species. Raw reads were submitted to the European nucleotide archive under the study accession PRJEB20959. RNA-seq reads were assembled into transcriptomes using Trinity (version v2.2.0) (Grabherr et al., 2011). For further analysis, only the first reported isoform per gene was selected and the longest complete or partial ORF (⩾60 amino acids) was called. Orthologous clusters were generated by orthAgogue (Ekseth et al., 2014) and protein sequences were aligned using the MUSCLE software (version 3.8.31) (Edgar, 2004). 2,092 high quality alignments containing at least 14 species (without any duplication), with at least 50 amino acid positions with coverage in all represented species, were concatenated into a supermatrix spanning 350,000 amino acids. On the basis of the previous analysis of dozens of gene families (Baskaran et al., 2015), we chose the LG substitution model to reconstruct a maximum-likelihood tree using RA × ML (version 8.2.9, options –m PROTGAMMAILG –f a –N 100) (Stamatakis, 2014).

The majority of Pristionchus species are cryptic and typologically similar to each other, including the two novel species described here. In general, Pristionchus species are distinguished from each other by stomatal and male tail characters only (for review see Ragsdale et al., 2015). Therefore, the general morphology, e.g., body shape and gonadal structures, is described first as common characters, and then the distinctive characters are described for each species.

Their characteristics include a cylindrical and stout body, a thick cuticle, with fine annulation and clear longitudinal striations. Lateral field consists of two lines, weakly distinguishable from body striation with the presence of deirid. Head exists without apparent lips and with six short and papilliform labial sensillae. Four small, papilliform cephalic papillae are present in males, as typical for diplogastrid nematodes. Amphidial apertures are located on the lateral sector, slightly dorsally shifted, at level of margin of cheilo- and gymnostom. Stomatal dimorphism is present, with stenostomatous (narrow mouthed) and eurystomatous (wide mouthed) forms occurring in both males and females, but male eurystomatous form is not as common as females. Detailed stomatal morphology is described below. Dorsal pharyngeal gland is clearly observed, with a penetrating dorsal tooth to gland opening. The anterior part of pharynx (=pro and metacorpus) is 1.5 times as long as the posterior part (=isthmus and basal bulb). Procorpus is very muscular, stout, occupying half to two-thirds of the corresponding body diameter. Metacorpus is very muscular, forming a well-developed median bulb. Isthmus is narrow, not muscular. Basal bulb is glandular. Pharyngo-intestinal junction is clearly observed; it is well-developed. Nerve ring is usually surrounding the middle region of isthmus. Excretory pore is not conspicuous, ventrally located at the level of isthmus to pharyngo-intestinal junction; excretory duct extends anteriad and is reflexed back to the position of pore. Deirid is observed laterally, located from the level of pharyngo-intestinal junction to one body diameter and posterior to the junction. Hemizonid is not clearly observed. Lateral glands, small pores connected to secretory cell, are present and observed on the lateral body surface, with positions inconsistent among individuals, numbering 5 to 8 for males and 9 to 13 for females. Because the structure is very small and indistinctive, the consistency of their position and number was not confirmed by light microscopy.

Cheilostom consists of six per- and interradial plates. Incision between plates is not easily distinguished by light microscopy. The anterior end of each plate is usually rounded and elongated to project from stomatal opening and forms a small flap. Some more details are described for each species below. The anterior end of each plate sometimes splits into two, forming two narrow flaps, i.e., number of cheilostomatal flaps varies from 6 to 12, regardless of the number of plates (=6). Gymnostom is short, with a cuticular ring-like anterior end overlapping cheilostom internally. Dorsal gymnostomatal wall is slightly thickened compared to the ventral side. Stegostom is divided into three subsections: pro-meso, meta and telostegostom. Pro-meso stegostom forms a weak cuticularized ring surrounding the anterior edge of pharynx. Metastegostom bears a conspicuous and movable triangular or flint-shaped dorsal tooth with strongly sclerotized surface, giving an appearance of an inverted V-shape in light microscopy in lateral view; a pointed left subventral ridge with three minute adventitious denticles on a plate is present; a pointed right subventral ridge exists, often with distinct distal adventitious denticle(s). Telostegostom forms a weakly sclerotized cup-like cavity connecting stoma and pharynx.

Cheilostom is divided into six distinctive per- and interradial plates. The anterior end of each plate is usually rounded and elongated to project from stomatal opening, forming a small flap. The anterior part of each plate is sometimes split into two, forming two narrow flaps, i.e., number of cheilostomatal flaps varies from 6 to 12, regardless of the number of plates (=6). Gymnostom is present with a thick cuticle, forming a short, ring-like tube being thicker posteriorly. The anterior end of gymnostom is internally overlapping the posterior end of cheilostomatal plates. Stegostom is divided into three subsections: pro-meso, meta and telostegostom. Pro-mesostegostom forms a weak cuticularized ring surrounding the anterior edge of pharynx. Metastegostom bears large claw-like dorsal tooth, large claw-like right subventral tooth, ridge of left subventral denticles or cusps, varying in number and size. Dorsal and right subventral teeth are movable. No movement is observed for left subventral denticles. Telostegostom forms a weak sclerotized cup-like cavity connecting stoma and pharynx.

They are ventrally arcuate and become strongly ventrally curved at tail region when killed by heat. Testis is single and ventrally located; the anterior part is reflexed to the right side. Spermatogonia is arranged in three to five rows in the reflexed part; then well-developed spermatocytes are arranged in three to four rows in anterior two-thirds of the main branch; then mature amoeboid spermatids are arranged in multiple rows in remaining, proximal part of gonad. Vas deferens is not clearly separated from other parts of gonad. Three (two subventral and one dorsal) cloacal gland cells are observed at distal ends of testis and intestine. Spicules are paired and separate. Spicules are smoothly curved in ventral view, adjacent to each other for distal third of their length, smoothly tapering to pointed distal end. Spicule in lateral view is smoothly ventrally arcuate, giving spicule about 100° curvature; oval manubrium is present at the anterior end; lamina/calomus complex (blade) is clearly expanded slightly posterior to manubrium (ca 1/4 of blade length from anterior), smoothly tapering to pointed distal end. Gubernaculum conspicuous, about one-third of spicule length, is broad anteriorly such that dorsal wall is slightly recurved and that dorsal and ventral walls separate at 50 to 60° angle at the posterior end. The dorsal side of gubernaculum possesses a single, membranous, anteriorly directed process and a lateral pair of more sclerotized, anteriorly and obliquely ventrally directed processes. In lateral view, anterior half of gubernaculum is present with two serial curves separated by anteriorly and obliquely ventrally directed process, with anterior terminal curvature being highly concave and almost closed, and with deep posterior curvature being one-third of the gubernaculum length; posterior half forms a tube-like process enveloping spicules. A thick cuticle exists around tail region, falsely appearing as a narrow leptoderan bursa in ventral view. Cloacal opening (co) is slit-like in ventral view. One small, ventral, single genital papilla (vs) exists on anterior cloacal lip. Nine pairs of genital papillae (v1-v7, ad, pd) and a pair of phasmids (ph) are present, with three precloacal and six postcloacal pairs. Within those pairs, three distal ventral pairs (v5, v6, and v7) and a dorsal pair (pd) are close to each other around the posterior end of tail (just anterior to the root of spike). Anterior five pairs of papillae (v1-4 and ad) are present, almost equal in size, rather large and conspicuous; v7 and pd papillae are obviously smaller than anterior five pairs; v5 and v6 are very small, sometimes difficult to observe with light microscope. Anterior two pairs of the ventral triplet papillae (v5 and v6) papilliform are present and born from a socket-like base, v7 is simple or typical thorn-like in shape. The tip of v6 papillae is split into two small papilla-like projections. A detailed arrangement of papillae and phasmid will be described for each species below. The tail is conical, with a long spike about two to three cloacal body diameter. Bursa or bursal flap is absent.

They are relaxed or slightly ventrally arcuate when killed by heat. Gonad is didelphic, amphidelphic. Each gonadal system is arranged from vulva/vagina as uterus, oviduct, and ovary. The anterior gonad is in 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 on left of intestine. Oocytes are mostly arranged in three to four rows in distal two-thirds of ovary and in double or single row in rest of the ovary, with distal tips of each ovary reaching oviduct of the opposite gonad branch. The anterior end of oviduct (= junction tissue between ovary and oviduct) consists of rounded cells. The anterior part of oviduct consists of rounded cells forming simple tube. The middle part of oviduct serves as spermatheca and consists of roundish and relatively large cells. Eggs in single to multiple-cell stage or even further stages are developed at the posterior part of oviduct (= uterus), which in young female is composed of squared or angular cells, long enough to contain one well-developed oocyte. Receptaculum seminis is not observed, i.e., the organ is not independent and is a part of oviduct/uterus. Vaginal glands are present but obscure. Vagina is perpendicular to the body surface, surrounded by sclerotized tissue. Vulva is slightly protuberant in lateral view and pore-like in ventral view. Rectum is about one anal body diameter long, intestine/rectum junction is surrounded by a well-developed sphincter muscle. Three anal glands (two subventral and one dorsal) are present but not obvious. Anus is in the form of dome-shaped slit, posterior anal lip slightly protuberant. Phasmid is about one to two anal body diameter long, posterior to anus. Tail is long, smoothly tapered to distal end variable from filiform to long and conical.

Pristionchus paulseni* sp. n. (Figs 1, 2; Table 1)

Figure 1

Figure 2

*MH dedicates the species name to Dr. MJ Paulsen of Lincoln, Nebraska, entomologist expert, friend, long-term expedition fellow and secret twin brother.

Summarized in Table 1.

The anterior end of cheilostomatal plates is slightly elongated to form a small rounded flap in most cases. However, in a few cases, the anterior end splits into two tips having two small flaps, i.e., regardless of the number of plates (=6) resulting 6 to 12 flaps. Metastegostom bears a conspicuous and movable triangular or flint-shaped dorsal tooth with strongly sclerotized surface, giving an appearance of an inverted V-shape in light microscopy in lateral view; a pointed distal end, often curved to direct anteriorly; a pointed left subventral ridge with three minute adventitious denticles on a plate; and a pointed right subventral ridge, often with a distinct distal adventitious denticle.

Cheilostomatal plates are very thick. Anterior 1/3 to 1/2 sometimes split into two small tips. The anterior end of each plate is rounded and elongated to project from stomatal opening, forming a small flap; thus, the stoma has 6 to 12 flaps (=tips), regardless of the number of plates (=6). Each cheilostomatal plate is slightly inclined inwards, giving an appearance that the whole stoma is narrowing anteriorly. Gymnostom with very thick cuticle is present, forming a short, ring-like tube being thicker posteriorly and whole gymnostomatal ring widening anteriorly. The anterior end of gymnostom is internally overlapping the posterior end of cheilostomatal plates. Thus, the cheilo- and gymnostomatal regions form a barrel shape in lateral view. Metastegostom bears large claw-like dorsal tooth; large claw-like right subventral tooth; ridge of left subventral denticles or cusps, varying in number and size, i.e., three large cusps, the tip of these cusps is usually split into three or more small tips, sometimes forming serrated plate. The dorsal and right subventral teeth are movable. No movement is observed for left subventral denticles.

The paired papillae and the phasmid are arranged as <v1, v2, v3d, co, v4, ad, ph, (v5, v6, v7), pd>, where v1 is located at ca 1 CBD anterior to co; v2 and v3d are close to each other, but not overlapping, and v2 is located at less than 1/2 CBD anterior to co; v3d is at the midway between v2 and co; v4 ca 1/2 CBD is posterior to co; laterally located ad is at the midway between CO and the root of tail spike; ph is subventrally located in between ad and v5, but closer to v5; ventral v5 to v7 is forming triplet just anterior to the root of tail spike; and subdorsal pd is overlapping or slightly posterior to v7.

The species-specific (diagnostic) characters are described above. The new species is molecularly characterized by the barcoding sequence (NCBI accession number MH114982). In addition, the new species is typologically distinctive from many other Pristionchus spp with its barrel-shaped stoma in the eurystomatous form, i.e., the eurystomatous stoma of the P. pacificus, P. maupasi and P. lhertieri-groups has wide tube shapes, and the elegans-group has a tube-shaped stoma with membranous cheilostomatal plates (summarized in Ragsdale et al., 2015). A similar stomatal shape has been reported in the eury- and megastomatous forms of the Pristionchus triformis group (P. triformis, P. hoplostomus and P. fukushimae), and in the eurystomatous form of P. fissidentatus (Kanzaki et al., 2012a, b; Ragsdale et al., 2013). Although the new species share the partially split cheilostomatous plates with the megastomatous form of P. fukushimae and P. hoplostomus (and P. yamagatae n. sp., see below), P. paulseni sp. n. is distinguished from the P. triformis group by its anterior end of the gymnostom and pro-mesostegostom, and by the arrangement of the genital papillae, i.e., the most anterior sublaterally directed papillae are the third pair (v3d) vs. the second pair (v2d) (Ragsdale et al., 2013). In addition, P. paulseni sp. n. is distinguished from P. triformis by the reproductive mode, gonochoristic vs. hermaphroditic (Ragsdale et al., 2013). P. paulseni sp. n. is morphologically and phylogenetically closest to P. fissidentatus, but it is distinguished from P. fissidentatus by the cheilostomatous plates in both stenostomatous and eurystomatous forms, the arrangement of male genital papillae, especially the anterior four pairs, at ca 1 CBD anterior to co; v2 and v3d close to each other, but not overlapping, and v2 located at less than 1/2 CBD anterior to co; v3d at the midway between v2 and co; v4 ca 1/2 CBD posterior to co vs. v1 at 1.5 to 2 CBD anterior to co, and adcloacal v2 to v4 are gathered within 1/2 CBD from co, and finally the reproductive mode (Kanzaki et al., 2012a).

The culture from which the type specimens were obtained was originally isolated by M. Herrmann from an adult Lucanus dybowski taiwanus collected at Pilu Sacred tree, Taroko National Park Taiwan in May 2017.

One slide with holotype male and two slides, each with paratypes one male and one female, 28533-28535, were deposited in the University of California Riverside Nematode Collection (UCRNC), CA. Two slides, each with paratypes one male and one female (SMNHType-8991 and 8992) were deposited in the Swedish Natural History Museum, Stockholm, Sweden. Two slides, each with paratypes one male and one female (SMNK-NEMA-T-0145 and 0146) were deposited in the Natural History Museum Karlsruhe, Germany. The strain is available as living culture and as frozen stocks under culture code RS5918 in the Department of Evolutionary Biology, Max Planck Institute (MPI) for Developmental Biology, Tübingen, Germany, and it can be provided to other researchers upon request. The new species binomial has been registered in the ZooBank database (zoobank.org) under the identifier [urn:lsid:zoobank.org:act:6E8BF33F-6F9E-4A6E-99C2-5576242EC26E].

Pristionchus yamagatae* sp. n. (Figs 3, 4; Table 1).

Figure 3

Figure 4

*The species name is derived from the type locality of the nematode, Yuza, Yamagata Prefecture, Japan.

Summarized in Table 1

The anterior ends of gymnostomatal and pro-mesostegostomatal rings are weakly serrated, but they are difficult to observe under light microscopy. Metastegostom bears conspicuous and movable triangular or flint-shaped dorsal tooth with strongly sclerotized surface, giving an appearance of an inverted V-shape in light microscopy in lateral view; a pointed distal end often curved to direct anteriorly; a pointed left subventral ridge with three minute adventitious denticles on a plate; bluntly or sharply pointed right subventral ridge, often with a distinct distal adventitious denticle.

Cheilostomatal plates are very thick. The anterior end of each plate is rounded and elongated to project from stomatal opening, forming a small flap. In the anterior part, only the tip to anterior half of each cheilostomatal plate sometimes splits into two small flaps, i.e., stomatal opening bearing 6 to 12 flaps (= tips), regardless of the number of plates (=6). Each cheilostomatal plate is slightly inclined inwards, giving an appearance that the whole stoma is narrowing anteriorly. Gymnostom with a very thick cuticle is present, forming a short, ring-like tube being thicker posteriorly, and whole gymnostomatal ring widening anteriorly. The anterior end of gymnostom is weakly serrated and it internally overlaps the posterior end of cheilostomatal plates. Thus, the cheilo and gymnostomatal regions form a barrel shape in lateral view. The anterior edge of pro-mesostegostom is clearly serrated. Metastegostom bears a large claw-like dorsal tooth; a large claw-like right subventral tooth; ridge of left subventral denticles or cusps, varying in number and size, i.e., three large cusps, the tip of these cusps is usually split into three or more small tips, sometimes forming serrated plate. The dorsal and right subventral teeth are movable. No movement is observed for left subventral denticles.

The paired papillae and the phasmid are arranged as <v1, v2d, v3, co, v4, ad, ph, (v5, v6, v7), pd>, where v1 is located at ca 1 CBD or slightly more anterior to co; v2d and v3 close to each other, but not overlapping, and v2 is located at ca 1/3 CBD anterior to co; v3 at the midway between v2d and co; v4 is less than 1/3 CBD posterior to co; thus, the distance between v2d and v4 is less than 1 CBD; laterally located ad is at the midway between co and the root of tail spike; ph is subventrally located just anterior to or overlapping with v5; ventral v5 to v7 is forming triplet just anterior to the root of tail spike; and subdorsal pd is overlapping or slightly posterior to v7. In addition to regular nine pairs of papillae, a few individuals have an extra pair of subventral papillae anterior to v1 (= v0).

The diagnostic characters are described above. This new species is molecularly characterized by the barcoding sequence (NCBI accession number MH114983). Also, the new species is typologically distinctive from many other Pristionchus spp. with its barrel-shaped stoma with weakly serrated anterior end of gymnostomatal ring and clearly serrated anterior end of pro-mesostegostomatal ring. These specific characters suggest that P. yamagatae n. sp. belongs to the P. triformis group including P. triformis, P. hoplostomus and P. fukushimae. These three species share the stomatal structure with the new species (Ragsdale et al., 2013, 2015). However, P. yamagatae n. sp. is readily distinguished from P. triformis by its reproductive mode, gonochoristic vs hermaphroditic, the cheilostomatal plates in the stenostomatous and the eurystomatous forms, the arrangement of male genital papillae, v2d and v3 being close to each other vs clearly separated, and relative position of phasmid, located just anterior to or overlapping with v5 vs being located midway between ad and pd paired papillae, or more anteriorly, and clearly separated from v5 (Ragsdale et al., 2013). The new species is distinguished from P. hoplostomus by the cheilostomatal plates in the stenostomatous form, i.e., the twelve-plated stenostomatous form was not found, the arrangement of genital papillae, the level of v4 being clearly apart from cloacal opening vs close to cloacal opening, and the relative position of phasmid, located just anterior to or overlapping with v5 vs being located midway between ad and pd paired papillae, or more anteriorly, and clearly separated from v5 (Ragsdale et al., 2013). P. yamagatae sp. n. is also distinguished from P. fukushimae by the cheilostomatal plates in the stenostomatous form, and the relative position of phasmid, located just anterior to or overlapping with v5 vs being located midway between ad and pd paired papillae, or more anteriorly, and clearly separated from v5 (Ragsdale et al., 2013). In addition to these typological characters, P. yamagatae sp. n. is distinguished from its close relatives by its molecular phylogenetic status and mating experiments.

The culture from which the type specimens were obtained was originally isolated by N. Kanzaki from an adult Holotrichia kyotoensis collected at a Mamurogawa, Yamagata, Japan in July 2017.

One slide with holotype male and two slides, each with paratypes one male and one female, 28536–28538, were deposited in the University of California Riverside Nematode Collection (UCRNC), CA. Two slides, each with paratypes one male and one female (SMNHType-8993 and 8994), were deposited in the Swedish Natural History Museum, Stockholm, Sweden. Two slides, each with paratypes one male and one female (SMNK-NEMA-T-0147 and 0148), were deposited in the Natural History Museum Karlsruhe, Germany. The strain is available as living cultures and as frozen stocks under culture code RS5964 in the Department of Evolutionary Biology, MPI for Developmental Biology, Tübingen, Germany, and it can be provided to other researchers upon request. The new species binomial has been registered in the ZooBank database (zoobank.org) under the identifier [urn:lsid:zoobank.org:act:2A1FAEB0-0D51-4563-A043-34B1515206A4].

The phylogenetic positions of the two novel species were determined by phylogenetic analysis of transcriptomes from all cultivable Pristionchus species (Rödelsperger et al. 2018). The resulting phylogeny of the genus, as shown in Figure 5, places P. yamagatae sp. n. into the subclade consisting of P. triformis, P. hoplostomus, and P. fukushimae. More importantly, this phylogeny positions P. paulseni sp. n. as sister species to the hermaphroditic P. fissidentatus. Thus, except for P. mayeri and P. boliviae, all hermaphroditic species in the Pristionchus genus have at least one relatively close gonochoristic sister species.

Figure 5