Fig 1.
Line drawings of Pellioditis pelhamensis n. sp., strain EM434.
(A–E, G) Adult female: (A) anterior region, left side view, excretory pore (ep) at the level of the grinder. (B, C) Tail region: (B) left side view, (C) ventral view (compare with Fig 2F), showing prominent phasmids approximately halfway down the tail from the anus, protruding through the thick cuticle and terminating on or slightly outside of the external surface. (D) Anterior end showing stoma region, ventral view (compare with Fig 2B showing a left side view). (E) Metarhabdion showing tiny wart-like protrusions. (F) Stoma region of J2, left side view, showing that the stoma is relatively longer and narrower than in the adult. (G) Lateral field of adult female showing ridges (compare with Fig 2A showing same for male). (H–L) Male: ventral view (H) and left side view (I) of adult male tail with genital papillae (GPs) labeled according to the homology system developed for Rhabditina [28, 29] based on common developmental origins of the GPs in the lateral epidermis [23, 27, 58, 59]. Accordingly, there are two GPs—the anterior dorsal (ad) and posterior dorsal (pd)—with cell lineages originating more dorsally than the seven others (v1–v7) (compare Fig 1J). The small ray-like phasmids (ph) are only seen in ventral view. (J) Adherens junctions of the ray cell and lateral seam cells in the left lateral epidermis of a J4 male (anterior up, dorsal to the right) as visualized by immunostaining with MH27 monoclonal antibody at the time when external ray papillae are forming (compare Fig 2D). (K) Right spicule, left side view; notice the complex tip (compare with Fig 2G). (L) Gubernaculum, ventral view (compare Fig 2G).
Fig 2.
DIC photomicrographs of P. pelhamensis n. sp.
(A) Lateral field of an adult male. (B) Adult male head with stoma, sagittal plane, left side view. (C) Adult male pharynx region, left side view. (D) J4 male tail, left side view, showing developing genital papillae and phasmid (ph) as it is drawn out in the wake of tail tip morphogenesis in this peloderan species; ad and pd indicate the anterior dorsal and posterior dorsal genital papilla (compare Fig 1H–1J). (E) Posterior oviduct and part of posterior uterus of adult female filled with sperm, left side view (anterior up). (F) Adult female tail, ventral view, showing phasmids (ph) projecting through the thick cuticle. (G) Isolated spicules (sp) and gubernaculum (gub) of adult male in ventral view. (H, I) Two different focal planes of the same male tail in ventral view, genital papillae labeled as in Fig 1H–1J, showing a typical GP arrangement (i.e. ad+v4 close together and adcloacal). (J) Male tail in right side view showing variant with ad closer to v5 than to v4. Scale bars 10 μm for A–B, 50 μm for C–J.
Fig 3.
Scanning electron micrographs of Pellioditis pelhamensis n. sp. strain EM434 female.
(A) Habitus. (B–D) Lip region, en face view; note that the "bristle-like" features in C and D are bacteria. (E) Anterior region, left side view. (F) Lip region, ventral view. (G) Vulval region with slit-like vulva, sublateral view. (H) Vulva, ventral view. (I) Lateral field, mid-body left-subventral view, vulva at left. (J–M) Posterior region showing conical tail, anus and slightly protruding phasmids, right side views (J, K), ventral view (L) and left side view (M). Abbreviations: dors. = dorsal, vent. = ventral, am = amphid, cs = cephalic sensillum, ils = inner lip sensillum, ols = outer lip sensillum, ph = phasmid.
Fig 4.
Scanning electron micrographs of Pellioditis pelhamensis n. sp. strain EM434 adult male (A–K) and juvenile (sex unknown) (L–P). (A) Anterior portion, left-subventral view, showing lateral field; excretory pore (ep) at lower left and deirid (dei) at lower right. (B, C) Anterior end, en face view, showing lips separated into pairs: one dorsal and two sublateral. (D) Right-subventral view of anterior. (E–G) Lateral field. (H–K) Male tail, showing bursa, genital papillae, phasmids, spicules protruding from the cloaca, postcloacal sensilla. (H) Left sublateral view. (I) Ventral view. (J) Right lateral view. (K) Left lateral view. (L) En face view of juvenile mouth area, dorsal to the upper left. (M) Anterior of juvenile, left side view, dorsal to the right. (N) Lateral field of juvenile. (O, P) Juvenile tail, ventral and sublateral right side view, respectively, showing anus and slightly protruding phasmids. Scale bars 1 μm in B, C, L; 10 μm in other panels. Abbreviations: dors. = dorsal, vent. = ventral, am = amphid, cs = cephalic sensillum, ils = inner lip sensillum, ols = outer lip sensillum, ad = anterior dorsal genital papilla, pd = posterior dorsal genital papilla, ph = phasmid.
Table 1.
Morphometrics of Pellioditis pelhamensis n. sp. strain EM434 and Pellioditis pellio strain SB361.
Fig 5.
Drawings of Pellioditis pellio strain SB361 female (A-E) and male (F-H). (A) Anterior portion showing stoma, pharynx and excretory pore, left side view. (B, C) Female tail showing positions of anus and phasmids; left side (B) and ventral (C) views. (D) Stoma region, right side view. (E) Lateral field near the midbody. (F) Ventral view of adult male tail with genital papillae labeled according to the rhabditid homology system [28, 29] based on common developmental origins of the GPs in the lateral epidermis [23, 27, 58, 59]. (G) Spicule (lateral view) and (H) gubernaculum (ventral view). Scale bars as shown.
Fig 6.
DIC photomicrographs of P. pellio.
(A) Anterior uterus and oviduct of a young female and two laid embryos, arrow points to the position of the vulva; left side view, anterior up. (B) Female lateral field. (C) Female tail in right side view. (D) Pharynx region of an adult male, right side view; arrow points to the location of the excretory pore. (E) Stoma of an adult female in right side view; arrow points to the border between gymnostom and stegostom. (F) Isolated spicules in lateral view and gubernaculum in ventral view. (G) Male tail in right side view. Anterior (ad) and posterior dorsal (pd) genital papillae are indicated with arrows. (H) Male tail in ventral view; the base of the postcloacal sensilla is in focus here (unlabeled arrows). (I) Male tail in ventral view, focused closer to the body than in H and showing phasmids (ph) and the distal end of the gubernaculum (gub). Scale bars 10 μm in B and E, all others 50 μm.
Fig 7.
SEM micrographs of Pellioditis pellio females (A, B, E, J, K, O, P) and males (C, D, F-I, L-N). (A, B) Lip region of female, en face view; in A, the right dorsal lip is abnormal. (C, D) Lip region of male, en face view. (E) Anterior portion of a female in subventral right view. (F) Anterior portion of a male in subventral left view, (G–H) enlarged detail views corresponding to areas delineated in F. (I) Lateral field at mid-body in male. (J) Vulval region of a female with mating plug; inset shows detail of lateral field. (K) Female vulva. (L–N) Male tail in ventral or subventral view showing narrow bursa with genital papillae and cloacal opening with precloacal sensillum and postcloacal sensilla. (O, P) Female tail in ventral and left side views. The phasmids (ph) form small papillae. Abbreviations: am = amphid opening, cs = cephalic sensilla, ep = excretory pore, ad = anterior dorsal genital papilla, pd = posterior dorsal genital papilla, ph = phasmid. Scale bars 10 μM.
Fig 8.
DIC photomicrographs comparing buccal tube and spicules of Pellioditis pelhamensis n. sp. and Pellioditis pellio.
P. pelhamensis n. sp. and P. pellio differ in the shape of the stoma and in the shape of the spicule tip. The stoma of P. pelhamensis n. sp. is shorter and wider—and the spicule tip is narrower—than those of P. pellio. (A–D) P. pelhamensis n. sp. (E–H) P. pellio. (A) Stoma of an adult female in subventral left view. (B) Stoma of an adult male in subventral left view. (E) Stoma of an adult female right side view. (F) Stoma of an adult male right side view. (C, D, G, H) Spicules in lateral view; gubernaculum in ventral view. Scale bars for all stoma images 10 μm (A, B, E, F), for all spicule images 20 μm (C, D, G, H).
Fig 9.
Comparison of male and female tails of Pellioditis pellio redescribed here with those depicted in figures from the original description (Schneider, 1866) [18], subsequent plausible reisolates and isolates mistakenly claimed to be P. pellio.
Schneider [18] only provided a single drawing in the original description: a female tail in ventral view. In the description of P. pellio, he noted that the species was in all aspects similar to his directly preceding description of P. papillosa (of which the male bursa is redrawn here from Taf. XI, Fig IIIc [18]), except that the female tail of P. papillosa was cupola-shaped whereas that of P. pellio was cone-shaped (redrawn here from Taf. XI, Fig X [18]). Neither Schneider nor any of the other authors depicted the phasmids in the males, which are admittedly not very prominent in this species and could have easily been overlooked. In all of the depictions of species compatible with the original description (e.g., from the current paper, Reiter [68], Völk [69], Örley [67], and Hertwig [9]), the male tail is peloderan and GPs 4 and 5 (i.e., v4 and ad) are situated close together, at about the same anteroposterior location as the cloaca. Also, wherever female tails are depicted in these descriptions, all show conical forms, though of varying lengths consistent with variation present in strain SB361 (Table 1). In contrast, the male tails from other depictions mistakenly identified as P. pellio (bottom row) have clearly different male tail tip morphologies and papilla arrangements. For example, the drawings by Bütschli [70] and Thorne [71] show species with leptoderan tail tips; those by Bütschli [70] and Aubertot [72] show GPs 2–3 bracketing the cloaca and those by Thorne [71] and Stock & Camino [73] show GPs 3–4 bracketing the cloaca.
Table 2.
Interfertility tests1.
Fig 10.
Results of phylogenetic analyses (A) and evolutionary reconstructions (B). (A) Molecular phylogenetic analyses. Left tree: WP analysis of all parsimony-informative sites in the supermatrix, which consists of sequences from the nuclear ribosomal RNA gene cluster, the RNA polymerase large subunit gene, and mitochondrial sequences spanning the cytochrome oxidase I gene and the mitochondrial ribosomal RNA (and intervening) gene sequences (see S1 Fig). Numbers at nodes are percentages of 1000 jackknife replicates (25% of characters randomly deleted per replicate) that supported those bipartitions. Synrhabditis and Pellioditis clades [11] are strongly supported. Right tree: Clann [36] supertree of bootstrapped ML trees from 7 subsets of the supermatrix (see S1 Fig). Numbers at the nodes represent the proportions of bipartitions among individual ML trees that were consistent with the branches shown. In both trees, branches are collapsed where bipartition support is less than 70% or 0.7, such that only robustly supported branches are shown. Four species groups are shown with different colors: Pellio group, Papillosa group, Kenyensis group and a group containing P. huizhouensis and P. meridionalis. Obligate parasitism evolved twice, once in the Angiostomatidae (represented here by Angiostoma norvegicum) from within Pellioditis, and in the lineage to Agfa (represented here by Agfa flexilis), the sister group to Pellioditis. (B) Character evolution traced by parsimony onto the WP phylogeny. Cupola-shaped female tails have evolved four times independently from conical-shaped tails (red lineages), and possibly again in the "slow" isolate of P. akhaldaba, not shown. Hermaphroditism evolved twice: in the lineages to P. hermaphrodita and P. californica.