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 : 5-9

Prosthiostomum auratum Kato, 1937b ( Fig. 1 )

Prosthiostomum auratum Kato, 1937b: 363–364 , pl. 22, fig. 8, text-figs. 23–24; Kato 1938a: 572; Kato 1938b: 589, pl. 39, fig. 7; Kato 1939b: 152; Kato 1944: 307; Prudhoe 1985: 191; Hagiya and Gamo 1992: 18, pl. 1, fig. 9, pl. 2, fig. 9.

Euprosthiostomum auratum – Faubel 1984: 234.

Material examined: 10 specimens (2 series of sagittal sections [ ICHUM 6149, 6150]; 8 unsectioned specimens [AT2019033110, 2019022104– 2019022110]), all collected by T. Miura, K. Oguchi, and H. Kohtsuka in Arai-hama (35.1609°N, 139.6105°E), Misaki, Kanagawa, Japan. ICHUM 6149, 5 slides, March 25, 2019; ICHUM 6150, 4 slides, March 25, 2019; AT2019033110, 70% ethanol, March 25, 2019; AT2019022104–AT2019022110, 70% ethanol, February 19, 2019.

Type locality: Misaki, Kanagawa, Japan.

Description: Body elongated, tapered posteriorly, 7.3–12.0 mm long and 2.3–3.4 mm wide at its widest point while alive (n = 10); anterior margin rounded; mid-point of posterior margin acute. Tentacles absent. Dorsal surface smooth, uniformly golden-yellow except cerebral-eyespot area; yellowish in color, a little darker along midline; a few reddish-brown spots present in front of brain in one individual ( Fig. 1A ). Ventral surface translucent without color pattern ( Fig. 1B, C ). Pair of linear cerebral-eyespot clusters, each consisting of five to 10 eyespots (n = 10); anterior end of cluster located at distance of 0.80 mm posterior to anterior margin of body ( Fig. 1D ). About 12 marginal eyespots (n = 10) arranged in single row along frontal margin, extending anterior to brain ( Fig. 1D, E ). One pair of ventral eyespots present near front end of brain ( Fig. 1E ). Anterior branch of main intestine short, extending to position 0.33 mm posterior from anterior margin of body ( Fig. 1F, G ). Plicated pharynx tubular in shape, 1.46 mm in length (about one-third of body), located in anterior half of body ( Fig. 1B, F ). Mouth situated at anterior end of pharynx, located at 1.10 mm posterior from anterior margin of body ( Fig. 1B, F ). Male copulatory apparatus consisting of large seminal vesicle, pair of prostatic vesicles, and armed penis papilla, located immediately posterior to pharyngeal pocket ( Fig. 1F ). Pair of spermiducal vesicles forming single row on each side of midline, separately entering into seminal vesicle laterally ( Fig. 1C, F ). Ejaculatory duct with thick muscular layer, entering penis papilla. Prostatic ducts with thin muscular layer, separately connected to ejaculatory duct behind proximal end of penis papilla. Pair of spherical prostatic vesicles, each coated with 0.05-mm-thick, non-nucleated muscular wall, located on each side of ejaculatory duct. Seminal vesicle oval, coated with 0.04-mm-thick muscular wall. Diameter of prostatic vesicle (0.16 mm) as long as dorsoventral diameter of seminal vesicle (0.13 mm) (n = 1). Penis papilla armed with pointed tubular stylet (0.08 mm in length; n = 1), enclosed in penis pouch, protruding into male atrium ( Fig. 1F, H ). Male atrium elongated anteriorly from gonopore to penis pouch (3.1 mm in length; n = 1). Female gonopore situated at 0.25 mm behind male gonopore (n = 1) ( Fig. 1B ). Female copulatory apparatus posterior to male reproductive system. Female gonopore leading to vagina across cement pouch; proximal end of vagina anteriorly curved ( Fig. 1F ). Cement glands developed, concentrated around vagina and releasing their contents in cement pouch ( Fig. 1H ). Oviducts not observed. Sucker large (0.46 mm in diameter; n = 1), situated at center of body (0.32 mm length from female atrium; 4.10 mm length from posterior extremity; n = 1) ( Fig. 1B ).

Distribution: So far, this species has only been confirmed along Japanese coasts, from the northmost Honshu Island to the southwestern Kyushu: Yuno-shima near Asamushi, Aomori; Ohtsuchi, Iwate; Nanao, Noto, Ishikawa; Misaki, Kanagawa; Manazuru, Kanagawa; Suzaki, Shimoda, Shizuoka; Shirahama, Wakayama; and Tomioka, Amakusa, Kumamoto.

Habitat: In the original description, this species was found on the surfgrass Phyllospadix in Misaki ( Kato 1937b). In Shirahama, numerous specimens were obtained under stones ( Kato 1938b). Our specimens were collected from the seaweed Corallinales spp. and holdfasts of the kelp Eisenia bicyclis subtidally in Misaki.

Sequences: Partial 28 S rRNA (1010 bp) and COI (585 bp) sequences from two individuals. LC625886 (28 S rRNA) and LC625892 ( COI) from AT2019033110; LC625893 ( COI) from ICHUM 6149.

Remarks: Although P. auratum was once placed in Euprosthiostomum ( Faubel 1984) , our morphological examination of the present topotypes confirmed that it is part of Prosthiostomum , primarily due to the presence of a frontal branch of the main intestine, a character state that was not mentioned in the original description by Kato (1937b). Our specimens are consistent with the original description in that i) the dorsal body is colored uniformly golden-yellow, ii) each cerebral-eyespot cluster is formed in a linear shape, and iii) a pair of prostatic vesicles are moderately large. No mention was made as to the ventral eyespots in the original description, but these were clearly present in a photograph of a specimen from Shirahama ( Kato 1938b, pl. 39, fig. 7) as well as in our specimens ( Fig. 1E ).

As was shown in P. auratum based on topotypes, there is room for examination in the adequacy of classifying the other five species, P. angustum Bock, 1913 , P. bellum Kato, 1939a , P. laetum Kato, 1938a , P. matarazzoi Marcus, 1950 , and P. pulchrum Bock, 1913 , in the genus Euprosthiostomum , because these were listed under the latter genus by Faubel (1984) without sound basis. The genus Euprosthiostomum was established by Bock (1925) based on E. adhaerens Bock, 1925 , which was characterized by i) the location of the sucker relatively near the caudal end of the body and ii) the absence of a frontal median branch of the intestine ( Bock 1925; Marcus 1948; Hyman 1953). Subsequently, E. viscosum Palombi, 1936 , E. mortenseni Marcus, 1948 , and E. pakium Du Bois-Reymond Marcus and Marcus, 1968 were established in this genus. Also, P. molle Freeman, 1930 was transferred to Euprosthiostomum by Hyman (1953). Later, Faubel (1984) proposed the presence/ absence of the frontal median branch of the intestine as a determination key to distinguish Prosthiostomum (present) from Euprosthiostomum (absent). As a result, Faubel (1984) transferred P. angustum , P. auratum , P. bellum , P. exiguum Hyman, 1959 , P. laetum , P. matarazzoi (= Lurymare matarazzoi ), and P. pulchrum to Euprosthiostomum . In fact, however, in the original descriptions of P. angustum , P. auratum , P. bellum , P. laetum , P. matarazzoi , and P. pulchrum , the presence/ absence of this branch was not clearly shown ( Bock 1913; Kato 1937b 1938b 1939a; Marcus 1950), although Faubel (1984) apparently assumed as if the frontal median branch was absent in these species. In the same work, Faubel (1984) categorized those species for which the presence/absence of the frontal branch was unknown and placed them in Prosthiostomum . Among them, P. matarazzoi was redescribed based on freshly collected material ( Bahia 2016); a lectotype was subsequently designated for this species ( Bahia and Schrödl 2018). Still, the presence/absence of the frontal branch in P. matarazzoi was not mentioned in these works ( Bahia 2016; Bahia and Schrödl 2018), although a common muscle bulb was confirmed to wrap up the prostatic and seminal vesicles, a character that was alleged to distinguish Lurymare from Prosthiostomum ( Faubel 1984) , but has been said to vary ontogenetically ( Prudhoe 1989).

Prosthiostomum hibana sp. n. Tsuyuki, Kohtsuka, and Kajihara [New Japanese name: hibana-hoso-hiramushi] ( Figs. 2–4 ) urn:lsid:zoobank.org:act:9E5FA6BC-F6A2-4FD8-9E11-42250240FE25

Material examined: Two specimens ( ICHUM 6147, holotype, 6 slides; ICHUM 6148, paratype, 4 slides), both 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.

Etymology: The new specific name hibana is a Japanese noun, meaning fire sparks. It was named after the dorsal color pattern of the orange maculae, which look like sparks flying.

Type locality: Arai-hama, Misaki, Kanagawa, Japan.

Diagnosis: Body elongated; anterior margin rounded; dorsal surface translucent, covered with numerous orange maculae, some of which being agglutinated and forming larger maculae; pair of linear cerebral-eyespot clusters composed of relatively few eyespots; 3–4 pairs of ventral eyespots, embedded in parenchyma; marginal eyespots distributed antero-ventrally; inner wall of male atrium deeply ruffled; lumen of seminal vesicle narrow and elongated in shape; sucker large, occupying about 3% of body length, situated on body center.

Description of holotype: Body elongated, tapered posteriorly, 14 mm long and 3 mm wide at its widest point while alive ( Fig. 2A ); anterior margin rounded; mid-point of posterior margin acute. Tentacles absent. Dorsal surface smooth, translucent, uniformly covered with numerous orange maculae, some of which being agglutinated and forming larger maculae; the larger maculae scattered throughout ( Fig. 2A ); orange pigments more abundant medially. Ventral surface translucent, without color pattern ( Fig. 2B, C ). Pair of cerebral-eyespot clusters, each consisting of nine (left) and eight (right) eyespots; each cluster forming an antero-posteriorly elongated, curved line; anterior end of clusters located at distance of 0.85 mm posterior to anterior margin of body ( Fig. 2D ). About 20 marginal eyespots distributed antero-ventrally in front of brain ( Fig. 2E, F ). Four pairs of ventral eyespots, embedded in parenchyma ( Fig. 2G ); four eyespots on each side arranged at corner of parallelogram ( Fig. 2E ). Intestine highly branched, spreading all over body; anterior branch of main intestine extending to position 0.4 mm posterior from anterior margin of body. Plicated pharynx tubular in shape, 4.1 mm in length (about two-sevenths of body), located in anterior half of body ( Fig. 2A, B ). Mouth situated at anterior end of pharynx, located at 1.04 mm posterior from anterior margin of body ( Fig. 2B ). Male gonopore, female gonopore, and sucker closely set on body center ( Fig. 2B, C ); distance between male and female gonopores being 0.34 mm; distance between female gonopore and sucker being 0.39 mm. Male copulatory apparatus consisting of large seminal vesicle, pair of prostatic vesicles, and armed penis papilla, located immediately posterior to pharyngeal pocket ( Fig. 3A ). Spermiducal vesicles forming single row on each side of midline, each running from posterior to anterior, then bending posteriorly and separately entering into seminal vesicle. Ejaculatory duct wide, with thick muscular layer, entering penis papilla. Prostatic ducts with muscular layer, connected to ejaculatory duct separately at proximal end of penis papilla. Pair of spherical prostatic vesicles coated within 0.05-mm-thick, non-nucleated muscular wall, located on both sides of ejaculatory duct ( Fig. 3A, B ). Seminal vesicle oval, coated with 0.11-mm-thick muscular wall; its lumen narrow and elongated in shape ( Fig. 3A, C ). Without common muscular bulb enclosing male copulatory apparatus. Seminal vesicle (long axis 0.34 mm, short axis 0.23 mm) more than twice as large as prostatic vesicle (0.14 mm in diameter) ( Fig. 3A, B ). Penis papilla armed with pointed tubular stylet (0.14 mm in length), enclosed in penis pouch, protruding into male atrium ( Fig. 3D ). Penis sheath present between penis pouch and male atrium ( Fig. 3A, B ). Male atrium elongated anteriorly from male gonopore to penis pouch (0.40 mm in length); inner wall deeply ruffled, lined with ciliated and muscularized epithelium ( Fig. 3A, B ). Immature female reproductive system immediately posterior to male copulatory apparatus. Female gonopore leading to vagina across cement pouch ( Fig. 3A, E ); proximal end of vagina anteriorly curved ( Fig. 3A ). Cement glands and oviducts undeveloped and not observed. Lang’s vesicle absent. Sucker large (0.40 mm in diameter; 2.9% of body length), situated immediately behind female gonopore ( Fig. 3E ), at 4.2 mm anterior from posterior margin of body.

Description of paratype: Body 7.8 mm long and 2.9 mm wide at its widest point when slightly contracted while alive. Body coloration almost same as holotype. Pair of cerebral-eyespot clusters, each consisting of seven (left) and eight (right) eyespots ( Fig. 4A ). About 20 marginal eyespots, distributed ventrally along anterior margin ( Fig. 4B ). Ventral eyespots, 3–4 pairs in number, embedded in parenchyma ( Fig. 4B ). Frontal branch of main intestine extending anterior to brain. Pharynx 2.82 mm in length. Male and female reproductive systems undeveloped. Sucker large (0.20 mm in diameter; 2.6% of body length), situated on body center (3.6 mm anterior from posterior margin of body).

Distribution: So far only from the type locality, Misaki, Kanagawa, Japan.

Habitat: Among branching coralline algae Corallinales spp.

Sequences: Partial 28 S rRNA gene (1008 bp) and COI (585 bp) sequences from two individuals. LC625887 (28 S rRNA) and LC625894 ( COI) from the holotype ( ICHUM 6147); LC625888 (28 S rRNA) and LC625895 ( COI) from the paratype ( ICHUM 6148).

Remarks: Among ~60 species in Prosthiostomum , our new species is unique in having 3–4 pairs of ventral eyespots ( Figs. 2E and 4B ) and thus can easily be distinguished from the other congeners, where the ventral eyespots are mostly absent or at most single pair in number, if present. Only P. bellum has been known to possess two pairs of ventral eyespots ( Kato 1939a), but it is quite different from P. hibana sp. n. in the body coloration (white background with numerous brown spots scattered over the body in P. bellum ; translucent with orange maculae in P. hibana sp. n.) as well as the number of cerebral eyespots in each cluster (about 40 in P. bellum ; 7–9 in P. hibana sp. n.).

Nine other congeners are known to show a similar character state to that in P. hibana sp. n. pertaining to either dorsal coloration or cerebral-eyespot arrangement ( Table 2). Prosthiostomum capense Bock, 1931 , P. dohrnii Lang, 1884 , and P. grande resemble our new species in having yellow to orange maculae or spots scattered all over the body; P. dohrnii and P. grande are different from the new species in the number and distribution of the cerebral eyespots; P. capense is separated from P. hibana sp. n. by the size and position of the sucker (small, situated at four-fifths of the body in P. capense ; large, situated at the middle of the body in P. hibana sp. n.) ( Table 2). The five species P. auratum , P. cynarium Marcus, 1950 , P. purum Kato, 1937b , P. siphunculus , and P. vulgare Kato, 1937b have cerebral-eyespot arrangements similar to that in our new species, i.e., a pair of linear cerebral-eyespot clusters composed of relatively few (≤ 15) eyespots, but can be easily distinguished from P. hibana sp. n. by the dorsal coloration ( Table 2). Prosthiostomum parvicelis Hyman, 1939b also has this type of cerebral-eyespot arrangement; although the dorsal coloration is not known for this species, it can be distinguished from P. hibana sp. n. by the pyriform lumen of the seminal vesicle ( Hyman 1939b), whereas the seminal-vesicle lumen is narrow and elongated in P. hibana sp. n. ( Fig. 3A, C ).

Noticeably, in P. hibana sp. n., the inner wall of the male atrium is deeply ruffled ( Fig. 3A, B ). The morphology of the inner wall of the male atrium has so far attracted little attention as taxonomic features in Prosthiostomidae . This feature is not mentioned in the original descriptions or re-descriptions for most of the prosthiostomid species, but a slightly ruffled inner wall of the male atrium has been illustrated for P. gilvum Marcus, 1950 , P. latocelis Hyman, 1953 , and P. ostreae Kato, 1937b ( Kato 1937b; Marcus 1950; Hyman 1953).

Figures

Fig. 1. Prosthiostomum auratum Kato, 1937b; photographs taken in life (ICHUM 6150) (A–E), schematic diagram (F), and photomicrographs of sagittal sections (anterior to the left) (ICHUM 6149) (G, H); A, entire body, dorsal view; B, entire body, ventral view; C, magnification of the black edged area on B; D, magnification of head, dorsal view; E, magnification of head, ventral view, showing ventral eyespots (arrowheads); F, anterior half of the body, lateral view, anterior to the left; G, anterior end of body; H, middle portion of body, showing male and female copulatory apparatuses. Abbreviations: ab, anterior branch of main intestine; br, brain; ce, cerebral eyespots; cg, cement glands; 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; D, E = 100 µm; F–H = 300 µm.

Fig. 2. Prosthiostomum hibana sp. n., ICHUM 6147 (holotype); photographs taken in life (A–E) and photomicrographs showing eyespots observed in sagittal sections (anterior to the left) (F, G); A, entire body, dorsal view; B, entire body, ventral view; C, magnification of the white edged area on B; D, magnification of head, dorsal view; E, magnification of head, ventral view (ventral eyespots indicated by arrowheads); F, anterior portion of body, showing marginal eyespot; G, anterior portion of body, showing cerebral and frontal eyespots. Abbreviations: ce, cerebral eyespots; fg, female gonopore; me, marginal eyespot(s); mg, male gonopore; mo, mouth; ph, pharynx; su, sucker; ve, ventral eyespot. Scale bars: A = 5 mm, B = 1 mm, D– G = 500 µm.

Fig. 3. Prosthiostomum hibana sp. n., ICHUM 6147 (holotype); schematic diagram (A) and photomicrographs of sagittal sections (anterior to the left) (B–D); A, copulatory complex and sucker; B, male copulatory apparatus through male atrium lumen; C, male copulatory apparatus through seminal vesicle lumen, indicated by arrow; D, proximal portion of male atrium; E, female gonopore and sucker.Abbreviations: cp, cement pouch; ed, ejaculatory duct; fg, female gonopore; it, intestine; ma, male atrium; mg, male gonopore; ph, pharynx; pp, penis papilla; ps, penis sheath; pv, prostatic vesicle; spv, spermiducal vesicle; st, stylet; su, sucker; sv, seminal vesicle; va, vagina. Scale bars:A = 300 µm; B, C, E = 100 µm; D = 50 µm.

Fig. 4. Prosthiostomum hibana sp. n., ICHUM 6148 (paratype); photographs taken after being cleared in xylene; A, magnification of head, dorsal view; B, magnification of head, ventral view (ventral eyespots indicated by arrowheads). Abbreviations: ce, cerebral eyespots; me, marginal eyespots; mo, mouth. Scale bars: 500 µm.