Cerebratulus orochi Kajihara 2020, sp. nov.
Creators: name: Kajihara, Hiroshi
Publisher: name: Zenodo
Year: 2020
Subjects: Biodiversity
Taxonomy
Animalia
Nemertea
Anopla
Heteronemertea
Lineidae
Cerebratulus
Cerebratulus orochi

description: Cerebratulus orochi sp. nov.
[Japanese vernacular name: orochi-himomushi]
(Figs 1–6)
urn:lsid:zoobank.org:act: 4D83D504-9E2D-4782-9FF6-4C0B186D32F1
Cerebratulus marginatus: Yamaoka (1940), p. 222, pl. II, figs 6–8, text-fig. 9; Iwata (1954a), p. 14; Schwartz (2009), p. 79 [in part], figs 6E, 25E.
Heteronemertea sp. 13: Hiebert (2016), p. 266, figs 7.4, 7.5.
Diagnosis. A Cerebratulus with saddlebrown body colour with whitish lateral edges; several layers of diagonalmuscle meshwork coated with connective tissue, proximo-distally ranging from distal portion of body-wall outer longitudinal muscle layer to cutis-gland zone in precerebral and foregut regions; no connective tissue layer between cutis glands and body-wall outer longitudinal muscle layer; no diagonal muscle between body-wall outer longitudinal and middle circular muscle layers; heterotype proboscis with two muscle crosses; cephalic vascular system consisting of lateral and mid-dorsal vessels; sub-rhynchocoelic vessel possessing a pair of antero-lateral diverticula in post-cerebral, pre-oral region; no eyes; frontal organs present.
Material examined. Four specimens photographed, of which one was designated as the holotype; all from the type locality. Holotype: ICHUM 6078, 21 June 2019, collected by R. Rajagopal and R. Harada; serial transverse sections of the anterior end (from anterior tip to anterior foregut region), 6 µm, 80 slides, Mallory (except two, stained with HE); horizontal sections of the foregut region, 5 µm, 22 slides, Mallory; transverse sections of the intestinal region, 7 µm, 18 slides, Mallory; horizontal sections of the intestinal region, 5 µm, 35 slides, Mallory; transverse sections of the proboscis, 8 µm, 2 slides, HE. The other three specimens were collected in July 1999, May 2010, and June 2012; the 1999 specimen was studied by Schwartz (2009), the remaining two are kept in HK’s personal collection.
Type locality. About 43.0523°N, 144.8442°E, Shinryu, Akkeshi, Hokkaido, Japan; intertidal, muddy beach.
Sequences. LC538101, 16S, 504 bp; LC538102, COI, 639 bp; LC538103, 18S, 1682 bp; LC538104, 28S, 1282 bp; LC538105, H3, 331 bp.
Etymology. The specific name orochi is an indeclinable noun, taken from the Japanese vernacular name first appeared in Okuda (1947, p. 1472), referring to a giant serpent or an eight-headed dragon in Japanese mythology.
Description. External features. Holotype 32 cm in length, 1.3 cm in maximum width, saddlebrown in colour, with whitish lateral margins; latter becoming thinner in intestinal region while swimming. Head ovate, slightly pointed anteriorly, 7 mm in maximum width at middle portion of precerebral region, 6 mm in width at level of anterior end of mouth in anaesthetized state (Fig. 1A, B). Rhynchodaeum opening ventrally at anterior tip of head; body surface on periphery of rhynchodaeal pore tinged with olive colour (Fig. 1B, C). Lateral cephalic slits extending to level of anterior end of mouth. Mouth aperture 5 mm in antero-posterior length in anaesthetized state. Intestinal region flattened, with thin paler transverse lines encircling body, arranged at more-or-less regular intervals. Posterior end of body paler than anterior portion for about 1 cm; caudal cirrus about 1 mm long [indicating recent posterior regeneration; caudal cirrus in an intact specimen reached 15 mm]. Proboscis yellowish brown when everted in living state.
Body wall and musculature. Epidermis 20–30 µm tall in precerebral region, 30–40 µm tall in foregut region (Fig. 2A, B), and 40–50 µm tall in anterior intestinal region. Subepidermal muscles consisting of circular, diagonal, and longitudinal muscles in foregut region (Fig. 2C); no diagonal muscles found in intestinal region (Fig. 2D). Cutis glands chiefly acidophilic in foregut region (Fig. 2A, B), but predominantly basophilic in intestinal region (Fig. 3A); cutis glands not separated by connective-tissue layer from body-wall outer longitudinal muscle layer in any portion of body. Body-wall musculature consisting of outer longitudinal, middle circular, and inner longitudinal muscle layers. Bundles of diagonal muscles forming meshwork coated with connective tissue present as four to eight distinct layers, proximo-distally ranging from distal portion of body-wall outer longitudinal muscle layer to just above cutis-gland zone, antero-posteriorly distributed from precerebral region to foregut region. No diagonal muscle layer present between body-wall outer longitudinal muscle and middle circular muscle layers. Oblique-muscle meshwork present within outer portion of body-wall longitudinal muscle layer in precerebral region (Fig. 2E). Radial muscles running through body-wall outer longitudinal muscle layer in foregut region (Fig. 2A). Dorsoventral muscles powerfully developed between intestinal diverticula and gonads through body-wall inner longitudinal muscle layer (Fig. 3A, B); dorsoventral muscles also present in body-wall outer longitudinal muscle layer, but sparsely (Fig. 3B). Body-wall middle circular muscle layer not apposed to rhynchocoel outer circular muscle, but always separated from latter by body-wall inner longitudinal muscles in foregut and intestinal regions. Muscle cross between bodywall middle circular layer and rhynchocoel outer circular layer not found. Epidermis on lateral edge of body in intestinal region indented proximally in non-swimming state (Fig. 3B).
Proboscis and rhynchocoel. Rhynchodaeum anteriorly ciliated, not glandular throughout its length. Rhynchodaeal sphincter present just in front of proboscis insertion, latter situated in front of brain. Proboscis heterotype, its middle portion consisting of glandular epithelium, outer longitudinal muscle layer, neuroplexus, circular muscle layer, diagonal muscle layer, inner longitudinal muscle layer, connective tissue, and endothelial lining containing circular muscles (Fig. 3C); two muscle crosses present (Fig. 3D). Pseudocnidae about 5 µm in length, arranged as bundles, each containing about 15 in number (Fig. 3E) on top of proboscis epithelium mostly on same side as small-sized muscle cross. Rhynchocoel under vascular plug anteriorly interwoven with body-wall musculature (Fig. 4 A–F); posteriorly, rhynchocoel wall consisting of separate inner longitudinal and outer circular muscle layers in foregut region (Fig. 4G, H).
Vascular system. Cephalic lacuna beginning above rhynchodaeal opening, posteriorly soon divided into three vessels, each assuming radial, dendritic, or amoeboid shape in cross section, lying dorsal and lateral to rhynchodaeum (Fig. 2E); lateral vessels often further subdivided by muscle bundles; just anterior to proboscis insertion, dorsal vessel fuses with lateral ones and disappears. After passing through cerebral ring, two lateral cephalic vessels fuse ventrally with each other to form U-shaped vessel, from which mid-dorsal vessel arises to enter rhynchocoel. U-shaped vessel soon divides into two, upper lateral vessels and single, median, sub-rhynchocoelic vessel (Figs 4A, 5A). Posteriorly, median sub-rhynchocoelic vessel flanked with anteriorly blind-ending vessels on both sides for about 100 µm in length (Fig. 4B) before fused with each other (Fig. 4C). Further posteriorly, median sub-rhynchocoelic vessel horizontally forked into two, further posteriorly running as lower lateral vessels (Fig. 4D). Upper lateral vessels surrounding posterior margins of cerebral organs (Fig. 4A, B), posteriorly connecting for sometimes to lower lateral vessels on each side immediately anterior to mouth opening (Fig. 4E). Further posteriorly, lower lateral vessels subdivided to form vascular plexus surrounding foregut. Blood corpuscles present, about 10 µm in diameter (Fig. 4G, H).
Nervous system. Conforming to general heteronemertean nervous system (cf. Beckers 2015). Brain and lateral nerves with both inner and outer neurilemmata (Fig. 6A); glial cells abundant between ganglionic cells and outer neurilemma. Dorsal and ventral commissures 90 µm and 120 µm in thickness, respectively, in terms of fibrous tissues. Bürger type I cells with orange-G staining nucleus and little cytoplasm, most frequently found above neuropil of dorsal lobe (Fig. 5A, B). Bürger type II with nucleus having various staining affinities also frequently found above neuropil of dorsal lobe as well as ventral to neuropil of ventral lobe (Fig. 5 A–C). Bürger type III mainly distributed medially to neuropil of dorsal lobe as well as medioventrally to neuropil of ventral lobe; cytoplasm usually staining greyish with Mallory’s staining (Fig. 5C), but occasionally reddish (Fig. 5B). Neurochord cell, up to 70 µm in long axis, present medially to neuropil of ventral lobe (Fig. 5C). Neurochord (i.e., innervation from neurochord cell) running anterolaterally for 170 µm within neuropil of ventral ganglion, but its ultimate fate not traceable.
Lateral nerve with longitudinal muscle fibres in ganglionic portion abutting laterally on inner neurilemma of fibrous core (Fig. 6B). No neurochord found in lateral nerve. Radial muscles from body-wall middle circular muscle piercing upper ganglionic portion of lateral nerve (Fig. 6B).
Digestive system. Conforming to normal heteronemertean digestive system. Longitudinal muscle plate present below rhynchocoel in foregut region; transverse muscles present below longitudinal muscle plate in foregut region (Fig. 4G, H). Somatic muscles consisting of isolated longitudinal and circular fibres laterally and ventrally to foregut (Fig. 6C).
Sensory system. Frontal organs present, three in number (Fig. 5D). Basophilic subepidermal glandular cells distributed from rear of frontal organs to short distance posterior to rhynchodaeal opening; glandular contents not discharged via frontal organs. Epidermis on lateral cephalic slits not fixed well in histological preparation; epidermal specialization on cephalic slits not observable. Cerebral sensory organs posteriorly bathed in upper lateral vessels (Fig. 4A). No eyes.
Excretory system. Collecting tubules present on distal wall of vascular plexus around foregut (Fig. 6C). Nephridiopores not included in serial sections.
Reproductive system. Holotype female. Ovaries alternating with intestinal lateral diverticula (Fig. 3A) with interval being about 3 ovaries per 1 mm antero-posterior length; one randomly chosen ovary contained 62 oocytes. Oocytes teardrop-shaped, with mean (standard deviation) size being 122 (15) µm in long-axis length and 67 (10) µm in short-axis length (n = 10). Germinal vesicle with mean (standard deviation) diameter being 45 (2) µm (n = 10).
Phylogeny and species delimitation. The resulting maximum-likelihood and Bayesian trees based on the multi-locus dataset were identical in topology. In the resulting trees (Fig. 7), Cerebratulus orochi sp. nov. was sister to C. cf. marginatus from the US Pacific coast; these two species differed by 0.061 uncorrected p -distance and 0.064 K2P in terms of 16S (506 bp), and 0.173 p -distance and 0.178 K2P in terms of COI (639 bp).
*Conspecific with Cerebratulus marginatus of Andrade et al. (2012).
The MegaBLAST search at the NCBI website with the 16S sequence of C. orochi sp. nov. detected four spe-cies—all tentatively identified or unidentified—that showed less than 0.060 p -distance and 0.063 K2P to the query sequence (Table 2). Both the ABGD and PTP analyses based on the 16S dataset resulted in the same delimitation, in which C. orochi sp. nov. was conspecific with the Heteronemertea sp. 13 of Hiebert (2016) (Fig. 8). The same search and analyses with the COI dataset detected five species (Table 3, Fig. 9), where C. orochi sp. nov. was most closely related to Cerebratulus sp. “spade head” of Hiebert (2016).
description: Published as part of Kajihara, Hiroshi, 2020, Redescription of Cerebratulus marginatus auct. (Nemertea: Pilidiophora) from Hokkaido, Japan, as a new species, pp. 295-315 in Zootaxa 4819 (2) on pages 298-306, DOI: 10.11646/zootaxa.4819.2.4, http://zenodo.org/record/4396930