Rhombonemertes rublinea, Hookabe & Moritaki & Jimi & Ueshima, 2022

Hookabe, Natsumi, Moritaki, Takeya, Jimi, Naoto & Ueshima, Rei, 2022, A new oerstediid discovered from wood falls in the Sea of Kumano, Japan: Description of Rhombonemertes rublinea gen. et sp. nov. (Nemertea: Eumonostilifera), Zoologischer Anzeiger 301, pp. 154-162 : 156-157

3.2. Rhombonemertes rublinea gen. et sp. nov.

[New Japanese name: hishimochi-himomushi]

Material examined. Holotype: NSMT-Ne-003, unsectioned complete specimen except for the posterior tip, fixed in Bouin’ s fluid and later preserved in 70% ethanol, posterior tip preserved in 99% ethanol, male, collected on January 10 2022 by NJ, at depths of 150–200 m, off Owase (approx. 34 ◦ 06 ′ 49.0 ′′ N, 136 ◦ 30 ′ 35.0 ′′ E), the Sea of Kumano , Japan, NW Pacific. Paratype: NMST-Ne-004, serial transverse sections from anterior tip to intestinal region (6 slides), everted proboscis (5 slides), and serial frontal sections of intestinal region (5 slides), posterior tip used for DNA extraction, rest of body fixed in Bouin’ s fluid and later preserved in 70% ethanol, female, collected on February 7 2022 by TM, at depths of 150 m, off Kumano (33 ◦ 52 ′ 18.4 ′′ N, 136 ◦ 11 ′ 54.8 ′′ E), the Sea of Kumano, Japan, NW Pacific .

Diagnosis. Same as for generic diagnosis.

Description. External morphology: body 0.8 cm in length and 0.1 cm in width in male (holotype); 3.2 cm in length and 0.7 cm in width in female (paratype) ( Fig. 2A ); single median brownish-red stripe extending from transverse band in head region to posterior tip of body ( Fig. 2A, D ). Four eyes present ( Fig. 2B, D ). Head demarcated from body, anteriorly pointed, and diamond shaped ( Fig. 2A ); brownish-red-colored transverse band present between head and trunk (= dorsally between anterior and posterior cephalic furrows ( Fig. 2B ) while ventrally overlapped with posterior cephalic furrows ( Fig. 2C )). Anterior cephalic furrows not meeting mid-dorsally, each slightly curving posteriorly in lateral margin ( Fig. 2B ); ventrally transverse, meeting at mid-line without reaching to proboscis pore ( Fig. 2C ). Posterior cephalic furrows meeting at mid-line both dorsally ( Fig. 2B ) and ventrally ( Fig. 2C ); gently V-shaped in dorsal surface ( Fig. 2B ) and steeply A-shaped in ventral surface ( Fig. 2C ). Blood not red, probably uncolored. Cerebral ganglia red colored in squeezed preparation ( Fig. 2D ), laterally leading from cerebral organs via cerebral canals ( Fig. 2E ). Intestinal caecum with lateral diverticula, anteriorly not reaching cerebral ganglia ( Fig. 2D ); lateral diverticula of intestinal caecum and intestinal diverticula deeply branched ( Fig. 2D ). Pinkish ovary or pale testes visible thorough body wall ( Fig. 2A ).

Internal morphology: Internal morphology. Epithelium 45–60 μm thick, with red-stained acidophilic gland cells in precerebal region ( Fig. 3A ) and with yellow- and red-stained gland cells in foregut region ( Fig. 3B ). Dermis up to 20 μm, cup-shaped in precerebal region ( Fig. 3A ) and flattened in foregut region ( Fig. 3B ). Cephalic glands anteriorly with red-stained acidophilic cells ( Fig. 3D and E ) and posteriorly replaced with blue-stained basophilic cells ( Fig. 3G ). Body-wall musculature with outer longitudinal and inner circular muscles ( Fig. 3A and B ). Some diagonal muscle fibers present as a lattice between outer circular and inner longitudinal muscle layers ( Fig. 3C ). Dorsoventral muscles present between intestinal diverticula ( Fig. 3L ). Precerebral vessel bifurcated ( Fig. 3E and F ). Mid-dorsal vessel without protruding into rhynchocoel ( Fig. 3H ). Oesphagus short, replaced with stomach in precerebral region (around cerebral organ opening) ( Fig. 4 ); stomach with ciliated cells, well-developed basophilic glands, and acidophilic glands ( Fig. 3G ). Intestinal caecum developed beneath pylorus, 2 or 3 pairs of lateral diverticula deeply branched, not reaching brain region anteriorly ( Fig. 3G ).

Proboscis pore subterminal, opening ventrally ( Fig. 2B ). Rhynchocoel short, less than half of body length ( Fig. 2D ). Rhynchocoel musculature with outer circular and inner longitudinal muscles ( Fig. 3L ). Proboscis epithelium with developed papillae up to 70 μm thick, basophilic, and acidophilic glands ( Fig. 3I ); outer circular, middle longitudinal, and inner circular muscles ( Fig. 3I ); 8 proboscis nerves ( Fig. 3I ). Stylet basis oval, 22 μm in length and 13.0 μm in maximum width; central stylet smooth, 26.4 μm in length; stylet-to-basis-length ratio 1.20 ( Fig. 2E ). Two accessory stylet pouches present, each containing 2–3 stylets.

A single frontal organ present ( Fig. 3D ). Cerebral organs opening ventrolaterally; cerebral canals without branching and replaced with acidophilic glands anterior to precerebral septum ( Fig. 4 ). Brain with outer neurilemma ( Fig. 3J ); glomerular structures present in dorsal cerebral ganglia ( Fig. 3J ). Lateral nerves without accessory nerves ( Fig. 3K ).

Nephridial tubules convoluted and opening dorsolaterally at intestinal caecum ( Fig. 3K ). Each ovary containing as many as 10 oocytes; each oocyte containing a germinal vesicle; oocyte 150–200 μm in diameter ( Fig. 3L and M ).

Etymology. The specific name is derived from a composite of the Latin ruber (= red coloured) + linea (= line), referring to the mid-dorsal stripe of the present species.

Type locality and distribution. The present species was found from the type locality, off Owase to Kumano, the Sea of Kumano, Japan .

2. Molecular phylogeny and interspecific genetic distances

Our molecular phylogenetic analyses indicated that R. rublinea gen. et sp. nov. constituted a clade together with Monostilifera sp. Genroku Seamount collected from a gentle slope of Genroku Seamount at a depth of 2084 m along the Nishi-Shichito ridge, Japan, with 72% bootstrap value and 0.99 posterior probability ( Fig. 5 ). Although R. rublinea appeared to be included in the clade formed by Eumonostilifera sp. Vema2B (Vema Fracture Zone), Monostilifera sp. Genroku Seamount ( Japan), Kurilonemertes dilutebasisae ( Kulikova, 1987) , Kurilonemertes papilliformis ( Korotkevitsch, 1977) ( Russia), Kurilonemertes phyllospadicola ( Stricker, 1985) ( Canada), Pseudotetrastemma sp. ( Russia), and Tetrastemma bilineatum Coe, 1904 ( USA), phylogenetic relationships within the clade were not well resolved except for monophyly of Kurilonemertes Chernyshev, 1993 .

An uncorrected p- distance based on 635 bp of COI was 11.9% between R. rublinea gen. et sp. nov. and Monostilifera sp. Genroku Seamount.

Figures

Fig. 2. Rhombonemertes rublinea gen. et sp. nov, living specimens. (A) Whole body, dorsal view, holotype (male) and paratype (female). (B) Magnification of head, dorsal view, holotype. (C) Magnification of head, ventral view, paratype. (D) Microphotograph of a living specimen, squeezed with a coverslip after anesthetization, holotype. (E) Magnification of stylet apparatus in everted proboscis, a black arrowhead pointing to a central stylet and white arrowheads to accessory stylet pouches. Abbreviations: af, anterior cephalic furrows; cc, cerebral canal, cg, cerebral ganglia; co, cerebral organ; ic, intestinal caecum; id, intestinal diverticulum; pb, proboscis; pf, posterior cephalic furrows; pp, proboscis pore; rc, rhynchocoel, te, testis. Scale bars: 5 mm (A), 500 μm (B, C), 750 μm (D), 100 μm (E).

Fig. 3. Rhombonemertes rublinea gen. et sp. nov. (paratype). Photomicrographs of transverse (A, B, D–M) and frontal (C) sections. (A) Body wall in precerebral region; (B) Body wall in foregut region; (C) diagonal muscle fibers (arrowheads) between outer circular and inner longitudinal muscle layers of body wall, intestinal region; (D) frontal organ; (E, F) precerebral vessels (arrowheads); (G) cephalic glands; (H) mid-dorsal vessel (arrowhead); (I) proboscis, arrowheads pointing to proboscis nerves; (J) brain; (K) nephridial canals (arrowhead); (L) rhynchocoel musculature and dorsoventral muscles; (M) ovaries. Abbreviations: br, brain; co, cerebral organ; dvm, dorso-ventral muscle of body wall; ep, epidermis; ic, intestinal caecum; ilm, body-wall inner longitudinal muscle; ocm, body-wall outer circular muscle; pe, proboscis epithelium; pic, proboscis inner circular muscle; pml, proboscis middle longitudinal muscle; poc, proboscis outer circular muscle; py, pylorus, rcm, rhynchocoel circular muscle; rlm, rhynchocoel longitudinal muscle; ov, ovary. Scale bars: 45 μm (A, B); 50 μm (C–E); 100 μm (F–K), 75 μm (L, M).

Fig. 4. Rhombonemertes rublinea gen. et sp. nov. (paratype). Photomicrographs of serial transverse sections, showing cerebral organ ventrolaterally opening and replaced with acidophilic glands anterior to precerebral septum. Abbreviations: oc, ocelli; ps, precerebral septum; st, stomach. Scale bar: 75 μm.

Fig. 5. A maximum likelihood (ML) tree based on concatenated sequences of 16S (400 bp), COI (602 bp), 18S (1750 bp), and 28S (1043 bp). Numbers near each node are support values generated by a separate partitioned ML bootstrap analysis with 1000 replicates and posterior probability by the BI analyses. Solid triangles indicate full support values (100% of bootstrap value/1.00 of posterior probability).