The genus Pristionchus (Kreis, 1932) is emerging as a model system for biodiversity research in nematodes and animals in general (Sommer, 2015). As Pristionchus species are often found in association with scarab beetles, detailed sampling of these insects has provided a comprehensive picture of the diversity of their associated nematodes (Ragsdale et al., 2015). Specifically, more than 30 species from around the world are known to form four well-defined groups of the genus. Two major strategies can be taken to explore Pristionchus taxonomy and phylogeny to eventually reach the saturation level of species identification. First, broad collecting efforts have to be conducted in continents where taxonomic discovery of Pristionchus is unsaturated. For example, samplings in East Asia and Taiwan in particular, revealed the identification of novel species in every new field trip as indicated in the accompanying publication (Yoshida et al., 2018). In contrast, the Pristionchus diversity in Europe has apparently reached saturation with collections in Western and Eastern Europe repeatedly finding a total of seven species in the “lheritieri” species-complex from soil and scarab beetle samplings (Herrmann et al., 2006). Similarly, collections in Africa and South-America have so far only identified the known hermaphroditic species of the genus (M.H., pers. Obs.). Thus, future studies should focus on East Asia, both looking at continental and island settings. Second, previous research had indicated that Pristionchus evolution involves host switching from scarab beetles to other insects including the Colorado potato beetle and fig wasps (D’Anna and Sommer, 2011; Susoy et al., 2016). Therefore, sampling efforts should include other arthropod taxa as potential sources of novel species.
Here, we describe five new Pristionchus species that result from field trips in 2016 and 2017 that aimed to explore new areas in East Asia and sampling of new arthropod species. Specifically, we found three new species on millipedes sampled in Japan, an Island that had previously shown saturation with regard to scarab beetle-associated Pristionchus species (Kanzaki et al., 2012; Kanzaki et al., 2014). In addition, we found two new Pristionchus species on scarab beetles in Hongkong, which represents the first scarab beetle sampling for Pristionchus nematodes in China. We describe all five new species by morphology, morphometrics, mating experiments, and molecular sequence analysis.
Materials and methods
Beetle sampling and nematode isolation
In addition to collecting millipedes in different parts of Japan, a field trip to Hongkong was conducted in June 2017. Basically all major areas in Hongkong, including the new territories have been visited and more than 250 specimens of scarabaeoid beetles belonging to 16 species in 13 genera were collected. Nematodes were isolated using standard procedures as previously described (Herrmann et al., 2006). In total, we isolated and characterized 12 strains of the Diplogastridae family with eight of these strains representing members of the genus Pristionchus. Three of the Pristionchus strains were hermaphroditic and were shown by sequencing of the full length SSU rRNA of 1.6kb using the primers RH5401 (5′- AAAGATTAAGCCATGCATG -3′) and SSU R2 (5′- GGTTCAAGCCACTGCGAT-3′) for PCR amplification, and RH5403 (5′- AGCTGGAATTACCGCGGCTG-3′), SSU R4 (5′- TGGTGCCCTTCCGTCAATTC-3′) and SSU R2 (5′- GGTTCAAGCCACTGCGAT-3′) for sequencing and mating experiments (see below) to represent new isolates of P. pacificus. Thus, these strains are not further considered in this study. Five strains of Pristionchus were gonochoristic and represent new species that will be described in this study. All new isolates are available as frozen stocks in the Department of Evolutionary Biology, Max Planck Institute (MPI) for Developmental Biology, Tübingen, Germany and can be provided to other researchers upon request.
Nematode cultivation
P. laevicollis sp. n. was isolated from an adult of Aegus subnitidus (Waterhouse, 1873) (Coleoptera: Lucanidae), formerly a subspecies of A. laevicollis Saunders, 1854, collected in Nagoya, Japan. The strain of P. hongkongensis sp. n. and P. neolucani sp. n. were isolated from two adults of Neolucanus championi, (Parry, 1864) (Coleoptera: Lucanidae) collected at Mui Tsz Lam, Hongkong. Pristionchus degawai sp. n. was isolated from the millipede Parafontaria laminata armigera (Verhoeff, 1936) (Diplopoda: Xystodesmidae) in Nagano, Japan and P. riukiariae sp. n. came from a millipede of the species Riukiaria holstii (Pocock, 1895) (Diplopoda: Xystodesmidae) from Okinawa, Japan. All strains have been kept in laboratory culture on NGM agar plates seeded with Escherichia coli strain OP50 under the following culture code and freezing voucher numbers RS5939 (P. laevicollis sp. n.), RS5957 (P. hongkongensis sp. n.), RS5949 (P. neolucani sp. n.), RS5938 (P. degawai sp. n.), and RS5937 (P. riukiariae sp. n.).
Morphological observation and preparation of type material
Light microscopic observations for drawings and morphometrics were conducted using live nematode material, which was handpicked from culture plates as described previously (Kanzaki, 2013).
Remarks concerning morphometric measurements: based on our repeated findings that measurements of certain nematode dimensions can change over time as a result to different culture conditions (Fonderie et al., 2013; Kanzaki et al., 2012a,2013; Ragsdale et al., 2015; Herrmann et al., 2016), we combine molecular, morphological, biological, and ecological information to characterize new material as broadly as possible.
Mating experiments
To examine reproductive isolation of the new species we crossed them with the most closely related species in the phylogeny. In the case of the four new species P. hongkongensis sp. n., P. neolucani sp. n., P. degawai sp. n., and P. riukiariae sp. n., we tested all possible combinations of all four species since they are closely related to one another. Mating experiments were performed as previously described with some modifications (Herrmann et al., 2006). Three virgin females of a strain were tested with six males of another strain on a mating plate that contained a small amount of E. coli OP50 bacteria. All crosses were performed reciprocally and in quadruplicate. To test for the production of viable F1 hybrids, crosses were set up between all possible pairs and F1 hybrids were tested for their ability to produce F2 progeny. We considered strains to belong to the same species if they produced viable and fertile offspring.
Molecular characterization and phylogenetic analysis
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 to 3 mixed-stage plates per species using standard Trizol extraction following the manufacturers’ instructions (Zymo Research, CA, USA). RNAseq 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 14 million 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). A total of 2,092 high quality alignments containing at least 14 species (without any duplication) and with at least 50 amino acid positions with coverage in all represented species were concatenated into a supermatrix spanning 350,000 amino acids. Based on previous analysis of dozens of gene families (Baskaran et al., 2015), we chose the LG substitution model to reconstruct a maximum-likelihood tree using RAxML (version 8.2.9, options -m PROTGAMMAILG -f a -N 100) (Stamatakis, 2014).
Results
Arthropod samplings in East Asia identify nine new Pristionchus species
We carried out field trips to Taiwan, Japan, and Hongkong in 2016 and 2017 as described in detail in “materials and methods” and in the accompanying publication (Yoshida et al., 2018). In Japan and Hongkong, we isolated a total of 12 gonochoristic Pristionchus strains, 10 strains from Japan, and 2 strains from Hongkong. These strains were first subjected to SSU rRNA sequence analysis, which suggested that they represent five new species of Pristionchus. Mating experiments with reference strains of available Pristionchus species with identical or closely related SSU sequences were performed confirming that these species are indeed reproductively isolated. Also, transcriptome analysis reported below confirmed these strains to represent novel species.
Mating experiments
We performed mating experiments to confirm reproductive isolation between P. laevicollis sp. n. and the related species P. maxplancki (Fig. 1). We observed some viable but sterile F1 individuals between the two species. In contrast, P. maxplancki did not produce viable F1 with species other than P. laevicollis sp. n. Next, we performed mating experiments between the other new species, P. neolucani sp. n., P. degawai sp. n., P. hongkongensis sp. n., and P. riukiariae sp. n., which form a new clade in the phylogenetic analysis (see below). All combinations of mating experiments produce no F1 hybrids. Thus, in contrast to the species in the pacificus species group sensu stricto, the species considered in this study largely do not form F1 hybrids.
Figure 1
Results of mating experiments of all five new species with P. maxplancki. A: Mating between P. laevicollis sp. n. and P. maxplancki; B: Mating between the four new species, P. honkongensis sp. n., P. degawai sp. n., P. neolucani sp. n. and P. riukiariae sp. n. Numbers indicate the results of four replicates of mating experiments. Production of no F1 progeny is indicated as “1,” the formation of viable, sterile F1 progeny is indicated as “2.”
Description
Description of common characters
Most Pristionchus species are typologically similar to each other and generally distinguished based on stomatal and male tail characters. 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. It is important to note that these common characters in the novel species are also identical to the typological characters of other Pristionchus species, such as those described in the accompanying publication (Yoshida et al., 2018). Therefore, the following description of common characters is highly redundant, but included here for completeness.
Adult
The adult body can be described as follows: 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; stomatal dimorphism present, with stenostomatous (narrow mouthed) and eurystomatous (wide mouthed) forms occurring in males and females, the latter (i.e. eurystomatous form), however, less frequent in males. Detailed stomatal morphology is described below. Dorsal pharyngeal gland clearly observed, penetrating dorsal tooth to gland opening; anterior part of pharynx (= pro and metacorpus) 1.5 times as long as posterior part (= isthmus and basal bulb); procorpus very muscular, stout, occupying half to two-thirds of corresponding body diameter; metacorpus very muscular, forming well-developed median bulb; isthmus narrow, not muscular; basal bulb glandular; pharyngo-intestinal junction clearly observed, well developed; nerve ring usually surrounding middle region of isthmus; excretory pore not conspicuous, ventrally located at level of isthmus to pharyngo-intestinal junction, excretory duct extending anteriad and reflexed back to position of pore; deirid observed laterally, located from slightly anterior to pharyngo-intestinal junction to a half body diameter posterior to the junction; hemizonid not clearly observed; lateral glands, small pores connected to secretory cell, 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.
Stenostomatous form
Cheilostom consisting of six per- and interradial plates. Incision between plates is not easily distinguished by light microscopy. Anterior end of each plate rounded and elongated to project from stomatal opening and form a small flap; gymnostom short, cuticular ring-like anterior end overlapping cheilostom internally; dorsal gymnostomatal wall slightly thickened compared to ventral side; stegostom separated into three subsections: pro-meso, meta, and telostegostom; pro-meso stegostom forming a weakly cuticularized ring surrounding the anterior edge of pharynx. Metastegostom bearing 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; pointed left subventral ridge with three minute adventitious denticles on a plate; pointed right subventral ridge, often with distinct distal adventitious denticle(s). Telostegostom forming a weakly sclerotized cup-like cavity connecting stoma and pharynx.
Eurystomatous form
Cheilostom divided into six distinctive per- and interradial plates. Anterior end of each plate rounded and elongated to project from stomatal opening, forming a small flap; gymnostom with thick cuticle, forming a short, ring-like tube being thicker posteriorly; anterior end of gymnostom internally overlapping posterior end of cheilostomatal plates; stegostom separated into three subsections: pro-meso, meta, and telostegostom; pro-mesostegostom forming a weakly cuticularized ring surrounding the anterior edge of pharynx; metastegostom bearing large claw-like dorsal tooth; right subventral tooth; ridge of left subventral denticles or cusps, varying in number and size; dorsal and right subventral teeth movable; no movement observed for left subventral denticles; telostegostom forming a weakly sclerotized cup-like cavity connecting stoma and pharynx.
Male
The male body can be described as follows: 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 two-thirds of the main branch, then mature amoeboid spermatids arranged in multiple rows in remaining, proximal part of gonad; vas deferens not clearly separated from other parts of gonad; three (two subventral and one dorsal) cloacal gland cells observed at distal end of testis 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 1/4 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. Thick cuticle around tail region, falsely appearing as a narrow leptoderan bursa in ventral view; cloacal opening (co) slit-like in ventral view; 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, 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) almost equal in size, rather large and conspicuous, v7 and pd papillae obviously smaller than anterior five pairs, v5 and v6 very small, 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. Detailed arrangement of papillae and phasmid will be described for each species below. Tail conical, with long spike, about two to three cloacal body diameters (CDB) long; bursa or bursal flap absent.
Female
The female body can be described as follows: 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 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 on left of intestine; oocytes mostly arranged in three to four rows in distal two-thirds of ovary and in double or single row in rest of ovary, distal tips of each ovary reaching oviduct of opposite gonad branch; anterior end of oviduct (= junction tissue between ovary and oviduct) consists of rounded cells; anterior part of oviduct consists of rounded cells, forming a simple tube; middle part of oviduct serving as spermatheca, consists of roundish and relatively large cells. Eggs in single to multiple-cell stage or even further developed at posterior part of oviduct (= uterus), in young females being composed of squared or angular cells, long enough to contain one well developed oocyte. Receptaculum seminis not observed, i.e., the organ is not independent, and a part of oviduct/uterus works as the organ; vaginal glands present but obscure; vagina perpendicular to body surface, surrounded by sclerotized tissue; vulva slightly protuberant in lateral view, pore-like in ventral view; rectum about one anal body diameter long, intestine/rectum junction surrounded by well-developed sphincter muscle; three anal glands (two subventral and one dorsal) present but not obvious; anus in form of dome-shaped slit, posterior anal lip slightly protuberant; phasmid about one to two anal body diameters posterior to anus; tail long, smoothly tapered, distal end variable from filiform to long and conical.
Description of species-specific characters
Diagnostic characters, i.e., stomatal morphology and male tail characters (arrangement of genital papillae) are herein described for each species including measurements for all species (Tables 1–3). Additionally, these typological characters applicable to species diagnosis of the pacificus group are summarized in Table 4.
Table 1
Morphometrics of P. laevicollis sp. n.
| Character | P. laevicollis RS5939 stenostomatous male | Stenostomatous female | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| n | 10 | 10 | ||||||||||||
| L | 834 ± 104 (662–971) | 1104 ± 209 (746–1325) | ||||||||||||
| L’ | 705 ± 89.6 (566–836) | 908 ± 179 (595–1126) | ||||||||||||
| a | 15 ± 1 (13–17) | 13 ± 0.8 (12–15) | ||||||||||||
| b | 6.5 ± 0.5 (5.9–7.4) | 7.6 ± 0.8 (5.8–8.6) | ||||||||||||
| c | 6.5 ± 0.4 (5.9–7.2) | 5.7 ± 0.7 (4.6–6.9) | ||||||||||||
| c’ | 3.9 ± 0.4 (3–4.3) | 5.8 ± 0.7 (4.9–7.1) | ||||||||||||
| T or V | 59 ± 2 (56–62) | 46 ± 2.1 (42–49) | ||||||||||||
| Maximum body diam. | 57 ± 7.4 (47–66) | 83 ± 14.8 (57–97) | ||||||||||||
| Stoma length | 9.4 ± 1 (7.7–10.6) | 11.1 ± 1.1 (9–12.5) | ||||||||||||
| Stoma diam. | 5.6 ± 0.7 (4–6.4) | 6.4 ± 0.7 (5.5–8.0) | ||||||||||||
| Pharynx length (head to base of pharynx) | 118 ± 9.5 (103–132) | 137 ± 15.5 (113–154) | ||||||||||||
| Anterior pharynx (pro– + metacorpus) | 72 ± 6.7 (61–81) | 84 ± 8.7 (71–93) | ||||||||||||
| Posterior pharynx (isthmus + basal bulb) | 46 ± 2.9 (42–51) | 53 ± 7.1 (42–63) | ||||||||||||
| Ant/total pharynx % | 61 ± 1 (58–61) | 61 ± 1.2 (59–63) | ||||||||||||
| Median bulb diam. | 20 ± 2.1 (16–22) | 27 ± 3.1 (22–30) | ||||||||||||
| Terminal bulb diam. | 19 ± 1.7 (16–21) | 25 ± 3.8 (19–31) | ||||||||||||
| Testis length | 490 ± 72.5 (370–570) | – | ||||||||||||
| Ant. end to vulva | – | 504±86.2 (365–613) | ||||||||||||
| Vulva to anus distance | – | 410 ± 78.9 (275–523) | ||||||||||||
| Cloacal or anal body diam. | 33 ± 2.3 (28–36) | 34 ± 4.5 (32–60) | ||||||||||||
| Tail length | 129 ± 17.4 (96–152) | 196 ± 47.1 (138–285) | ||||||||||||
| Spicule length (curve) | 44 ± 2.5 (40–47) | – | ||||||||||||
| Spicule length (chord) | 35 ± 2.1 (31–38) | – | ||||||||||||
| Gubernaculum length | 16 ± 1 (14–17) | – | ||||||||||||
| Character | P. hongkongensis RS5957 stenostomatous male | Stenostomatous female | P. neolucani RS5949 stenostomatous male | Stenostomatous female | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| n | 10 | 10 | 10 | 10 | ||||||||||
| L | 1014 ± 185 (700–1302) | 1181 ± 315 (918–2008) | 871 ± 49.6 (775–951) | 1060 ± 111.1 (930–1297) | ||||||||||
| L’ | 847 ± 184 (560–1125) | 940 ± 288 (703–1700) | 745 ± 50.7 (658–826) | 864 ± 103.2 (743–1080) | ||||||||||
| a | 14 ± 3.7 (11–24) | 13 ± 0.9 (12–15) | 15 ± 1 (14–16) | 14 ± 0.7 (13–15) | ||||||||||
| b | 6 ± 1 (4.6–7.7) | 6.6 ± 1.1 (5.6–9.5) | 5.4 ± 0.6 (4.7–6.6) | 5.5 ± 0.4 (4.8–6.5) | ||||||||||
| c | 6.1 ± 1.3 (5–8.5) | 4.8 ± 0.7 (4.3–6.5) | 7 ± 0.9 (6.1–9.2) | 5.4 ± 0.5 (4.5–6.1) | ||||||||||
| c’ | 4.2 ± 0.9 (3–5.9) | 6.3 ± 0.5 (5.1–6.9) | 3.7 ± 0.7 (2.6–4.9) | 6.1 ± 0.9 (5–7.8) | ||||||||||
| T or V | 53 ± 4.8 (51–68) | 45 ± 2.6 (42–50) | 53 ± 7.5 (33–59) | 47 ± 1.3 (45–48) | ||||||||||
| Maximum body diam. | 73 ± 14.9 (51–96) | 92 ± 29.4 (67–170) | 57 ± 5.8 (49–67) | 75 ± 9 (63–95) | ||||||||||
| Stoma length | 12.6 ± 1 (11.4–14.6) | 13.3 ± 1.6 (10.3–16) | 10.4 ± 1 (8.7–12) | 11 ± 0.9 (10.2–13) | ||||||||||
| Stoma diam. | 7.2 ± 0.7 (6.1–8.5) | 7.7 ±1.1 (6.5–9.7) | 6.0 ± 0.7 (5.3–7.6) | 7.1 ± 0.7 (6.3–8.4) | ||||||||||
| Pharynx length (head to base of pharynx) | 155 ± 9.7 (140–172) | 168 ± 14 (154-202) | 151 ± 11.9 (129-169) | 182 ± 14.9 (149-194) | ||||||||||
| Anterior pharynx (pro- + metacorpus) | 98 ± 5.4 (90–106) | 106 ± 7.4 (94–117) | 95 ± 10.2 (76–112) | 116 ± 9.2 (97–128) | ||||||||||
| Posterior pharynx (isthmus + basal bulb) | 57 ± 5.9 (49–66) | 62 ± 8.4 (56–85) | 56 ± 5.3 (51–69) | 66 ± 7.1 (52–75) | ||||||||||
| Ant/total pharynx % | 63 ± 2 (60–66) | 63 ± 2.5 (58–66) | 63 ± 3.2 (59–67) | 64 ± 1.8 (61– 66) | ||||||||||
| Median bulb diam. | 27 ± 2 (24–31) | 31 ± 3.6 (26–38) | 25 ± 1 (24–27) | 31 ± 2.9 (27–36) | ||||||||||
| Terminal bulb diam. | 26 ± 2.2 (22–28) | 29 ± 6.6 (24–47) | 25 ± 1.3 (22–27) | 29 ± 1.7 (27–33) | ||||||||||
| Testis length | 540 ± 141 (366–765) | – | 467±43 (425–561) | – | ||||||||||
| Ant. end to vulva | – | 539 ± 172 (384–995) | – | 493 ± 50.9 (420–581) | ||||||||||
| Vulva to anus distance | – | 419 ± 149 (320–815) | – | 371 ± 47 (290–447) | ||||||||||
| Cloacal or anal body diam. | 41 ± 5.4 (30–50) | 39 ± 8 (32–60) | 35 ± 4.8 (27–43) | 32 ± 3.8 (27–38) | ||||||||||
| Tail length | 167 ± 21.6 (140–206) | 241 ± 30 (215–308) | 125 ± 13.2 (99–148) | 196 ± 19 (175–228) | ||||||||||
| Spicule length (curve) | 53 ± 4.2 (44–57) | – | 47±2.3 (43–51) | – | ||||||||||
| Spicule length (chord) | 45 ± 3.7 (37–49) | – | 36 ± 1.7 (34–39) | – | ||||||||||
| Gubernaculum length | 19 ± 1.8 (17–22) | – | 17 ± 1.5 (14–19) | – | ||||||||||
| Character | P. riukiariae RS5937 stenostomatous male | Stenostomatous female | P. degawai RS5938 stenostomatous male | Stenostomatous female | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| n | 10 | 10 | 10 | 10 | ||||||||||
| L | 918 ± 162 (663–1180) | 1362 ± 211 (1025–1700) | 872 ± 142.2 (710–1105) | 1184 ± 310.5 (811–1853) | ||||||||||
| L’ | 780 ± 151 (553–1010) | 1142 ± 201 (835–1466) | 727 ± 134.6 (585–961) | 986 ± 282 (652–1600) | ||||||||||
| a | 12 ± 2.5 (9.4–15) | 14 ± 1.5 (11–16) | 16 ± 2 (13–19) | 16 ± 3 (132–23) | ||||||||||
| b | 5.7 ± 0.8 (5–7.5) | 8.4 ± 1.5 (5.9–10.8) | 5.3 ± 0.6 (4.6–6.4) | 6.6 ± 1.2 (5.1–8.6) | ||||||||||
| c | 6.8 ± 1.2 (5.4–9.3) | 6.2 ± 0.7 (5–7.3) | 6 ± 0.8 (5.4–7.7) | 6 ± 0.9 (4.7–7.3) | ||||||||||
| c’ | 3.4 ± 0.4 (2.7–4.2) | 5.2 ± 0.6 (4.5–6.1) | 4.1 ± 0.5 (3.3–4.7) | 5.2 ± 1 (3.8–7.3) | ||||||||||
| T or V | 51 ± 5.3 (41–59) | 45 ± 11.1 (32–72) | 45 ± 5 (40–56) | 51 ± 5.5 (45–63) | ||||||||||
| Maximum body diam. | 78 ± 13.2 (62–97) | 99 ± 19.4 (65–128) | 56 ± 14.4 (40–80) | 80 ± 28.6 (37–138) | ||||||||||
| Stoma length | 11.1 ± 1 (9.7–12.5) | 11.8 ± 1.3 (9.2–14) | 11.4 ± 0.8 (10–12.3) | 12.1 ± 0.9 (11–13.4) | ||||||||||
| Stoma diam. | 6.5 ± 0.8 (5.4–7.6) | 7.3 ±0.7 (6.2–8.4) | 6.7 ± 0.3 (6.3–7) | 7.6 ± 0.6 (6.9–8.7) | ||||||||||
| Pharynx length (head to base of pharynx) | 143 ± 17.6 (111–167) | 156 ± 12.2 (136–178) | 152 ± 10.1 (143–176) | 169 ± 16.3 (148–207) | ||||||||||
| Anterior pharynx (pro– + metacorpus) | 94 ± 11 (78–111) | 97 ± 8.8 (86–116) | 95 ± 4.2 (89–103) | 106 ± 8.4 (96–122) | ||||||||||
| Posterior pharynx (isthmus + basal bulb) | 54 ± 5.8 (43–63) | 58 ± 7 (40–66) | 58 ± 7.1 (47–73) | 62 ± 9.6 (51–85) | ||||||||||
| Ant/total pharynx % | 67 ± 11.9 (59–100) | 62 ± 3.4 (60–71) | 62 ± 2.4 (59–67) | 64 ± 2.3 (60– 68) | ||||||||||
| Median bulb diam. | 27 ± 2.2 (23–31) | 31 ± 1.4 (29–33) | 26 ± 3 (22–32) | 30 ± 4.6 (24–41) | ||||||||||
| Terminal bulb diam. | 24 ± 2.8 (19–28) | 28 ± 2.3 (24–32) | 25 ± 4 (19–33) | 28 ± 4.8 (22–39) | ||||||||||
| Testis length | 468 ± 116.4 (295–677) | – | 397 ± 105.7 (285–580) | – | ||||||||||
| Ant. end to vulva | – | 624 ± 199 (415–1040) | – | 601 ± 173.3 (416–1043) | ||||||||||
| Vulva to anus distance | – | 510 ± 94.6 (332–665) | – | 365 ± 72.8 (255–487) | ||||||||||
| Cloacal or anal body diam. | 41 ± 4.5 (32–46) | 43 ± 6.1 (31–52) | 35 ± 5.6 (30–44) | 39 ± 8.7 (22–55) | ||||||||||
| Tail length | 137 ± 21.2 (104–170) | 220 ± 14.3 (190–241) | 145 ± 14.5 (125–180) | 198 ± 36.8 (150–253) | ||||||||||
| Spicule length (curve) | 53 ± 5.5 (40–60) | – | 47±7.6 (37–57) | – | ||||||||||
| Spicule length (chord) | 43 ± 3.4 (36–47) | – | 40 ± 6.9 (32–50) | – | ||||||||||
| Gubernaculum length | 12 ± 2.5 (9.4–15) | – | 16 ± 2.4 (13–19) | – | ||||||||||
| Stenostomatous form | Eurystomatous form | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Species | Reproductive mode | Right subventral plate | Left subventral plate | Right subventral tooth | Left subventral cusps | Genital papillae | Other characteristic feature(s) | |||||||
| pacificus | H | Single peak | Three bumps | Claw-like | Three large cusps each sometimes split into two (= 3-6 peaks) | Type 1: v1-v2d = v2d-v4; v2d and v3 are close to each other | ||||||||
| Exspectatus | G | Single peak | Three bumps | Claw-like | three large cusps each sometimes split into two (= 3-6 peaks) | Type 1: v1-v2d = v2d-v4; v2d and v3 are close to each other | ||||||||
| Occultus | G | single peak | three bumps | claw-like | Three large cusps each sometimes split into two to three (= 3-9 peaks) | Type 1: v1-v2d = v2d-v4; v2d and v3 are close to each other | ||||||||
| Sikae | G | Single peak | Three bumps | Claw-like with or without blunt projection | Three large cusps each sometimes split into two (= 3-6 peaks) | Type 1: v1-v2d = v2d-v4; v2d and v3 are close to each other | ||||||||
| Arcanus | G | Single peak | Three bumps | Claw-like | Three large cusps each sometimes split into two (= 3-6 peaks) | Type 2: v1-v2d > v2d-v4; v2d and v3 are close to each other | ||||||||
| Kurosawai | G | Single peak | Three bumps | Claw-like | Three large cusps each sometimes split into two (= 3-6 peaks) | Type 1: v1-v2d = v2d-v4; v2d and v3 are close to each other | ||||||||
| Taiwanensis | G | Single peak | Three bumps | Claw-like with blunt projection | Three large cusps each sometimes split into two (= 3-6 peaks) | Type 1: v1-v2d = v2d-v4; v2d and v3 are close to each other | ||||||||
| Laevicollis n. sp. | G | Single peak | Three bumps | Claw-like | Three large cusps each sometimes split into two (= 3-6 peaks) | Type 3: v1-v2 = v2-v4; v2 and v3d are separated | ||||||||
| maxplancki | G | 1-2 peaks | with minute denticles | claw-like | three large cusps each sometimes split into two (= 3-6 peaks) | Type 3: v1-v2 = v2-v4; v2 and v3d are separated | ||||||||
| Japonicus | G | Single peak | Three bumps | Claw-like | Three large cusps each sometimes split into two (= 3-6 peaks) | Type 4: v1-v2 < v2-v4; v2 and v3d are separated | ||||||||
| Riukiariae n. sp. | G | Two peaks | Three bumps | Two large ridges or multiple small denticles | Three large cusps each sometimes split into two to three (= 3-9 peaks) | Type 1: v1-v2d = v2d-v4; v2d and v3 are close to each other | ||||||||
| Neolucani n. sp. | G | Single peak | Three bumps | Claw-like | Three large cusps each sometimes split into two (= 3-6 peaks) | Type 1: v1-v2d = v2d-v4; v2d and v3 are close to each other | ||||||||
| Degawai n. sp. | G | Single peak | Three bumps | Claw-like | Three large cusps each sometimes split into two (= 3-6 peaks) | v1-v2d = v2d-v4; v2d and v3 are close to each other | v2 and v3 are very close, and sometimes third pair is directed sublaterally (v3d) | |||||||
| Hongkongensis n. sp. | G | Single peak | Three bumps | Claw-like | Three large cusps each split into three or more (= more than 9 peaks) | v1-v2 < v2-v4; v2 and v3d are separated | Comparatively large and slightly more barrel-shaped stoma in eurystomatous form | |||||||
| Quatrusdecimus | G | 1-2 peaks | Three bumps | Claw-like | Three large cusps each sometimes split into two (= 3-6 peaks) | v1-v2 < v2-v4; v2 and v3d are separated | ||||||||
| P. pacificus | P. exspectatus | P. occultus | P. sikae | P. arcanus | P. kurosawai | P. taiwanensis | P. laevicollis sp. n. | P. maxplancki | P. riukiariae sp. n. | P. neolucani sp. n. | P. degawai sp. n. | P. hongkongensis sp. n. | P. quartusdecimus | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| P. pacificus | 0 | 8 | 5 | 10 | 6 | 3 | 6 | 21 | 16 | 35 | 25 | 31 | 42 | 36 |
| P. exspectatus | – | 0 | 6 | 12 | 8 | 5 | 8 | 23 | 16 | 34 | 27 | 33 | 43 | 38 |
| P. occultus | – | – | 0 | 1 | 4 | 2 | 5 | 18 | 14 | 27 | 21 | 27 | 38 | 33 |
| P. sikae | – | – | – | 0 | 10 | 1 | 4 | 18 | 14 | 32 | 21 | 27 | 38 | 33 |
| P. arcanus | – | – | – | – | 0 | 3 | 4 | 19 | 14 | 31 | 24 | 30 | 41 | 35 |
| P. kurosawai | – | – | – | – | – | 0 | 3 | 18 | 13 | 28 | 22 | 28 | 39 | 33 |
| P. taiwanensis | – | – | – | – | – | – | 0 | 19 | 14 | 29 | 24 | 30 | 41 | 35 |
| P. laevicollis sp.n. | – | – | – | – | – | – | – | 0 | 18 | 35 | 34 | 36 | 47 | 42 |
| P. maxplancki | – | – | – | – | – | – | – | – | 0 | 29 | 28 | 30 | 41 | 39 |
| P. riukiariae sp.n. | – | – | – | – | – | – | – | – | – | 0 | 5 | 4 | 14 | 19 |
| P. neolucani sp.n. | – | – | – | – | – | – | – | – | – | – | 0 | 6 | 17 | 18 |
| P. degawai sp.n. | – | – | – | – | – | – | – | – | – | – | – | 0 | 14 | 19 |
| P. hongkongensis sp.n. | – | – | – | – | – | – | – | – | – | – | – | – | 0 | 23 |
| P. quartusdecimus | – | – | – | – | – | – | – | – | – | – | – | – | – | 0 |
