Prosthiostomum hibana, Tsuyuki & Kohtsuka & Kajihara, 2021

Tsuyuki, Aoi, Kohtsuka, Hisanori & Kajihara, Hiroshi, 2021, Fig. 4. A in Saphonecrus globosus Schweger and Tang 2015, Zoological Studies (Zool. Stud.) 60 (29), pp. 1-20 : 14-16

Prosthiostomum vulgare Kato, 1938b ( Fig. 6 )

Prosthiostomum vulgaris [sic] Kato, 1938b: 589–590, pl. 39, figs. 3–4; Kato 1938a: 578; Kato 1944: 308; Faubel 1984: 232; Prudhoe 1985: 192; Hagiya and Gamo 1992: 18, pl. 1, fig. 10, pl. 2, fig. 10; Tsunashima et al. 2017: fig. 2B.

Prosthiostomum siphunculus – Yeri and Kaburaki 1918: 41, pl. 2, fig. 13; Kato 1937a: 230.

Material examined: Three specimens ( ICHUM 6036, 3 slides; ICHUM 6154, 4 slides; ICHUM 6155, 5 slides), all collected by T. Miura, K. Oguchi, and H. Kohtsuka in Arai-hama (35.1609°N, 139.6105°E), Misaki, Kanagawa, Japan, on March 25, 2019.

Type locality: Yuzaki, Shirahama, Wakayama, Japan.

Description: Body elongated, tapered posteriorly, 6.8–8.8 mm long and 1.4–2.1 mm wide at its widest point when slightly contracted while alive (n = 3); anterior margin rounded ( Fig. 6A–D ). Tentacles absent. Dorsal surface smooth, buffy; cinnamon pigments medially abundant, forming wide midline ( Fig. 6A ). Ventral surface translucent, without color pattern ( Fig. 6B ). Pair of cerebral-eyespot clusters, each consisting of seven eyespots (n = 3); each cluster forming antero-posteriorly elongated line; anterior end of clusters located at distance of 0.58 mm posterior to anterior margin of body ( Fig. 6C ). Marginal eyespots distributed along frontal margin rather irregularly but largely arranged into two or three rows, extending backward to half position of brain ( Fig. 6C ). One pair of ventral eyespots present near front end of brain ( Fig. 6D ). Anterior branch of main intestine extending anterior to brain. Plicated pharynx tubular in shape, 3.0 mm in length (about one-third of body), located in anterior half of body ( Fig. 6B ). Mouth situated at distance of 0.81 mm posterior to anterior margin of body ( Fig. 6B ). Male copulatory apparatus consisting of large seminal vesicle, pair of prostatic vesicles, and armed penis papilla, located immediately posterior to pharyngeal pocket ( Fig. 6E–H ). Pair of spermiducal vesicles forming single row on each side of midline, separately entering into seminal vesicle at point being close to proximal end of ejaculatory duct ( Fig. 6E, G ). Ejaculatory duct with thick muscular layer, entering penis papilla. Prostatic ducts with thin muscular layer, connected to ejaculatory duct separately posterior to proximal end of penis papilla. Pair of spherical prostatic vesicles coated with 0.03-mm-thick, non-nucleated muscular wall, located on both sides of ejaculatory duct ( Fig. 6E, G, H ). Seminal vesicle oval, coated with 0.009-mm-thin muscular wall ( Fig. 6G, H ). Seminal vesicle (long axis 0.17 mm, short axis 0.09 mm) twice as large as prostatic vesicle (0.09 mm in diameter) ( Fig. 6E ) (n = 1). Penis papilla armed with pointed tubular stylet, enclosed in penis pouch, protruding into male atrium ( Fig. 6F ). Male atrium elongated, lined with ciliated and muscularized epithelium ( Fig. 6H ). Female copulatory apparatus immature; only female gonopore developed, located at distance of 0.20 mm behind male gonopore ( Fig. 6E, H ). Sucker large (0.21 mm in diameter; n = 1), situated immediately (0.19 mm in length; n = 1) behind female gonopore, at distance of 3.47 mm anterior to posterior margin of body (n = 1) ( Fig. 6E, H ).

Distribution: This species was confirmed along Japanese coasts, from the Noto Peninsula of Honshu Island to the southwestern Kyushu: Nozaki, Noto, Ishikawa; Misaki, Kanagawa; Manazuru, Kanagawa; Suzaki, Shimoda, Shizuoka; Suga-shima, Mie; Shirahama, Wakayama; and Tomioka, Amakusa, Kumamoto.

Habitat: The information about habitats of this species was not mentioned in Yeri and Kaburaki (1918), Kato (1937a 1938a b), or Hagiya and Gamo (1992). Our specimens were collected from branching coralline algae Corallinales spp. in Misaki, Kanagawa, Japan.

Sequence: Partial 1008-bp 28S rRNA ( LC 625891) and 585-bp COI ( LC 625898) gene sequences from ICHUM 6036.

Remarks: Kato (1938b) originally described this species from Shirahama, Wakayama, Japan. He also pointed out that the specimens from Misaki identified as P. siphunculus by Yeri and Kaburaki (1918) should represent P. vulgare , assuming that these would possess a pair of spermiducal vesicles that open into the seminal vesicle at its anterior part near the ejaculatory duct. With this character, Kato (1938b) speculated that P. vulgare could be differentiated from P. siphunculus . Our specimens are consistent with the original description by Kato (1938b) in this characteristic position of the junction of the spermiducal vesicles into the seminal vesicle ( Fig. 6E, G ) in addition to the body coloration and the arrangement of the cerebral-eyespot clusters. We were not able to compare our specimens with Yeri and Kaburaki’s (1918) and Kato’s (1937a 1938a b) specimens, which had been lost ( Kawakatsu 2004).

Molecular phylogeny

The resulting tree ( Fig. 7 ) showed the genus Prosthiostomum to be monophyletic (with 76% bootstrap [BS] support), with P. lobatum Pearse, 1938 being sister to all the other species of the genus included in this analysis. All remaining Prosthiostomum species except for P. lobatum formed a clade supported with a 90% BS value. Furthermore, except for the unidentified Prosthiostomum in Litvaitis et al. (2019), the remaining Prosthiostomum clade had 92% BS support. Included in this latter clade were all the species for which sequences were generated de novo in this study, i.e., P. auratum , P. grande , P. hibana sp. n., P. cf. ostreae , P. torquatum , and P. vulgare . Euprosthiostomum mortenseni ’s position as sister to the Prosthiostomum clade did not receive high nodal support (76% BS value), while all the Enchiridium species included in the analysis formed a highly supported clade (96% BS value).

Figures

Fig. 6. Prosthiostomum vulgare Kato, 1938b, ICHUM 6036 (A, C), ICHUM 6154 (B, D, H), ICHUM 6155 (E–G); photographs taken in life (A– D), schematic diagram (E), and photomicrographs of sagittal sections (anterior to the left) (F–H); A, entire body, dorsal view; B, entire body, ventral view; C, magnification of head, dorsal view; D, magnification of head, ventral view, showing ventral eyespots (arrowheads); E, copulatory complex and sucker; F, stylet; G, male copulatory apparatus; H, copulatory complex and sucker. Abbreviations: ce, cerebral eyespots; fg, female gonopore; it, intestine; ma, male atrium; me, marginal eyespots; mg, male gonopore; mo, mouth; ph, pharynx; pv, prostatic vesicle; spv, spermiducal vesicle; st, stylet; su, sucker; sv, seminal vesicle. Scale bars: A, B = 1 mm; C, D = 0.5 mm; E, G, H = 300 µm; F = 100 µm.

Fig. 7. Maximum likelihood phylogenetic tree based on the 28S rRNA (939 bp) and COI (585 bp) gene sequences. Numbers near nodes are the bootstrap values (≥ 70) (%). The names of species for which morphological description are provided in this study are indicated in boldface.