The genus
The three major radiations include the “
Here, we describe two new species.
One strain of
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
The majority of
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.
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.
Morpometrics
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Character | stenostomatous male | stenostomatous female | stenostomatous male | stenostomatous female |
n | 10 | 10 | 10 | 10 |
L | 1208±95.4 (990–1315) | 1560±230 (1222–1895) | 848±42.2 (796–916) | 1162±121.9 (937–1385) |
L’ | 1032±92.5 (813–1138) | 1298±209.6 (978–1560) | 681±34.7 (616–724) | 908±103.7 (717–1096) |
a | 14±1,3 (12–16) | 14±0.6 (13–15) | 14±1.4 (13–18) | 14±1.2 (12–17) |
b | 7.2±0.8 (5.8–9.0) | 8.7±1.0 (7.3–10.1) | 5.8±0.3 (5.3–6.2) | 6.8±0.6 (5.8–7.8) |
c | 6.9±0.8 (5.6–8.3) | 6.0±0.6 (5.0–6.9) | 5.1±0.4 (4.4–5.9) | 4.6±0.4 (4.1–5.3) |
c’ | 4.0±0.6 (3.4–4.9) | 6.0±0.5 (5.3–6.8) | 5.1±0.6 (3.9–6) | 7.3±0.7 (6.1–8.2) |
T or V | 58±2.9 (53–62) | 47±0.9 (45–48) | 51±3.3 (45–54) | 45±1.3 (44–48) |
Maximum body diam. | 86±4.7 (75–91) | 114±19.3 (84–138) | 60±6.4 (47–70) | 84±9.6 (68–98) |
Stoma length | 13.0±0.5 (12.0–13.8) | 14.4±0.4 (13.9–15.0) | 11.5±0.7 (10–12.2) | 13.3±0.5 (12.3–14) |
Stoma diam. | 5.7±0.5 (5.0–6.8) | 6.8±0.4 (6.2–7.5) | 6±0.5 (5.4–6.7) | 7.4±0.5 (6.7–8.3) |
Pharynx length (head to base of pharynx) | 156±13.8 (122–168) | 169±7.2 (158–178) | 135±5.4 (124–143) | 156±7.3 (146–170) |
Anterior pharynx (pro- + metacorpus) | 96±8.3 (75–105) | 106±2.9 (102–110) | 83±3.9 (77–88) | 98±4.2 (93–106) |
Posterior pharynx (isthmus + basal bulb) | 61±6.1 (47–68) | 64±4.8 (56–69) | 52±2.2 (47–55) | 58±3.9 (52–64) |
Ant/total pharynx % | 61±1.3 (59–63) | 62±1.6 (60–65) | 63±4.2 (59–74) | 63±1.2 (61–65) |
Median bulb diam. | 28±2.1 (25–31) | 34±2.8 (30–36) | 22±0.7 (21–23) | 30±3.8 (22–36) |
Terminal bulb diam. | 26±1.8 (23–28) | 30±3.9 (25–34) | 21±1.3 (18–22) | 27±3.7 (20–33) |
Testis length | 698±69.9 (532–787) | – | 433±37.9 (372–475) | – |
Ant. end to vulva | – | 730±101.3 (592–860) | – | 527±48.8 (446–610) |
Vulva to anus distance | – | 570±103.2 (424–710) | – | 380±52.7 (281–470) |
Cloacal or anal body diam. | 44±3.4 (36–48) | 44±4.3 (37–50) | 33±3.6 (28–40) | 35±1.9 (32–38) |
Tail length | 176±19.3 (155–220) | 262±69.9 (235–335) | 167±17.5 (140–192) | 255±27.1 (212–289) |
Spicule length (curve) | 51±2.6 (47–54) | – | 43±1.2 (41–45) | – |
Spicule length (chord) | 41±2.5 (36–44) | – | 36±0.8 (34–37) | – |
Gubernaculum length | 14±1.3 (12–16) | – | 14±1.4 (13–18) | – |
*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
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
The culture from which the type specimens were obtained was originally isolated by M. Herrmann from an adult
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].
*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
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
The culture from which the type specimens were obtained was originally isolated by N. Kanzaki from an adult
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
In this study, we have described two new
Also, the species pair