Description of a New Species of the Marine Flatworm Prosthiostomum (Platyhelminthes: Polycladida) and its Three Known Congeners from Misaki, Japan, with Inference of Their Phylogenetic Positions within Prosthiostomidae

© 2021 Academia Sinica, Taiwan Open Access Description of a New Species of the Marine Flatworm Prosthiostomum (Platyhelminthes: Polycladida) and its Three Known Congeners from Misaki, Japan, with Inference of Their Phylogenetic Positions within Prosthiostomidae Aoi Tsuyuki1,* , Hisanori Kohtsuka2, and Hiroshi Kajihara3 1Graduate School of Science, Hokkaido University, N10W8, Sapporo, Hokkaido 060-0810, Japan. *Correspondence: E-mail: [email protected] (Tsuyuki) 2Misaki Marine Biological Station, The University of Tokyo, Koajiro 1024, Misaki, Miura, Kanagawa 238-0225, Japan. E-mail: [email protected] (Kohtsuka) 3Faculty of Science, Hokkaido University, N10W8, Sapporo, Hokkaido 060-0810, Japan. E-mail: [email protected] (Kajihara) Received 22 September 2020 / Accepted 6 April 2021 / Published 24 June 2021 Communicated by Benny K.K. Chan The present study provides morphological descriptions of four species of Prosthiostomum (Polycladida, Prosthiostomidae)—P. auratum Kato, 1937; P. hibana sp. n.; P. cf. ostreae Kato, 1937; and P. vulgare Kato, 1938—based on specimens collected among branching coralline algae and kelp holdfasts in Misaki, Japan. The new species P. hibana sp. n. is characterized by i) the dorsal surface of the body covered with numerous orange maculae, some of which coalesce together to form larger ones; ii) a pair of linear cerebral-eyespot clusters, each consisting of relatively few (7–9) cerebral eyespots; iii) 3–4 pairs of ventral eyespots embedded in parenchyma: iv) the inner wall of the male atrium deeply ruffled; v) the lumen of the seminal vesicle being narrow and elongated in shape; and vi) a large sucker situated in the center of the body. We remark on some morphological characters that were not mentioned in the original description of P. auratum. We infer the phylogenetic positions of these four species within Prosthiostomidae using the maximum-likelihood analysis based on partial 28S rRNA and COI gene sequences determined de novo, in addition to those that are currently available in public databases. In the resulting tree, the four species—P. auratum, P. hibana sp. n., P. cf. ostreae, and P. vulgare—were nested in a clade that was composed of all the other Prosthiostomum species included in the analysis. Key words: Cotylea, Phylogeny, Species inquirenda, Systematics, Taxonomy. BACKGROUND The order Polycladida consists of more than 800 species of marine flatworms (Tyler et al. 2006–2020) that live in variety of marine habitats, including tide pool, coral reefs, mud flat, and deep sea (Prudhoe 1985). Some species are known to associate with other invertebrates, such as corals (e.g., Rawlinson et al. 2011), hermit crabs (e.g., Lytwyn and McDermott 1976), innkeeper worms (Anker et al. 2005), and mollusks (e.g., Fujiwara et al. 2016). Of the about 150 species of Polycladida known to inhabit Japanese waters (Kato 1944), 130 were originally described from this area; ~100 of the latter are poorly known and thus can be regarded species inquirendae, or species of questionable taxonomic status. They were mostly established over 70 Citation: Tsuyuki A, Kohtsuka H, Kajihara H. 2021. Description of a new species of the marine flatworm Prosthiostomum (Platyhelminthes: Polycladida) and its three known congeners from Misaki, Japan, with inference of their phylogenetic positions within Prosthiostomidae. Zool Stud 60:29. doi:10.6620/ZS.2021.60-29. Zoological Studies 60:29 (2021) doi:10.6620/ZS.2021.60-29 1 © 2021 Academia Sinica, Taiwan years ago (e.g., Kato 1944) and have not been recorded since. As for the species that were either redescribed or originally described since the 1990s (Hagiya 1992; Oya and Kajihara 2017 2019a b; Oya et al. 2019; Tsuyuki et al. 2019; Oya et al. 2020; Tsuyuki and Kajihara 2020), morphological characters important for generic assignment have been clearly documented and/ or illustrated. However, because such key characters became incorporated to taxonomic revisions during the 1980s (Faubel 1984; Prudhoe 1985), it is no surprise that these features were scarcely mentioned in original taxonomic descriptions before that period (e.g., Stimpson 1857; Yeri and Kaburaki 1918 1920; Kato 1944) with some notable exceptions (e.g., Bock 1922 1923 1924). The genus affiliations of these species remain questionable, and they cannot be confirmed based on their name-bearing type specimens because those specimens are entirely non-existent. Stimpson’s material is said to have been destroyed during the Great Chicago Fire in 1871 (e.g., Evans 1967; Deiss and Manning 1981). The material used by Yeri and/or Kaburaki has not been found, likely because it was lost during the Great Kanto Earthquake and a subsequent disastrous fire in 1923 (cf. Kato 2018). Kato’s material was destroyed during the Bombing of Tokyo in 1945 (Kawakatsu 2004). Therefore, to precisely understand the polyclad biodiversity and systematics in not only Japanese waters but also the Northeast Pacific, morphological and molecular information is needed on these species inquirendae based on newly collected specimens, preferably from type localities. The cotylean polyclad family Prosthiostomidae Lang, 1884 is characterized by i) an elongated body with a ventral sucker posterior to the female gonopore, ii) a plicate tubular pharynx, and iii) paired prostatic vesicles. Monophyly of this family has been supported in previous molecular phylogenetic studies based on partial sequences of the 28S rRNA gene alone (Bahia et al. 2017; Tsunashima et al. 2017; Litavaitis et al. 2019) or in combination with the 18S rRNA gene (Dittmann et al. 2019). Aguado et al. (2017) argued that Prosthiostomidae is not monophyletic, but this is probably due to the fast-evolving gene markers that they utilized (i.e., the mitochondrial 16S rRNA and cytochrome c oxidase subunit I (COI) genes). Prosthiostomidae currently includes five genera: Enchiridium Bock, 1913; Enterogonimus Hallez, 1911; Euprosthiostomum Bock, 1925; Lurymare Du Bois-Reymond Marcus and Marcus, 1968; and Prosthiostomum Quatrefages, 1845 (Faubel 1984). Of these, Lurymare may be a junior synonym of Prosthiostomum (cf. Dittmann et al. 2019; Litvaitis et al. 2019; Tsuyuki et al. 2019), because the alleged morphological distinction between the two genera is based on a character that can vary ontogenetically (Prudhoe 1989), namely the presence/absence of a muscle bulb surrounding the seminal and prostatic vesicles (Faubel 1984). Indeed, Litvaitis et al. (2019) transferred two species formerly placed in Lurymare into Prosthiostomum primarily because their phylogenetic positions were nested within a clade comprising Prosthistomum species. While the separation of Lurymare from Prosthiostomum based on this character alone (i.e., presence/absence of a muscle bulb) appears systematically unsubstantiated, a definitive taxonomic act to formally synonymize Lurymare with Prosthiostomum should not proceed until an analysis is performed using reliably identified prosthiostomid specimens, including those representing the type species of the two genera (Prosthiostomum drygalskii Bock, 1931 for Lurymare; Planaria siphunculus Delle Chiaje, 1828 for Prosthiostomum). For the sake of conciseness, this Prosthiostomum– Lurymare complex is simply referred to as the genus Prosthiostomum in this paper. It currently contains about 60 species worldwide, which are characterized by i) the seminal and prostatic vesicles that are occasionally (but usually not) surrounded by a common muscle bulb, ii) the main intestine accompanied with a frontal branch over the pharynx, and iii) the penis armed with a pointed tubular stylet (cf. Faubel 1984). Congeners are distinguished chiefly based on body color pattern and eyespot arrangement (Bock 1913; Hyman 1939b). In Japan, 22 species of Prosthiostomum have been reported (Kato 1944; Tsuyuki et al. 2019). Twenty of these—all except for P. purum Kato, 1937b (Litvaitis et al. 2019 [Israel]) and P. trilineatum Yeri and Kaburaki, 1920 (Newman and Cannon 2003 [Australia]; Pitale et al. 2014 [India]; Litvaitis et al. 2019 [Guam])—have so far been found exclusively along the Japanese coasts, and thus may be endemic to this area. Misaki, situated on the western coast of the Miura Peninsula of Honshu, Japan, is one of the faunistically best-studied areas in terms of not only polyclads but also other marine organisms along Japanese coastal regions (Kajihara and Kakui 2017). So far, 29 polyclad species have been originally described based on specimens collected in shallow water around Misaki (Yeri and Kaburaki 1918; Bock 1922 1923 1924; Kato 1937b). Of these, five represent the genus Prosthiostomum: P. auratum Kato, 1937b; P. ostreae Kato, 1937b; P. purum Kato, 1937b; P. rubropunctatum Yeri and Kaburaki, 1918; and P. yerii Kato, 1937b. These five species are species inquirendae and have not been reported from Misaki since their original descriptions, although distribution of P. auratum and P. purum in localities other than Misaki have been reported (P. auratum from page 2 of 20Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan other Japanese coastal regions such as Asamushi, Noto, Shimoda, Shirahama, and Amakusa (Kato 1938a b 1939b 1944) and Manazuru (Hagiya and Gamo 1992); P. purum from the northern coast of the Red Sea (Litvaitis et al. 2019)). A faunal survey was conducted in Misaki in the hope of collecting topotypes of P. auratum, P. ostreae, P. purum, P. rubropunctatum, and P. yerii, as well as revealing undiscovered polyclad diversity in this region. As a result, we obtained polyclad specimens representing four species of Prosthiostomum, one of which turned out to be new to science. The purposes of this paper are to i) describe the new species and provide morphological accounts of the other three species with taxonomic remarks and ii) examine the generic placements of these species through molecular phylogenetic analysis using sequences of the 28S rRNA and COI genes. MATERIALS AND METHODS Polyclad specimens were collected in Araihama, Misaki, Kanagawa, Japan; nine specimens were collected from kelp holdfasts at 2 m deep by SCUBA on February 19, 2019; the others were collected subtidally from branching coralline algae by snorkeling on March 25, 2019. Worms were anesthetized in seawater containing menthol before fixation. The relaxed worms were photographed with a Nikon D5600 digital camera with external strobe lightning provided by a pair of Morris Hikaru Komachi Di flash units. For DNA extraction, a posterior piece of the body was removed and stored in 99.5% ethanol. The rest of the body was fixed in Bouin’s solution for 24 h and preserved in 70% ethanol for long-term storage. For histological examination, tissues were dehydrated in an ethanol series, cleared in xylene, embedded in paraffin wax, and sectioned serially at a thickness of 7 µm on a sagittal plane using a microtome. Sections were stained with hematoxylin and eosin, mounted on glass slides in Entellan New (Merck, Germany), and then observed and photographed under an Olympus BX51 compound microscope. All slides were deposited into the Invertebrate Collection of the Hokkaido University Museum (ICHUM), Sapporo, Japan. Other specimens were deposited in Aoi Tsuyuki’s personal polyclad collection (AT). All graphical treatments were done with Adobe Photoshop CC. Illustrations were prepared with Adobe Illustrator CC. Total DNA was extracted using a silica-based method (Boom et al. 1990) after specimens were incubated overnight at 55°C in 180 µl of ATL buffer (Qiagen, Germany) with 20 µl of proteinase K (> 700 U/ml; Kanto Chemical, Japan). A 585-bp fragment of the COI gene was amplified with primers Pros_COIF and Pros_COIR (Tsuyuki and Kajihara 2020). A fragment (ca. 1010 bp) of the 28S rRNA gene was amplified with primers fw1 and rev2 (Sonnenberg et al. 2007). Polymerase chain reaction (PCR) amplification conditions were 94°C for 5 min; 35 cycles of 94°C for 30 s, 52.5°C (28S rRNA) or 50°C (COI) for 30 s, 72°C for 1.5 min (28S rRNA) or 1 min (COI); and 72°C for 7 min. All nucleotide sequences were determined by direct sequencing with a BigDye Terminator Kit ver. 3.1 and a 3730 Genetic Analyzer (Life Technologies, California, USA). Following a protocol by Oya and Kajihara (2020), two internal primers—hrms_fw2 (Oya and Kajihara 2020) and rev4 (Sonnenberg et al. 2007)—were used in addition to fw1 and rev2. Sequences were checked and edited using MEGA ver. 7.0 (Kumar et al. 2016). In addition, the following were determined by the same methods described above: a 944-bp partial sequence of the 28S rRNA gene and a 553-bp partial sequence of the COI gene from a specimen of P. grande Stimpson, 1857 (ICHUM 6032), collected by A. Tsuyuki in Kagoshima, Japan, on July 25, 2018, as well as a 585-bp partial COI gene sequence from the holotype of P. torquatum Tsuyuki et al., 2019 (ICHUM 5563). All the edited sequences were deposited into DDBJ/EMBL/GenBank under accession numbers LC625886–LC625900 and LC635089. For a phylogenetic analysis, a concatenated dataset comprised of partial 28S rRNA and COI gene sequences was prepared. We employed the 28S rRNA gene as one of the two markers because it is the most commonly used for determining members of Polycladida (Bahia et al. 2017; Tsunashima et al. 2017; Dittmann et al. 2019; Litvaitis et al. 2019). In addition to the sequences determined for the four species from Misaki and for P. grande (see above), 24 other partial 28S rRNA gene sequences from 19 prosthiostomid species downloaded from GenBank were used in the analysis; multiple sequences derived from individuals representing geographically different local populations were used for Enchiridium periommatum Bock, 1913; Enchiridium sp. 2 of Litvaitis et al. (2019); and P. siphunculus (Table 1). The 28S rRNA gene sequences were aligned using MAFFT ver. 7.427 (Katoh et al. 2017), with the L-INS-i strategy selected by the “Auto” option; ambiguous sites were removed with Gblocks ver. 0.91b (Castresana 2002) using options for a less stringent selection. The COI sequences of the four species collected in Misaki were used, plus P. grande and P. torquatum (see above) and E. daidai Tsuyuki and Kajihara, 2020 (holotype, ICHUM 5993) (Table 1). COI was aligned manually with MEGA ver. 7.0 (Kumar et al. 2016). A concatenated dataset (1524 bp in total length, consisting page 3 of 20Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan of 939-bp 28S rRNA and 585-bp COI) was prepared, also with MEGA ver. 7.0. The phylogenetic analysis was performed with the maximum likelihood (ML) method using RAxMLNG ver. 0.9.0 (Kozlov et al. 2019) under a partition model. The optimal substitution models selected with Partitionfinder ver. 2.1.1 (Lanfear et al. 2016) under the Akaike Information Criterion (AIC) (Akaike 1974) using the greedy algorithm (Lanfear et al. 2012) were GTR+I+G (28S rRNA, second codon position in COI) and GTR+G (first and third codon positions in COI). Prostheceraeus crozieri (Hyman, 1939a) (Euryleptidae) and Pseudobiceros splendidus (Lang, 1884) (Pseudocerotidae) were selected as outgroups (Tsuyuki and Kajihara 2020) (Table 1). Nodal support within the ML tree was assessed by analyzing 100 bootstrap pseudoreplicates (Felsenstein 1985). We considered ML bootstrap (BS) values ≥ 70% as indicating clade support. Table 1. List of species used for the molecular phylogenetic analysis, sample locations, DDBJ/EMBL/GenBank accession numbers, and references Species Location Accession number Reference 28S rRNA COI Enchiridium daidai Tsuyuki and Kajihara, 2020 Bonotsu, Kagoshima, Japan LC504235 LC504240 Tsuyuki and Kajihara (2020) Enchiridium evelinae Marcus, 1949 Praia das Conchas, Rio de Janeiro, Brazil KY263683.2 - Bahia et al. (2017) Enchiridium japonicum Kato, 1943 Eilat, Israel MH700298 - Litvaitis et al. (2019) Enchiridium periommatum Bock, 1913 St. Ann’s Bay, Jamaica MH700299 - Litvaitis et al. (2019) Enchiridium periommatum Bock, 1913 Crawl Cay, Bocas del Toro, Panama MH700300 - Litvaitis et al. (2019) Enchiridium periommatum Bock, 1913 Tavernier Key, Florida, USA MH700301 - Litvaitis et al. (2019) Enchiridium sp. 1 Saint Helena Island, UK KY263665 - Bahia et al. (2017) Enchiridium sp. 2 New South Wales, Australia MH700302 - Litvaitis et al. (2019) Enchiridium sp. 2 Heron Island, Australia MH700303 - Litvaitis et al. (2019) Enchiridium sp. 3 Lizard Island, Australia MN384686 - Dittmann et al. (2019) Euprosthiostomum mortenseni Marcus, 1948 St. Ann’s Parish, Jamaica MH700304 - Litvaitis et al. (2019) Prosthiostomum acroporae (Rawlinson et al., 2011) Victorville, California, USA MH700370 - Litvaitis et al. (2019) Prosthiostomum auratum Kato, 1937b Misaki, Kanagawa, Japan LC625886 LC625892 this study Prosthiostomum cynarium Marcus, 1950 St. John, US Virgin Islands MH700371 - Litvaitis et al. (2019) Prosthiostomum grande Stimpson, 1857 Sakurajima, Kagoshima, Japan LC635089 LC625900 this study Prosthiostomum hibana sp. n. Misaki, Kanagawa, Japan LC625887 LC625894 this study Prosthiostomum katoi Poulter, 1975 Lizard Island, Australia MN384694 - Dittmann et al. (2019) Prosthiostomum lobatum Pearse, 1938 Missouri Key, Florida, USA MH700372 - Litvaitis et al. (2019) Prosthiostomum milcum Du Bois-Reymond Marcus and Marcus, 1968 Long Key, Florida, USA MH700373 - Litvaitis et al. (2019) Prosthiostomum cf. ostreae Kato, 1937b Misaki, Kanagawa, Japan LC625889 LC625896 this study Prosthiostomum purum Kato, 1937b Gulf of Aqaba, northern Red Sea MH700374 - Litvaitis et al. (2019) Prosthiostomum siphunculus (Delle Chiaje, 1828) Spain HQ659012 - Rawlinson et al. (2011) Prosthiostomum siphunculus (Delle Chiaje, 1828) Asturias, Spain MN384697 - Dittmann et al. (2019) Prosthiostomum siphunculus (Delle Chiaje, 1828) Barcelona, Spain MN421934 - Dittmann et al. (2019) Prosthiostomum torquatum Tsuyuki et al., 2019 Shirahama, Wakayama, Japan LC504234 LC625899 Tsuyuki and Kajihara (2020) Prosthiostomum trilineatum Yeri and Kaburaki, 1920 Ritidian Point, Guam MH700376 - Litvaitis et al. (2019) Prosthiostomum utarum Marcus, 1952 Piscadera Baai, Curaçao MH700377 - Litvaitis et al. (2019) Prosthiostomum vulgare Kato, 1938b Misaki, Kanagawa, Japan LC625891 LC625898 this study Prosthiostomum sp. Eilat, Israel MH700375 - Litvaitis et al. (2019) Outgroup Prostheceraeus crozieri (Hyman, 1939a) Long Key, Florida, USA HQ659013 - Rawlinson et al. (2011) Pseudobiceros splendidus (Lang, 1884) North Heron Island, Australia MH700388 - Litvaitis et al. (2019) page 4 of 20Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan RESULTS TAXONOMY Family Prosthiostomidae Lang, 1884 Genus Prosthiostomum Quatrefages, 1845 Prosthiostomum auratum Kato, 1937b (Fig. 1) Prosthiostomum auratum Kato, 1937b: 363–364, pl. 22, fig. 8, textfigs. 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 28S rRNA (1010 bp) and COI (585 bp) sequences from two individuals. LC625886 (28S 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 cerebraleyespot cluster is formed in a linear shape, and iii) a pair of prostatic vesicles are moderately large. No page 5 of 20Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan 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. A B D E mo ph mg fg su ce me me F GH ph br it mo br mg fg sv pv cg spv st sv cg pv it st ab ab ma C spv mama page 6 of 20Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan 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-F6A2-4FD8-9E1142250240FE25 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 anteroventrally; 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 page 7 of 20Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan 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. ce me me me ve ve ce ce ph mo mg fg su A B C D E F G page 8 of 20 Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan 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 28S rRNA gene (1008 bp) and COI (585 bp) sequences from two individuals. LC625887 (28S rRNA) and LC625894 (COI) from the holotype (ICHUM 6147); LC625888 (28S 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 cerebraleyespot 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 page 9 of 20Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan 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 ( LC625891) and 585-bp COI (LC625898) 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). DISCUSSION All the four Prosthiostomum species for which we gave morphological accounts in this study— P. auratum, P. hibana sp. n., P. cf. ostreae, and P. vulgare—were nested in the Prosthiostomum clade (Fig. 7), corroborating our morphology-based generic assignments. The reconstructed tree based on 24 species of Prosthiostomidae was largely in accordance with that in Litvaitis et al. (2019). In terms of the entire polyclad phylogeny, the analysis by Litvaitis et al. (2019) represented the densest sampling of Prosthiostomidae taxa before our study (cf. Aguado et al. 2017; Bahia et al. 2017; Tsunashima et al 2017; Dittmann et al. 2019). While a different taxonomic view was once proposed in terms of the generic affiliation (see Remarks for P. auratum), P. auratum was more closely related to P. siphunculus (type species of Prosthiostomum) than to Euprosthiostomum mortenseni in our tree (Fig. 7). This result supports the placement of the species in Prosthiostomum based on our morphological observation, given that the E. mortenseni specimen sequenced by Litvaitis et al. (2019) was actually more page 16 of 20Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan closely related to E. adhaerens Bock, 1925 (type species of Euprosthiostomum) than to Prosthiostomum species. Prosthiostomum katoi Poulter, 1975 was nested in the Prosthiostomum clade (Fig. 7), rendering further support for the taxonomic view that Lurymare is indeed a junior synonym of Prosthiostomum (cf. Dittmann et al. 2019; Litvaitis et al. 2019; Tsuyuki et al. 2019). Being originally described as a member of the subgenus Lurymare within the genus Prosthiostomum (Poulter 1975), P. katoi was then transferred into the genus Lurymare by Faubel (1984). Since then, the generic affiliation of the species has been controversial in relation to the validity of Lurymare (Prudhoe 1985; Marquina et al. 2015; Dittmann et al. 2019). Among the five species of Prosthiostomum for which Misaki being the type locality, only P. auratum was recovered in this study. Of the other four, P. purum has been reported from Israel (Gulf of Aqaba, northern Red Sea) along with a partial sequence of the 28S rRNA gene (Litvaitis et al. 2019). While the phylogenetic position of the P. purum of Litvaitis et al. (2019) undoubtedly indicates its reasonable genus assignment (Fig. 7), conspecificity should be confirmed in future studies using molecular techniques by comparing it with topotypic sequence(s), given that its geographic ranges in Israel and Japan are > 9,000 km apart. The other three—P. ostreae, P. rubropunctatum, and P. yerii—remain species inquirendae because their genus affiliation is open to question, due especially to the lack of information as to the presence/absence of a frontal branch of the main intestine over the pharynx. CONCLUSIONS In this study, we described the new species P. hibana sp. n. and presented morphological accounts on P. auratum, P. cf. ostreae, and P. vulgare based on specimens collected from branching coralline algae and kelp holdfasts in Misaki, Japan. Our examination of topotypic specimens of P. auratum suggested that this species should be placed in Prosthiostomum, not Euprosthiostomum, based on morphological and molecular phylogenetic evidence. The new species P. hibana sp. n. is characterized by i) the dorsal surface of the body covered with numerous orange maculae, some 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. 0.08 Enchiridium daidai Prosthiostomum cynarium Enchiridium japonicum Prosthiostomum hibana sp. n. Enchiridium periommatum (Jamaica) Prosthiostomum torquatum Prosthiostomum lobatum Prosthiostomum trilineatum Prosthiostomum sp. Enchiridium evelinae Enchiridium periommatum (USA) Prosthiostomum auratum Prosthiostomum cf. ostreae Prosthiostomum katoi Prostheceraeus crozieri Prosthiostomum purum Enchiridium periommatum (Panama) Euprosthiostomum mortenseni Prosthiostomum acroporae Enchiridium sp. 1 Prosthiostomum grande Prosthiostomum vulgare Prosthiostomum utarum Prosthiostomum milcum Enchiridium sp. 2 Pseudobiceros splendidus 100 90 89 92 98 70 84 100 98 76 100 100 76 76 74 100 96 100 Prosthiostomum siphunculus Enchiridium sp. 3 Prosthiostomum Euprosthiostomum Enchiridium Outgroup page 17 of 20 Zoological Studies 60:29 (2021) © 2021 Academia Sinica, Taiwan of which are agglutinated, ii) the pair of linear cerebraleyespot clusters composed of relatively few eyespots, iii) 3–4 pairs of ventral eyespots, iv) the inner wall of the male atrium deeply ruffled, v) the lumen of the seminal vesicle narrow and elongated in shape, and vi) the large sucker situated on body center. This new species can be easily distinguished from other congeners by the unique ventral-eyespot number. In addition to the ventraleyespot arrangement, the combination of the features in body coloration, cerebral-eyespot arrangement, and the size and position of the sucker allows separation of our new species from other similar-looking congeners (Table 2). Our specimens of P. cf. ostreae differed from the original description by the body-margin coloration and the frontal eyespots. In addition, our specimens had a pair of ventral eyespots, which might have been ignored in the original description. Further studies should test whether these two or three morphological differences are interor intraspecific. The reconstructed tree based on the partial 28S rRNA and COI gene sequences of 24 prosthiostomid species revealed that P. auratum, P. hibana sp. n., P. cf. ostreae, and P. vulgare are nested in the Prosthiostomum clade composed of the other 12 species, corresponding to their generic placements based on the morphological characteristics. The phylogenetic position of P. katoi in Prosthiostomum also supported a taxonomic view that Lurymare is indeed a junior synonym of Prosthiostomum (cf. Dittmann et al. 2019; Litvaitis et al. 2019; Tsuyuki et al. 2019). Acknowledgments: This work and the new species name have been registered with ZooBank under urn:lsid:zoobank.org:pub:32478B95-6577-465A9B0B-88E1973E4CE6. We are grateful to Prof. Toru Miura (Misaki Marine Biological Station, University of Tokyo) and Dr. Kohei Oguchi (National Institute of Advanced Industrial Science and Technology, Japan) for collecting materials. We thank Ms. Michiyo Kawabata (Misaki Marine Biological Station, the University of Tokyo) for their assistance in managing the samples. AT is thankful to Dr. Yuki Oya (Hokkaido University) for help in molecular phylogenetic analyses. We thank the anonymous reviewer and Mr. Noah Last (Third Draft Editing) for helping to improve the manuscript. This study was partially supported by the Research Institute of Marine Invertebrates (FY 2019, No. 15, for AT) and the Japanese Association for Marine Biology (JAMBIO). Authors’ contributions: AT designed the present study. HKo collected materials. AT conducted the morphological observation and molecular phylogenetic analyses as well as drafted the manuscript. HKa and HKo contributed to improvement of the manuscript. All authors read and approved the final manuscript. Competing interests: The authors declare that they have no conflict of interests. 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