Enchiridium daidai, Tsuyuki & Kajihara, 2020

Tsuyuki, Aoi & Kajihara, Hiroshi, 2020, A giant new species of Enchiridium (Polycladida, Prosthiostomidae) from southwestern Japan, ZooKeys 918, pp. 15-28 : 19-23

Enchiridium daidai sp. nov. New Japanese name: daidai-hoso-hiramushi Figures 2 , 3 , 4

Etymology.

The new specific name daidai is a Japanese noun, meaning the color orange. It was named after the thin marginal orange line surrounding the entire dorsal fringe.

Material examined.

Three specimens, all collected by A. Tsuyuki. Holotype: ICHUM 5993, sagittal sections through reproductive structures (22 slides), and the rest of the body, unsectioned, preserved in 70% ethanol, collected at 13-14 m depth off the coast of Bonomisaki (31.2542N, 130.2150E), Kagoshima, Japan, on 26 July 2018. Paratypes: ICHUM 5994, sagittal sections through head to reproductive structures (9 slides); ICHUM 5995, cross sections through reproductive structures (21 slides); both collected at 5 m depth in Nago (26.6013N, 127.9137E), Okinawa, Japan, on 22 May 2019.

Type locality.

Off the coast of Bonomisaki (31.2542N, 130.2150E), Kagoshima, Japan.

Description.

Body elongated, tapered posteriorly, 28-77 mm long (77 mm in holotype) and 4.6-14 mm maximum width (14 mm in holotype) in living state (Fig. 2A ); anterior margin rounded; mid-point of posterior margin acute. Tentacles absent. Dorsal surface smooth, translucent, fringed with thin marginal orange line (Fig. 2A ). Ventral surface translucent, without color pattern. Pair of cerebral-eyespot clusters, each consisting of 20-52 eyespots (left 20 and right 23 in holotype); each cluster of an antero-posteriorly elongated spindle shape (Fig. 2B ). Marginal-eyespot clusters forming single marginal band, extending to position of mouth (about anterior one-eighth of body) along margins on both sides; marginal eyespots abundant along anterior margin, diminishing posteriorly (Fig. 2B ). Ventral eyespots absent. Intestine highly branched, spreading all over body. Plicated pharynx tubular in shape, about one-fifth of body length, located in anterior one-third of body (Fig. 2A ). Oral pore situated at anterior end of pharynx, behind brain. Male gonopore and female gonopore closely set, both situated behind posterior end of pharynx. Male copulatory apparatus consisting of large seminal vesicle, pair of prostatic vesicles, and armed penis papilla (Fig. 3A ). Antero-posterior length of seminal vesicle more than twice as long as diameter of each prostatic vesicle. Spermiducal vesicles forming single row on each side of midline, separately entering into seminal vesicle. Ejaculatory duct 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 thin non-nucleated muscular wall, arranged anterodorsally to ejaculatory duct. Common muscular sheath enclosing two prostatic vesicles (Fig. 3B ). Seminal vesicle oval, coated with thick muscular wall, narrowing anteriorly and forming ejaculatory duct; latter almost immediately penetrating common muscular sheath (Fig. 3C ). Penis papilla armed with pointed tubular stylet, enclosed in penis pouch, protruding into male atrium (Fig. 3C ). Male atrium elongated anteriorly, lined with ciliated, muscularized epithelium (Fig. 3B ). Female reproductive system immediately posterior to male reproductive system. Cement glands numerous, concentrated around vagina and releasing their contents in cement pouch (Fig. 3D ). Vagina curving anteriorly, leading to two narrow lateral branches of uteri. Each branch of uteri turning laterally and then running backwards. Lang’s vesicle absent. Sucker set on body center (Fig. 2A ).

Habitat.

Subtidal (5-14 m depth), under rocks.

Variation.

Specimens from Kagoshima and Okinawa differed in body size. The holotype from Kagoshima was 77 mm long and 15 mm wide, whereas the paratype specimens from Okinawa were 28-37 mm long and 4.6-7.4 mm wide (Fig. 4 ).

Diagnosis.

Body elongated, usually rounded anteriorly; dorsal surface translucent, fringed by a thin marginal orange line; marginal eyespots present only anteriorly; plicated pharynx tubular in shape, about one-fifth of body length; pair of prostatic vesicles bound by common muscular sheath, the latter penetrated by ejaculatory duct.

Sequences.

Partial COI (585 bp) and 28S rDNA (1017 bp) sequences from three individuals: LC504240 (COI), LC504235 (28S rDNA) from ICHUM 5993 (holotype); LC504238 (COI), LC504236 (28S rDNA) from ICHUM 5994 (paratype); LC504239 (COI), LC504237 (28S rDNA) from ICHUM 5995 (paratype).

Molecular phylogeny and genetic distances.

In the phylogenetic tree, Enchiridium daidai sp. nov. was nested in a clade composed of Enchiridium species (Fig. 5 ). The genetic distances (uncorrected p -values) for the COI sequences among three specimens of Enchiridium daidai sp. nov. were 0.002-0.012. Genetic distances between individuals from different localities (Kagoshima vs. Okinawa), 0.010-0.012, were larger than that between individuals from the same locality (Okinawa), 0.002.

Remarks.

In spite of the noticeable difference in body size, specimens from Kagoshima and Okinawa - all having reached sexual maturity - were identified as conspecific. They shared the following morphological characteristics: i) body dorsally fringed with a thin orange line, ii) marginal-eyespot band extending to the position of the mouth (about anterior one-eighth of the body), iii) two prostatic vesicles covered by a common muscle sheath, and iv) common muscle sheath penetrated by ejaculatory duct. In addition, the COI p -distances among the specimens, 0.002-0.012, fell in a range of intraspecific values, 0.000-0.020, which was observed in four species of the acotylean leptoplanoid Notocomplana ( Oya and Kajihara 2017), thus rendering sup port for our interpretation of conspecificity. Within Polycladida , remarkable intraspecific variation in body size has been reported for the acotylean stylochoid Planocera reticulata (Stimpson, 1855), which was recorded to vary by 10-80 mm in length and 6-45 mm in width ( Yeri and Kaburaki 1918). Among the cotylean Proshiostomidae, sexually matured individuals of Prosthiostomum cyclops (Verrill, 1901) have been reported to vary a great deal (> × 10) in size by locality: 75-90 mm long × 10-15 mm wide in the Bermuda Islands ( Verrill 1901), whereas 6.5 mm long × 1.7 mm wide in the islands of Bonaire and Klein Bonaire ( Du Bois-Reymond Marcus and Marcus 1968). These observations may imply that these polyclads undergo an indeterminate growth, in which growth is not terminated after reaching adulthood, although other factors - such as geographical and ecological ones - must also be taken into account.

As for the taxon concept of Enchiridium , our results did not show a compatibility to Bock’s (1913) original view on the genus. The genus Enchiridium was established by Bock (1913) for E. periommatum based on two characteristics: i) two prostatic vesicles enclosed in a common muscle sheath, and ii) marginal eyespots completely surrounding the entire periphery of the dorsal surface. Subsequently, E. evelinae , E. japonicum , and E. punctatum were added to the genus ( Kato 1943; Marcus 1949; Hyman 1953) before Faubel (1984) re-defined Enchiridium . It was circumscribed so that "only the prostatic vesicles are bound into a common muscle bulb and oriented anterodorsal to the ejaculatory duct" ( Faubel 1984, p. 231); namely, the encircling marginal eyespots were not regarded as a necessary condition for Enchiridium . At the same time, Faubel ( 1984) transferred three Lurymare species, viz., L. delicatum , L. gabriellae , and L. russoi , into Enchiridium . As a result, seven species were included in Enchiridium in the taxonomic system of Faubel (1984). In contrast, Prudhoe (1985) supported Bock’s (1913) taxon concept of Enchiridium , retaining four species, E. evelinae , E. japonicum , E. periommatum , and E. punctatum , in Enchiridium and three species, L. delicatum , L. gabriellae , and L. russoi , in Lurymare . On the other hand, Cuadrado et al. (2017) followed Faubel’s (1984) redefinition when they established E. magec . The monophyly of Enchidirium sensu Faubel (1984) was strongly supported in a molecular phylogenetic analysis based on partial 28S rDNA ( Litvaitis et al. 2019). In our study, Enchidirium sensu Faubel (1984) received 80% bootstrap support with the exclusion of Enchiridium sp. 4 of Bahia et al. (2017); including the latter, the branch support decreased to 50% (Fig. 5 ). Also, Enchiridium in the sense of Bock (1913) and Prudhoe (1985) - represented by E. evelinae , E. japonicum , E. periommatum , Enchiridium sp. 3, and Enchiridium sp. 4 (cf. Bahia et al. 2017, Table 2 ) in our analysis - was not monophyletic. Therefore, the taxonomy of Enchiridium should be revised with further molecular phylogenetic analyses as well as careful examination of morphological characters among the constituent members. At the moment, however, we adopt Faubel’s (1984) redefinition and place our new species in the genus Enchiridium along with eight other species. We did so because our results indicated that the arrangement of the marginal eyespots should not be taken into account as generic diagnostic characters.

Enchiridium daidai sp. nov. is distinguished from E. evelinae , E. japonicum , E. periommatum , and E. punctatum by the arrangement of the marginal eyespots; the marginal-eyespot band in these four species completely encircles the periphery of the dorsal surface, whereas that of our specimens is present only along the anterior margin. Our new species is also easily distinguished from the other four congeners, E. delicatum , E. gabriellae , E. magec , and E. russoi , by the thin marginal orange line surrounding the entire dorsal fringe and by the lack of spots or maculae on the dorsal surface (Table 2 ).

Reaching 77 mm in body length, Enchiridium daidai sp. nov. is the largest species in the genus over E. punctatum (about 40 mm in body length; Hyman 1953, p. 386). Indeed, E. daidai is the second largest in the Prosthiostomidae after P. cyclops , which reaches 90 mm ( Verrill 1901). Among about 80 species of prosthiostomids, only E. daidai sp. nov. and P. cyclops are known to exceed 70 mm in body length, while most of the other species are less than 30 mm long. Therefore, our new species is considered to be unusually big in body size for a prosthiostomid.

Figures

Figure 2. Enchiridium daidai sp. nov., photograph taken in life and eyespots observed in fixed state after being cleared in xylene. A ICHUM 5993 (holotype), entire animal, dorsal view (left) and ventral view (right) B ICHUM 5994 (paratype), magnification of anterior body, showing arrangements of cerebral and marginal eyespots. Abbreviations: ce cerebral eyespots fg female gonopore me marginal eyespots mg male gonopore op oral pore ph pharynx su sucker. Scale bars: 10 mm (A); 1 mm (B).

Figure 3. Enchiridium daidai sp. nov., ICHUM 5993 (holotype), schematic diagram (A) and sagittal sections (B-D), anterior to the right. A Schematic diagram of copulatory complex B a common muscle sheath / bulb enclosing two prostatic vesicles and penis stylet C ejaculatory duct penetrating a common muscle sheath / bulb D female copulatory apparatus. Abbreviations: cg cement glands cp cement pouch ed ejaculatory duct fg female gonopore it intestine ma male atrium mg male gonopore ms muscle sheath / bulb pd prostatic duct pp penis papilla ppo penis pouch pv prostatic vesicle spv spermiducal vesicle st stylet sv seminal vesicle va vagina. Scale bars: 500 µm.

Figure 4. Difference in mature body size among Enchiridium daidai sp. nov. A ICHUM 5993 (holotype), from Kagoshima B ICHUM 5995 (paratype), from Okinawa C ICHUM 5994 (paratype), from Okinawa. Scale bar: 10 mm.

Figure 5. Maximum likelihood phylogenetic tree based on 935 - bp 28 S rDNA. Bootstrap support values are indicated near nodes.