Description of a new species of Prostheceraeus Schmarda, 1859 and redescription of a poorly known species, Phrikoceros sagamianus (Kato, 1937) comb. n., with inference of their phylogenetic positions within Pseudocerotoidea (Platyhelminthes: Polycladida: Cotylea) Author Tsuyuki, Aoi Faculty of Science, University of the Ryukyus, Okinawa, Japan; Author Kohtsuka, Hisanori Misaki Marine Biological Station, The University of Tokyo, Kanagawa, Japan; Author Okuno, Junji Coastal Branch of Natural History Museum and Institute, Chiba, Japan & Faculty of Science, University of the Ryukyus, Okinawa, Japan; text Journal of Natural History 2025 2025-01-29 59 5 - 8 303 330 http://dx.doi.org/10.6084/m9.figshare.28302561 journal article 10.1080/00222933.2024.2439032 1464-5262 14765230 Prostheceraeus fragum Tsuyuki, Kohtsuka, and Okuno sp. n. (New Japanese name: Ichigo-nagamimi-hiramushi) ( Figures 1 , 2 ) ?Euryleptid 19: Ryanskiy 2021: 97 , coloured unnumbered photographs. ZooBank: urn:lsid:zoobank.org:act: E0C83AD9-736A-4951-B4E8-5813C690C43F Material examined Holotype : NSMT-Pl 9505 , sagittal sections of the whole body (13 slides), dredged from a depth of 122–179 m in Sagami Bay , off Misaki , Kanagawa , Japan (between 35.140117°N , 139.546833°E and 35.135533°N, 139.545783°E ), on 10 June 2022 . Paratypes : NSMT-Pl 9507 , whole mount specimen, dredged from 101–105 m depth in Sagami Bay , off Misaki , Kanagawa , Japan (between 35.138555°N , 139.534663°E and 35.137662°N, 139.534970°E ), on the same day as holotype; NSMT-Pl 9506 , sagittal sections of the whole body (5 slides), the same collection data as NSMT-Pl 9507. Etymology The specific epithet fragum , a Latin term signifying ‘strawberry’, was chosen in reference to the dorsal colour pattern of the new species. This pattern is characterised by red maculae, resembling slices of a strawberry. Figure 1. Prostheceraeus fragum sp. n. , photographs of living animal (A, B, D) and fixed specimen (C). (A) NSMT-Pl 9505 (holotype; left) and NSMT-Pl 9507 (paratype; right), dorsal view. (B) NSMT-Pl 9507 (paratype), ventral view. (C) NSMT-Pl 9505 (holotype), cleared in xylene, ventral view. (D) NSMT-Pl 9505 (holotype), magnification of anterior body. Abbreviations: ce: cerebral eyespots; fg: female gonopore; mg: male gonopore; ph: pharynx; pte: pseudotentacle; su: sucker; te: tentacular eyespots; ut: uterus. Scale bars: A, B = 5 mm; C, D = 1 mm. Diagnosis Pair of pseudotentacles slender and pointed, reddish purple in colour; translucent creamwhite background; red maculae oval in shape, only distributed radially along body periphery and over midline on dorsal surface; pair of cerebral-eyespot clusters, each composed of about 30 eyespots; marginal and frontal eyespots absent; pseudotentacular eyespots numerous; multiple uterine vesicles present in each uterus. Description Body elongated oval, 11.1–12.5 mm in length ( 11 mm in holotype ) and 3.9–4.8 mm at its widest point ( 3.9 mm in holotype ) in living state ( Figure 1 (A,B)), 7.9 mm in length and 4.3 mm at its widest point after fixation ( Figure 1 (C)). Pair of pseudotentacles apparent, slender and pointed, coloured reddish purple, about 1 mm in length ( Figure 1 (D)). Dorsal surface smooth, translucent cream with specific colour pattern; four dark-red maculae ( 0.52–0.90 mm in diameter in holotype ) arranged in midline; around 30–50 oval red maculae ( 0.32–0.91 mm in diameter in holotype ), distributed radially along body margin; basically larger one and smaller one arranged alternately ( Figure 1 (A)); six triangular red maculae adjacent to each other distributed in anterior to posterior end of cerebral-eyespot clusters; red colouration fading towards centre in all maculae ( Figure 1 (A)). Marginal lines absent. Colour pattern on dorsal surface generally fading away after fixation ( Figure 1 (C)). Ventral surface translucent, reddish-purple pigments forming two dotted lines along body margin in specific area from anterior end of cerebral eyespots to level of 1/3 of pharynx; reddish-purple pigments sparsely distributed along body periphery posterior to dotted lines ( Figure 1 (B)). Cerebral eyespots in two elongated clusters ( 0.3–0.4 mm in length), each composed of about 30 eyespots ( Figure 1 (D)). Frontal and marginal eyespots absent. Pseudotentacular eyespots distributed at base of each tentacle, each cluster consisting of about 30 eyespots ( Figure 1 (C,D)). Intestine highly branched, anastomosing ( Figure 1 (B)). Pharynx plicated, tubular and bell-shaped, 0.82 mm in length in fixed specimen ( Figure 1 (B,C)). Mouth opening at anterior end of pharyngeal pouch. Male and female gonopores opening separately behind posterior end of pharynx ( 0.46 mm and 0.86 mm behind, respectively) ( Figure 1 (C)). Male copulatory apparatus with large seminal vesicle, spherical prostatic vesicle, and armed penis papilla ( Figure 2 (A,B)). Pair of sperm ducts forming common spermiducal vesicle before entering proximal end of seminal vesicle ( Figure 2 (A,B)). Seminal vesicle oval, 390 µm in long axis and 150 µm in short axis, coated with thick muscular wall, leading ejaculatory duct ( Figure 2 (A)). Prostatic vesicle spherical, 136 µm in diameter, coated with thin muscular wall; inner wall of prostatic vesicle lined with glandular epithelium consisting of tall cells (ca. 50 µm in length) ( Figure 2 (B)). Prostatic duct opening into penis papilla. Ejaculatory duct connecting to prostatic duct before proximal end of penis papilla ( Figure 2 (A)). Penis papilla bearing cuticular stylet 100 µm in length, surrounded by penis sheath ( Figure 2 (B)). Male atrium opening to exterior via male gonopore, 146 µm in diameter ( Figure 2 (A,B)). Female gonopore located at distance of 0.6 mm posterior to male gonopore ( Figures 1 (C), 2(A)). Pair of uteri filled with multiple eggs, each having 3–4 uterine vesicles ( Figure 2 (C)), running anteriorly on both sides from 1.4 mm distance from posterior end of body ( Figure 1 (B,C)), fusing before entering proximal end of vagina ( Figure 1 (C)). Lang’s vesicle absent. Vagina curving down, leading to cement pouch ( Figure 2 (A,D)). Cement glands well developed around female copulatory apparatus, releasing their contents in cement pouch ( Figure 2 (A,D)). Female atrium opening to exterior via female gonopore ( Figure 2 (A)). Sucker 0.3 µm in diameter, situated at distance of 1.7 mm posterior to female gonopore ( Figure 1 (C)). Figure 2. Prostheceraeus fragum sp. n. , holotype (NSMT-Pl 9505), reconstructed schematic diagram (A) and photomicrographs of sagittal sections (B–D) (anterior to the left). (A) Male and female copulatory apparatus. (B) Male copulatory apparatus. (C) Uterine vesicle and uterus. (D) Female copulatory apparatus. Abbreviations: cg: cement glands; cp: cement pouch; e: egg; ed: ejaculatory duct; fg: female gonopore; it: intestine; ma: male atrium; mg: male gonopore; po: penis pouch; pp: penis papilla; ps: penis sheath; pv: prostatic vesicle; sd: sperm duct; spv: spermiducal vesicle; st: stylet; sv: seminal vesicle; ut: uterus; uv: uterine vesicle; vg: vagina. Scale bars: A = 300 µm: B–D = 100 µm. Type locality and distribution Specimens were collected from the sublittoral zone at a depth of 122–179 m in Sagami Bay , off the coast of Miura Peninsula , Kanagawa , Japan (between 35.140117°N , 139.546833°E and 35.135533°N , 139.545783°E ), at 122–179 m depth . Tentatively identified euryleptid polyclads with a similar dorsal colour pattern have also been reported from the sublittoral zone in Indonesia ( Ryanskiy 2021 ). Habitat The collected specimens were found inhabiting coarse sand and seashell substrates on the seafloor at depths ranging from 100 to 180 m by dredging. An underwater photograph of a closely related species captured by a scuba diver in shallower waters ( Ryanskiy 2021 ) suggests the possibility of the species occurring in shallower habitats (see Discussion section). COI sequence The determined COI sequence ( LC856622 ) from the holotype ( NSMT-Pl 9505 ) was 715 bp in length. BLASTn of the sequence returned 84.01% identity with published Pseudobiceros stellae sequences (GenBank accession MT896282–896283 ) at maximum, followed by 83.93% of Pseudobiceros damawan sequence ( MN690539 ) and 83.79% of Prostheceraeus roseus ( MZ273078 ). The sequence of the holotype of Pr. fragum sp. n. did not show the most similarity to the published Prostheceraeus sequence, but this could be due to different rates in query cover among the compared sequences (41–42% for Pseudobiceros spp. ; 71% for Pr. roseus ). Remarks Based on the presence of key morphological features, the collected specimens were assigned to the genus Prostheceraeus . These features include: (1) a pair of slender, pointed pseudotentacles, (2) an intestine with a network of anastomosing branches, and (3) a pair of uteri with multiple uterine vesicles ( Faubel 1984 ; Prudhoe 1985 ). So far, Prostheceraeus species have been distinguished from each other based on external body colour patterns ( Cuadrado et al . 2021 ; Soutullo et al . 2021 ). Prostheceraeus fragum sp. n. can be distinguished from its congeners, Prostheceraeus boucheti Prudhoe, 1989 , Prostheceraeus fitae Soutullo et al ., 2021 , Pr. fuscolineatus , Prostheceraeus giesbrechtii Lang, 1884 , Prostheceraeus meleagrinus ( Kelaart, 1858 ) , Prostheceraeus pseudolimax Lang, 1884 , Prostheceraeus roseus Lang, 1884 , and Prostheceraeus vittatus ( Montagu, 1815 ) , by the complete absence of longitudinal lines on its dorsal surface ( Figure 1A ; Table 2 ). Our new species can further be distinguished from Prostheceraeus crozieri ( Hyman, 1939 ) and Prostheceraeus zebra Hyman, 1955a by lacking any transversal lines on its dorsal surface ( Figure 1A ; Table 2 ). Prostheceraeus albocinctus Lang, 1884 , Prostheceraeus crisotomum Cuadrado et al ., 2021 , Prostheceraeus maculosus ( Verrill, 1892 ) , Prostheceraeus moseleyi Lang, 1884 , Prostheceraeus nigricornus Schmarda, 1859 , and Prostheceraeus rubropunctatus Lang, 1884 possess spotted or maculated dorsal surfaces. These markings differ from those of Pr. fragum sp. n. in several aspects: colouration (red or reddish purple in Pr. fragum sp. n. ; brown to black in other species) and distribution (along the body periphery and midline in Pr. fragum sp. n. ; scattered across the dorsal surface in other species) ( Figure 1 (A); Table 2 ). Additionally, Prostheceraeus floridanus Hyman, 1955b and Prostheceraeus panamensis Woodworth, 1894 differ from Pr. fragum sp. n. by possessing an irregular meshwork on their dorsal surfaces. Furthermore, our new species lacks marginal lines, unlike Prostheceraeus flavomarginatus ( Ehrenberg, 1831 ) and Prostheceraeus violaceus Delle Chiaje, 1822 ( Table 2 ). Prostheceraeus fragum sp. n. has a somewhat similar colour pattern to Prostheceraeus argus ( Quatrefages, 1845 ) , both exhibiting strings of purple dots along the body margins. However, Pr. fragum sp. n. can be distinguished by the absence of (1) white dots scattered over the dorsal surface and (2) marginal eyespots extending posteriorly, both of which are present in Pr. argus ( Table 2 ). While information on the colouration of Prostheceraeus anomalus Haswell, 1907 is lacking, a distinct anatomical feature separates these species. Prostheceraeus anomalus possesses a unique vesicle, the ‘ receptacula seminalis ’, that connects to the vagina rather than the oviducts or uteri ( Haswell 1907 , p. 482), a feature absent in Pr. fragum sp. n. In conclusion, our new species Pr. fragum sp. n. can be morphologically distinguished from the 22 possible congeners.