Zoosyst. Evol. 99 (2) 2023, 363-373 | DOI 10.3897/zse.99.102604 Gave re BERLIN A new species of slender flatworm in the genus Eucestoplana and a record of FE. cf. cuneata (Platyhelminthes, Polycladida) from the Okinawa Islands, Japan, with an inference of their phylogenetic positions within Cestoplanidae Aoi Tsuyuki!:*, Yuki Oya?, Hiroshi Kajihara! 1 Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan 2 Creative Research Institute, Hokkaido University, Sapporo 001-0021, Japan 3. College of Arts and Sciences, J. F: Oberlin University, Machida 194-0294, Japan https://zoobank. org/D7ACA636-4B03-46F 4-AF77-D5DECS&EB7084 Corresponding author: Aoi Tsuyuki (tykamsp0430@gmail.com) Academic editor: Pavel Stoev # Received 24 February 2023 # Accepted 22 May 2023 @ Published 5 July 2023 Abstract In this study, we describe a new species of elongated marine flatworm, Eucestoplana ittanmomen sp. nov., collected from the inter- tidal zone of the Okinawa Islands, Japan. Eucestoplana ittanmomen sp. nov. is distinguished from other congeners based on the fol- lowing characteristics: 7) its translucent body lacking coloration, i/) its dome-shaped penis sheath, ii7) the absence of cilia on the inner wall of the male atrium except outside the penis sheath, and iv) the presence of an adhesive organ at the posterior end of the body. Additionally, we report the occurrence of EF. cf. cuneata (Sopott-Ehlers & Schmidt, 1975) in Japan; £. cuneata has previously been documented in the Galapagos and Fiji Islands. We conducted phylogenetic analyses to infer the positions of the two Eucestoplana species within Cestoplanidae using a concatenated dataset comprising partial 18S and 28S rDNA sequences from E. cf. cuneata and E. ittanmomen sp. nov. from Japan, as well as four known Cestoplana species with sequences available in public databases. Our phylogenetic analyses revealed that Cestoplana and Eucestoplana were reciprocally monophyletic. Furthermore, the genetic distance of the 16S rDNA sequences supported the genetic independence of the two sister species, E. cf. cuneata and E. ittanmomen sp. nov. Key Words Cotylea, histology, marine flatworms, marine invertebrates, molecular phylogeny, taxonomy Introduction 1884; Cestoplanella Faubel, 1983; Cestoplanides Faubel, 1983; Cestoplanoida Faubel, 1983; and Eucestoplana Polyclad flatworms in the family Cestoplanidae Lang, 1884 are distinguishable from other flatworms by i) their slender bodies without tentacles, i7) ruffled pharynx located posterior to the center of the body, ii) male copulatory apparatus directed anteriorly, and iv) adhesive organ at the posterior end of the body (Faubel 1983; Prudhoe 1985). Currently this family comprises six genera: Acestoplana Faubel, 1983; Cestoplana Lang, Faubel, 1983 (Faubel 1983). The genus Eucestoplana currently includes two spe- cies, Eucestoplana cuneata (Sopott-Ehlers & Schmidt, 1975) and Eucestoplana meridionalis (Prudhoe, 1982a), which are distinguished from other cestoplanids by 7) the presence of a tubular penis stylet housed in the male atri- um and i/) the absence of a Lang’s vesicle (Faubel 1983). These species have previously been reported in the Pacific Copyright Tsuyuki, A. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 364 Tsuyuki, A. et al.: A new species of Eucestoplana and E. cf. cuneata from Okinawa Islands Ocean, including the Galapagos Islands, the Fiji Islands, and South Australia (Sopott-Ehlers and Schmidt 1975; Prudhoe 1982a; Tajika et al. 1991). During a faunal sur- vey conducted as part of this study, we collected polyclad specimens of a new Eucestoplana species, along with E. cf. cuneata, from the intertidal zone of the Okinawa Islands, Japan. In this paper, we provide morphological descriptions of the new Eucestoplana species and E. cf. cuneata, based on the collected specimens. We calculated the genetic distances among the Japanese Eucestoplana specimens using partial 16S rDNA (16S) and cytochrome c oxidase subunit I (COI) sequences; the intraspecific ge- netic distances were also calculated based on partial 28S rDNA sequences among all cestoplanid species available in public databases. Additionally, we infer the phyloge- netic positions of these two Eucestoplana species among other cestoplanids using molecular phylogenetic analyses of partial 18S and 28S rDNA sequences of all currently available cestoplanids in public databases. Methods Specimen collection and fixation Specimens were collected from the Okinawa Islands, Japan, and processed using methods similar to those de- scribed in Tsuyuki et al. (2022, 2023). Gravel samples were collected at depths of about 20 cm from the water surface at low tide (down to about 15 cm from the sed- iment surface), then agitated in seawater to extract ani- mals. The supernatant was filtered using a dip net with about 1-mm mesh, and the remaining residue was trans- ferred into a bottle filled with fresh seawater. Before fix- ation, live worms were anesthetized in an MgCl, solution prepared with tap water to match the seawater salinity us- ing an IS/Mill-E refractometer (AS ONE, Japan). Spec- imens were photographed using a Nikon D5600 digital camera with external strobe lightning provided by a pair of Morris Hikaru Komachi Di flash units. A portion of the body was preserved in 99.5% ethanol for DNA ex- traction, whereas the rest of the body was fixed in Bouin’s solution for 24 h, then stored in 70% ethanol. Morphological observation For histological examination, specimens fixed in Bouin’s solution were prestained with acid fuchsin, dehydrat- ed in an ethanol series, cleared in xylene, embedded in paraffin wax, and sectioned serially at a thickness of 4 um using a microtome. The sections were stained with hematoxylin and eosin, mounted on glass slides, and em- bedded in Entellan New (Merck, Germany) under cover- slips. Specimens were observed and photographed using a Nikon D5600 digital camera under an Olympus BX51 compound microscope. zse.pensoft.net For comparison, we also examined the type series of Eucestoplana cuneata (as Cestoplana cuneata), which consists of the holotype ZMUG 25472 (3 slides) and the paratype ZMUG 25473 (5 slides), both of which have been deposited in the Biodiversity Museum Gottingen of the Georg-August-University Gottingen. In addition, we examined serial sagittal sections of Eucestoplana cuneata (as Cestoplana cuneata) collected from the Fiji Islands in Tajika et al. (1991). DNA extraction, polymerase chain reaction, and sequencing Total DNA was extracted using a DNeasy Blood & Tis- sue Kit (Qiagen, Germany). Prior to extraction, preserved tissues were incubated overnight in 180 ul of ATL buffer (Qiagen, Germany) with 20 ul of proteinase K (>700 U/ ml; Kanto Chemical, Japan) at 55 °C. Four gene markers were used for the analysis: a partial sequence (677 bp) of the COI gene and the 16S (444445 bp) for DNA barcod- ing, and fragments of the 18S rDNA (18S; 1,735 bp) and 28S rDNA (28S; 1,006 bp) for phylogenetic inference. Amplification of the four markers was performed using polymerase chain reaction (PCR) via a 2720 Thermal Cycler (Applied Biosystems, USA). The PCR reaction volume was 10 ul, including 1 ul of total DNA template, 1 ul of 10x ExTaq buffer (Takara Bio, Japan), 2 mM of each dNTP, | uM of each primer, and 0.25 U of Takara Ex Taq DNA polymerase (5 U/ul; Takara Bio, Japan) in deionized water. Specific forward and reverse primer pairs were used for each marker: Acotylea COL F and Acotylea COIL R (Oya and Kajihara 2017) for COI; 16SarL and 16SbrH (Palum- bi et al. 1991) for 16S; hrms18S_F and hrms18S_R (Oya and Kayihara 2020) for 18S; and fwl and rev4 (Sonnen- berg et al. 2007) for 28S. The PCR amplification proce- dures were as follows: 94 °C for 1 min; 35 cycles of 94 °C for 30 s, 50 °C (COI, 16S, and 18S) or 52.5 °C (28S) for 30 s, and 72 °C for 2 min (18S), 1.5 min (28S), or 1 min (COI and 16S); and 72 °C for 7 min. PCR products were purified enzymatically using ExoSAP-IT reagent. Nucleo- tide sequences were determined by direct sequencing with a BigDye Terminator Kit ver. 3.1 and a 3730 Genetic An- alyzer (Life Technologies, California, USA). Four internal primers were used for 18S: hrms18S_ Fil, hrms18S_Fi2, hrms18S_Ril, and hrmsl8S_Ri2 (Oya and Kajihara 2020), and two internal primers were used for 28S: hrms__ fw2 (Oya and Kajihara 2020) and rev4 (Sonnenberg et al. 2007). In addition to the specimens collected in the pres- ent study, a 1,735-bp partial sequence of 18S from the ho- lotype of Cestoplana nopperabo Oya & Kajihara, 2019 was obtained using the same methods described above. Sequences were checked and edited using MEGA ver 7.0 (Kumar et al. 2016). The edited sequences were deposit- ed in DDBJ/EMBL/GenBank, with accession numbers of LC740486—LC740495, LC745667, and LC745668. Zoosyst. Evol. 99 (2) 2023, 363-373 Molecular phylogenetic analyses For phylogenetic analyses, a concatenated dataset (2,834 bp) comprising partial 18S (1,735 bp) and 28S (1,099 bp) sequences was prepared (Table 1). Additional 18S and 28S sequences of three cotylean species, Pericelis flavomarginata Tsuyuki et al., 2020, Prosthiostomum siphunculus (Delle Chiaje, 1828) and Theama mediterranea Curini-Galletti et al., 2008, were used as outgroups (Table 1). Sequences were aligned using MAFFT ver. 7.427 (Katoh et al. 2017) with the L-INS-1 strategy selected using the “Auto” option. Ambiguous sites were trimmed using Clipkit ver. 1.0 via the “kpic” option (Steenwyk et al. 2020). The optimal substitution models selected using PartitionFinder ver. 2.1.1 (Lanfear et al. 2016) according to the Akaike Information Criterion (Akaike 1974) with the greedy algorithm (Lanfear et al. 2012), were GTR + I + G for both the 18S and 28S partitions. A maximum likelihood (ML) analysis was performed using RAXML ver. 8.2.10 (Stamatakis 2014). A Bayesian phylogenetic inference (BI) was performed using MrBayes ver. 3.2.6 (Ronquist and Huelsenbeck 2003; Altekar et al. 2004) with two independent runs of Metropolis-coupled Markov chain Monte Carlo, each consisting of four chains of 2,000,000 generations. All parameters (statefreq, revmat, shape, and pinvar) were unlinked between each position; trees were sampled every 100 generations. The first 25% of trees were discarded as burn-in before a 50% mayority- rule consensus tree was constructed. Convergence was confirmed based on an average standard deviation of split frequencies of 0.001138, potential scale reduction factors for all parameters of 1.000—1.025, and effective sample sizes for all parameters > 404. Nodal support within the ML tree was assessed using an analysis of 1,000 bootstrap (BS) pseudoreplicates (Felsenstein 1985). ML BS values >70% and posterior probability values >90% were considered to indicate clade support. Genetic distances (uncorrected p-distances) were calculated using MEGA ver. X (Kumar et al. 2018) with gaps/missing data deleted completely. 365 Results Molecular analyses Molecular phylogeny The resulting ML and BI trees were identical in terms of to- pology; all six examined species of Cestoplanidae formed a clade with full support (Fig. 1). Within the clade, Euces- toplana and Cestoplana were reciprocally monophyletic, each with support of 1.00PP/97% BS and 0.95PP/66% BS, respectively. Within Cestoplana, C. nopperabo was sister to the remaining three, C. rubrocincta (Grube, 1840), C. salar Marcus, 1949, and C. techa Du Bois-Rey- mond Marcus, 1957, which received full support. The lat- ter two C. salar and C. techa were sisters with low sup- port (0.67PP/62% BS). Genetic distances between cestoplanid species The interspecific genetic distances between our specimens representing E. cf. cuneata and E. ittanmomen sp. nov. were 3.153—3.378% for 16S and 1.107% for 28S, both of which were greater than the intraspecific ones (0.225% for 16S and 0.000% for 28S) observed within two specimens of E. cf. cuneata. We failed to amplify the COI sequence of the holotype of E. ittanmomen sp. nov. using the prim- er pair Acotylea_[COI_F and Acotylea COIR whereas that of the Japanese specimens of E. cf. cuneata was suc- cessfully amplified with the same primers (LC740486-— LC740488). The interspecific genetic distance for COI was 0.000—0.148% within three specimens of EF. cf. cuneata. The interspecific genetic distances for the 28S se- quences among five species of Cestoplanidae available in public databases are shown in Table 2. The minimum val- ue was 0.664% between C. salar and C. techa (both from Brazil), whereas the maximum value within this fam- ily was 6.977% between C. rubrocincta from Italy and Eucestoplana ittanmomen sp. nov. from Japan. Within the same genus, the maximum intraspecific genetic distance was 5.980% between C. rubrocincta and C. nopperabo. Table 1. List of species used for the molecular phylogenetic analysis, GenBank accession numbers, and references, respectively. Species Cestoplanidae Eucestoplana cf. cuneata (SopottEhlers & Schmidt, 1975) Eucestoplana ittanmomen sp. nov. Cestoplana nopperabo Oya & Kajihara, 2019 Cestoplana rubrocincta (Grube, 1840) Cestoplana salar Marcus, 1949 Cestoplana techa Du Bois-Reymond Marcus, 1957 Outgroup Pericelis flavomarginata Tsuyuki et al., 2020 Prosthiostomum siphunculus (Delle Chiaje, 1828) Theama mediterranea Curini-Galletti et al., 2008 GenBank accession Reference 18S rDNA 28S rDNA LC740491 LC740493 This study LC740492 LC740495 This study LC745668 LC322284 Oya and Kajihara (2019); this study MW376751 MW377504 Rodriguez et al. (2021) - KY263653.2 Bahia et al. (2017) - KY263654.2 Bahia et al. (2017) LC672041 LC568535 Tsuyuki et al. (2020); Tsuyuki et al. (2021) MZ292836 MZ292816 Rodriguez et al. (unpub.) MN384707 MN384705 Dittmann et al. (2019) zse.pensoft.net 366 Tsuyuki, A. et al.: A new species of Eucestoplana and E. cf. cuneata from Okinawa Islands 0.99/70 Prosthiostomum siphunculus 1.00/100 0.95/66 Theama mediterranea Eucestoplana ittanmomen sp. nov. 1.00/97 Eucestoplana cf. cuneata Cestoplana salar 0.67/62 Cestoplana techa Cestoplana rubrocincta Cestoplana nopperabo Pericelis flavomarginata Outgroup Figure 1. Maximum likelihood phylogenetic tree based on a concatenated dataset of partial 18S and 28S rDNA sequences. Numbers near nodes are posterior probabilities and bootstrap values. The species names of sequences which are newly determined in this study are indicated in the red. Table 2. Interspecific uncorrected p-distances (%) for the 28S gene fragments between cestoplanid species of which sequences are available in public databases. C. nopperabo C. nopperabo LC322284.1 - = C. rubrocincta MW377504.1 5.980 - C. salar KY263653.2 5.094 L772 C. techa KY263654.2 4.873 1.883 E. ittanmomen sp. nov. 4.430 6.755 E. cf. cuneata 4.651 6.977 Taxonomy Family Cestoplanidae Lang, 1884 Genus Eucestoplana Lang, 1884 Type species. Cestoplana cuneata Sopott-Ehlers & Schmidt, 1975. Eucestoplana cf. cuneata (Sopott-Ehlers & Schmidt, 1975) Figs 2, 3 ?Cestoplana cuneata Sopott-Ehlers & Schmidt, 1975: 210-212, figs 9, 10; Tajika et al. 1991: 335. ?Eucestoplana cuneata (Sopott-Ehlers & Schmidt, 1975): Faubel 1983: 95. Material examined. JAPAN ¢1; Okinawa Prefecture, the Okinawa Islands, Kouri Island, Tokei Beach; 26°42.86'N, 128°1.108'E; intertidal gravelly sediments; 7 Aug. 2021; zse.pensoft.net C. rubrocincta C. salar C. techa E. ittanmomen sp. nov. 0.664 - - 6.091 5.759 - 6.312 5.980 1.107 A. Tsuyuki and Y. Oya leg.; sagittal sections (3 slides); GenBank: LC740488 (COI) and LC740489 (16S); ICHUM 8440. JAPAN 1; same data as above, except for the date (11 Aug. 2021); sagittal sections (4 slides); Gen- Bank: LC740486 (COI), LC740491 (18S), LC740493 (28S); ICHUM 8441. JAPAN ¢1; Okinawa Prefecture, the Okinawa Islands, Okinawa Island, Nagahama Beach; 26°37.45'N, 128°11.06'E; under rocks; 9 Aug. 2021; A. Tsuyuki leg.; sagittal sections (4 slides); GenBank: LC740487 (COI), LC745667 (16S), LC740494 (28S); ICHUM 8442. For comparison, we also examined eight serial sec- tions of Eucestoplana cuneata (as Cestoplana cuneata) (ZMUG 25472 (holotype, three slides) and ZMUG 25473 (paratype, five slides)) and four serial sagittal sections of Eucestoplana cuneata (as Cestoplana cuneata) collected from the Fiji Island. Description. Body slender and elongated, 24-30 mm long and 0.71-0.82 mm wide in living state (Fig. 2A). Pair of eyespot-clusters, each composed of 11-19 eye- spots, distributed along midline in front of brain (Fig. 2B). Zoosyst. Evol. 99 (2) 2023, 363-373 367 Figure 2. Eucestoplana cf. cuneata (Sopott-Ehlers & Schmidt, 1975). A. ICHUM 8442, whole animal in living state, dorsal view; B. ICHUM 8442, magnification of anterior body in living state, dorsal view, showing eyespot distribution; C. ICHUM 8440, schematic diagram of male copulatory apparatus in sagittal view, anterior to the right; D, E. ICHUM 8440, photomicrographs of sagittal sections, anterior to the right, showing male copulatory apparatus; F. ICHUM 8441, photomicrograph of sagittal section, showing female copulatory apparatus, anterior to the right. Abbreviations: br — brain; eg — cement glands; ed — ejaculatory duct; fg — female gonopore; ma — male atrium; mg — male gonopore; ph — pharynx; pv — prostatic vesicle; st — stylet; sy — sem- inal vesicle; te — testicular follicle; va — vagina; 2 — female copulatory apparatus; 4 — male copulatory apparatus. Scale bars: 1 mm (A, B); 100 um (C-—F). Male copulatory apparatus composed of true seminal ves- icle, interpolated prostatic vesicle, and penis papilla with stylet (Fig. 2C—E). Testicular follicles arranged in two lateral, longitudinal rows, about half length of body, run- ning anteriorly from area in front of pharynx (Fig. 2A). Seminal vesicle antero-posteriorly elongated, posteriorly turning 180° right in front of female copulatory apparatus before running forward for short distance and then de- scending ventrally; thick muscular wall coating seminal vesicle, being thinner toward distal portion with forming ejaculatory duct seamlessly (Fig. 2C). Ejaculatory duct 942 um long, extending from proximal end of prostatic vesicle to proximal end of seminal vesicle. Prostatic vesi- cle oval, with 19-um thick muscular wall, lined with thick glandular epithelium (Fig. 2C—E). Penis papilla with wedged, strongly sclerotized stylet (about 60 um long) (Fig. 2C, E). Penis sheath cone-shaped (Fig. 2C, E). Male atrium lined with cilia (Fig. 2E), opening to exterior via male gonopore with depth of about 67 um (Fig. 2C, E). Pair of oviducts running posteriorly, then connecting to the proximal end of vagina independently. After receiving pair of oviducts, vagina curving dorsally and leading to female gonopore without evident cement pouch (Fig. 2F). Adhesive organ present at posterior end of body. Redescription of holotype of E. cuneata. Male cop- ulatory apparatus composed of true seminal vesicle, in- terpolated prostatic vesicle, and penis papilla with stylet (Fig. 3A). Seminal vesicle elongated, posteriorly turning 180° right, and then leading to ejaculatory duct; thick muscular wall coating seminal vesicle, being thinner zse.pensoft.net 368 Tsuyuki, A. et al.: A new species of Eucestoplana and E. cf. cuneata from Okinawa Islands K \ A . hy e eS oS eS a 3 : | 7 i Figure 3. Eucestoplana cuneata (Sopott-Ehlers & Schmidt, 1975), holotype (ZMUG 25472), schematic diagram (A) and photomi- crographs of sagittal sections (B—E) (anterior to the right). A. Male and female copulatory apparatuses; B—D. Male copulatory appa- ratus; E. Adhesive organ. Abbreviations: ad — adhesive organ; ed — ejaculatory duct; fa — female atrium; fg — female gonopore; ma — male atrium; mg — male gonopore; ps — penis sheath; pv — prostatic vesicle; spy — spermiducal vesicle; st —stylet; sv — seminal vesicle; va — vagina. Scale bars: 100 um (A-E). toward distal portion (Fig. 3A—D). Ejaculatory duct A455 um long, extending from proximal end of prostatic vesicle to proximal end of seminal vesicle. Prostatic vesi- cle oval, with 13-um thick muscular wall, lined with thick glandular epithelium (Fig. 3A, C). Penis papilla with sclerotized stylet (Fig. 3A, D). Penis sheath cone-shaped (Fig. 3A, D). Male atrium lined with cilia, opening to ex- terior via male gonopore with depth of about 95 um (Fig. 3D). Pair of oviducts running posteriorly, then connecting to proximal end of vagina independently. Vagina curving dorsally after receiving oviducts. Adhesive organ present at posterior end of body (Fig. 3E). Supplementary description of the specimen of E. cuneata from the Fiji Islands. Male copulatory appa- ratus composed of true seminal vesicle, interpolated pros- tatic vesicle, and penis papilla. Stylet not well observed possibly due to fixation state. Seminal vesicle elongated, posteriorly turning 180° right, and then leading to ejacu- latory duct. Ejaculatory duct running to anterior, curving posteriorly behind male atrium, then connecting to proxi- mal end of prostatic vesicle; part of ejaculatory duct from proximal end of seminal vesicle to proximal end of pros- zse.pensoft.net tatic vesicle ca. 1 mm long. Prostatic vesicle oval; inter- nal glandular epithelium not well observed possibly due to fixation state. Penis sheath cone-shaped. Male atrium lined with cilia, opening to exterior via male gonopore. Female reproductive organs and adhesive organ not avail- able to be observed possibly due to fixation state. Remarks. Eucestoplana cuneata was originally de- scribed from the Galapagos Islands. Our re-examination of the holotype revealed that the ejaculatory duct from proximal end of prostatic vesicle to proximal end of sem- inal vesicle was over twice as long as that in the original description (Sopott-Ehlers and Schmidt 1975, fig. 9). We tentatively identified the present specimens from Kouri Island as Eucestoplana cf. cuneata. The specimens were consistent with the type specimens of EF. cuneata in having: 7) the eyespots distributed only anterior to the brain, 7) the wedged sclerotized stylet, iii) an adhe- Sive organ at posterior end of body, iv) the conical penis sheath, and v) the fully ciliated inner wall of male atri- um (Sopott-Ehlers and Schmidt 1975). The following morphological differences between the specimens from Japan and the Galapagos Islands should be tested by Zoosyst. Evol. 99 (2) 2023, 363-373 genetic analyses if these are interspecific or intraspecif- ic: 7) body length (24-30 mm in our specimens; 10 mm in the original description), i7) eyespot number (about 30 in our specimens; 35—40 in the original description), and iii) length of ejaculatory duct from the proximal end of prostatic vesicle to proximal end of seminal vesicle (over 900 um in our specimens; 455 um in the holotype). The wide range of distribution of E. cuneata needs to be verified in future studies. So far, this species has been collected from the Galapagos Islands (Sopott-Ehlers and Schmidt 1975) and the Fiji Islands (Tajika et al. 1991). Our re-examination of a specimen collected from the Fiji Islands suggested that it corresponded to the holo- type of E. cuneata in having the 7) conical shape of penis sheath and ii) the fully ciliated inner wall of male atrium. However, the Fiji specimen might be identical to E. cf. cuneata from the Okinawa Islands because it was more similar to Japanese specimens by having a long ejacula- tory duct (about 1 mm). Future studies will resolve the doubt of the actual distribution of E. cuneata by compar- ing their morphology such as the body length, the eyespot number, and the male reproductive organs in more detail between different populations from the Galapagos Islands and the Fiji Islands. Eucestoplana ittanmomen sp. nov. https://zoobank. org/0D14C91F-156B-46C2-88C4-B 1 E63F94AC34 Figs 4, 5 Material examined. Holotype: JAPAN °1; Okinawa Pre- fecture, the Okinawa Islands, Kouri Island, Tokei Beach; 26°42.86'N, 128°1.108'E; intertidal gravelly sediments; 11 Aug. 2021; A. Tsuyuki and Y. Oya leg.; sagittal sec- tions (6 slides); GenBank: LC740490 (16S), LC740492 (18S), and LC740495 (28S); ICHUM 8443. Paratype: JAPAN °1; same data as for holotype; sagittal sections (4 slides); ICHUM 8444. 369 Type locality. Japan, Okinawa Prefecture, Kuniga- mi, Nakiin, Kouri Island, Tokei Beach (26°42 .86'N, 128°1.108'E). Diagnosis. Body slender and elongated; anterior mar- gin rounded; dorsal surface translucent white without any color pattern; pair of eyespot-clusters distributed along midline in front of brain; penis papilla with heavily scle- rotized stylet; penis sheath dome-shaped with external epithelium covered with cilia; cilia absent in inner wall of male atrium; adhesive organ present at posterior end of body. Description of holotype. Body slender and elongated, 26 mm long and 0.75 mm wide in living state (Fig. 4A); anteriorly rounded, spreading like fan; posteriorly ta- pered. Dorsal surface smooth, translucent, without any color pattern. Ventral surface translucent. Tentacles ab- sent. Pair of eyespot-clusters, each composed of 12—14 eyespots (12 on left; 14 on right), distributed along midline in front of brain (Fig. 4B), spreading out in fan shape anteriorly. Intestine highly branched without anas- tomosing, spreading throughout body, not reaching body margin. Pharynx ruffled, 1.94 mm long, situated on last fourth of body (Fig. 4A, C). Mouth opening at last third of pharyngeal pouch (Fig. 4C). Male gonopore opening at last ninth of body (Fig. 4A). Female gonopore situated posterior to male gonopore. Male copulatory apparatus consisting of true seminal vesicle, interpolated prostatic vesicle, and penis papilla with stylet (Fig. 4A). Testicular follicles arranged in single, lateral, longitudinal row on each side, about half length of body, running anteriorly from area in front of pharynx (Fig. 4A). Pair of sperm ducts separately entering proximal end of seminal vesi- cle; each duct forming spermiducal vesicle before enter- ing seminal vesicle (Fig. 5A, B). Seminal vesicle extend- ing posteriorly, about 700 um long and 90 um wide at its widest point, posteriorly turning 180° right in front of female copulatory apparatus before running anteriorly to lead to ejaculatory duct at position of proximal end of Ve on “as * at os _ . ~~ *« = ~ ed . ; ; rap : “ Aer . fog Vy » ea - x. ty wa - Sane fay i ee art . “so . - 2 oe ‘= > ° ‘, *, . sae v wy ™ 4 - sad a © on x 5, Vee me hry Le ond 4 > ta BSS CSR re ee a es sever 4 eo 2) get SESS ae fa-ie "= o>. ~ See re = < *. ah, iy a e* 5” , i ad bo A) =e. aK Le ws. ion eee Be ee hae) oe, raids ? a A Pe 4. + mae = * ee . Me ee 4.07 “a == Ate \ ¥y race Lace = oP eth 0 +7 = = — ot a Tecmo a ee om | = —— 5 a ‘Su. uti mY . ‘<2. k Figure 4. Eucestoplana ittanmomen sp. nov., holotype (ICHUM 8443). A. Whole animal in living state, dorsal view; B. Magnifica- tion of anterior body, dorsal view, showing eyespot distribution; C. Photomicrograph of sagittal section (anterior to the right), show- ing pharynx and mouth. Abbreviations: mo — mouth; ph — pharynx; te — testicular follicle; 2 — female copulatory apparatus; o — male copulatory apparatus. Scale bars: 1 mm (A, B); 100 um (C). zse.pensoft.net 370 Tsuyuki, A. et al.: A new species of Eucestoplana and E. cf. cuneata from Okinawa Islands Le Ee r' 4 ~~ Figure 5. Eucestoplana ittanmomen sp. noy., schematic diagram (A) and photomicrographs of sagittal sections (B—F) (anterior to the right). A. ICHUM 8443 (holotype), male and female copulatory apparatuses; B—D. ICHUM 8443 (holotype), male copulatory apparatus; E. ICHUM 8443 (holotype), female copulatory apparatus; F. ICHUM 8444 (paratype), adhesive organ. Abbreviations: ad — adhesive organ; eg — cement glands; ep — cement pouch; ed — ejaculatory duct; fg — female gonopore; ma — male atri- um; mg — male gonopore; po — penis pouch; pp — penis papilla; ps — penis sheath; pv — prostatic vesicle; spy — spermiducal vesicle; st —stylet; sy — seminal vesicle; va — vagina. Scale bars: 100 um (A-F). penis stylet; thick muscular wall, about 19 um thickness, coating seminal vesicle and ejaculatory duct (Fig. 5A, B). Prostatic vesicle oval, elongated, with about 18-um thick muscular wall, lined with thick glandular epithelium; dis- tal end of prostatic vesicle forming penis papilla (Fig. 5A, C). Penis papilla with wedged, strongly sclerotized stylet (131 um long), projecting into male gonopore (Fig. 5A, zse.pensoft.net D). Penis sheath dome-shaped, about 184 um wide at its widest point, housing penis stylet (Fig. SA, C, D); exter- nal epithelium being exposed to male atrium, former be- ing lined with cilia (Fig. 5C, D); penis pocket lined with non-ciliated epithelium. Male atrium lined with thin epi- thelium without cilia (Fig. 5C, D). Male gonopore about 27 um deep. Female copulatory organ lacking Lang’s Zoosyst. Evol. 99 (2) 2023, 363-373 vesicle. Pair of oviducts running posteriorly, then con- necting to proximal end of vagina independently. Vagina narrow, curved dorsoventrally, lined with ciliated epi- thelium, leading to female gonopore via narrow cement pouch (Fig. 5A, E). Numerous cement glands releasing their contents into cement pouch (Fig. SE). Adhesive or- gan located at posterior end of body. Description of paratype. Due to lack of anterior part of body, body length, width and eyespot arrangements unknown. Body coloration same as holotype. Pharynx ruffled, 1.27 mm in length; mouth opening at posterior region of pharyngeal pouch. Male copulatory apparatus composed of elongate seminal vesicle, interpolated pros- tatic vesicle, and penis papilla with wedged stylet (106 um long); penis stylet slenderer than that of holotype. Penis sheath dome-shaped, with external epithelium cili- ated; numerous eosinophilic glands piercing distal part of penis sheath. Male atrium covered with non-ciliated ep- ithelium. Female copulatory apparatus same as holotype except for shape of cement pouch being more expanded than that of holotype. Adhesive organs present at posteri- or end of body (Fig. 5F). Etymology. The specific name ittanmomen (Ittan-momen) is a Japanese noun, representing the name of one of the “yokai” (a class of supernatural entities and spirits in Japanese folklore). It is named after the long and narrow cloth-like white body of the flatworm, which evokes the similar-looking yokai, Ittan-momen. Distribution. To date, only from the Okinawa Islands, Japan. Remarks. Our specimens belong to Eucestoplana based on the following characteristics: 7) the evident sclerotized penis stylet and i) a female copulatory ap- paratus without any accessory ducts or Lang’s vesicle. Eucestoplana ittanmomen sp. nov. can be easily distin- guished from E. meridionalis by the following charac- teristics: 7) translucent body, ii) fewer eyespots distrib- uted only anterior to the brain, and iii) the presence of the adhesive organ (Table 3). Our new species is most Fil similar to E. cuneata in having the following character- istics: 7) around 30 eyespots distributed only anterior to the brain, i) a wedge-shaped stylet, and 777) the adhesive organ located on the posterior end of the body. However, E. ittanmomen sp. nov. is differentiated from EF. cuneata by the following characteristics: 7) the shape of the penis sheath (dome-shaped in EF. ittanmomen sp. nov.; cone- shaped in E. cuneata), ii) the arrangement of the cilia in the inner wall of the male atrium (only present along the outside of the penis sheath in E. ittanmomen sp. nov.; surrounding the whole male atrium in E. cuneata), and iii) the stylet length (106-131 um in E. ittanmomen sp. nov.; 70 um in FE. cuneata). Eucestoplana ittanmomen sp. nov. can be also distinguished from &. cf. cuneata col- lected from the same locality by the same morphological differences as mentioned above. In addition, the genetic distance for 16S and 28S sequences between them could support that the two entities are likely to be genetically independent. The values for 16S (3.153%—3.378%) were much larger than the three interspecific values 0.5—1.8%, which were observed among three species of Notocom- plana (N. hagiyai, N. japonica, and N. koreana) (Oya and Kajihara 2017). The p-distance between the 28S se- quences of E. ittanmomen sp. nov. and E. cf. cuneata was also much larger than that between C. salar and C. techa, which are clearly different species because of their mor- phological difference (Table 2). Discussion The molecular phylogeny presented here reveals that the two Eucestoplana species, E. cf. cuneata and E. ittan- momen sp. nov., were most closely related to each other (Fig. 1). This phylogenetic closeness suggests synapo- morphic traits within the cestoplanid lineage. Indeed, the 7) heavily sclerotized penis stylet, i7) reduced num- ber of eyespots, and iii) preference for gravelly intersti- tial habitats may be unique features of representatives Table 3. Comparison of the selected characteristics among the known Eucestoplana species and our new species. E. cuneata E. ittanmomen sp. nov. E. meridionalis Body length (mm) LO3 26 20 Body width (mm) slender, ribbon-shaped) 0.7 5 Anterior body shape Rounded Rounded Slightly pointed Eyespots 35-40", only anterior to the brain About 20-30, only anterior to the brain Numerous, distributed around brain Dorsal coloration ? @ translucent white> Translucent white Chocolate-brown Dorsal color pattern Pi Absent Absent Mouth position Near posterior end of pharynx Near posterior end of pharynx In posterior region of pharyngeal cavity Seminal vesicle Elongate, bending 180° Elongated, bending 180° at position posterior to female reproductive organ 106-131-um long; wedge-shaped Stylet 70-um long; wedge-shaped Penis sheath Cilia along inner Cone-shaped Surrounding the whole male atrium Elongate-oval Present Dome-shaped Cone-shaped Cilia along inner “Surrounding the whole male atrium Only present along the outside ofthe tS Adhesive organ Present Distribution penis sheath Present Absent South Australia The Galapagos Islands?; Fiji’ The Okinawa Islands, Japan Reference aSopott-Ehlers and Schmidt (1975); >Tajika et al. (1991) This study Prudhoe (1982a); Prudhoe (1982b) zse.pensoft.net Sie Tsuyuki, A. et al.: A new species of Eucestoplana and E. cf. cuneata from Okinawa Islands of Eucestoplana. Although we were unable to include Cestoplana nexa Sopott-Ehlers & Schmidt, 1975 in our phylogenetic analyses, future studies may show that this species should affiliate with Eucestoplana rather than Cestoplana because the latter two characteristics of eyespot number and habitats are also found in this species. Further investigations involving more cesto- planid species are necessary to confirm the monophyly of Cestoplana and Eucestoplana. Additional species such as E. meridionalis, the other four species of Cestoplana, and representatives of the other four genera, viz. Acestoplana, Cestoplanella, Cestoplanides, and Cestoplanoida should be included in future studies to gain a more comprehen- sive understanding of the relationships within this family. Acknowledgments AT and HK are grateful to Prof. Maria Teresa Aguado Molina (Biodiversitétsmuseum Gottingen) for kindly al- lowing us to borrow the type specimens of Eucestoplana cuneata and to Dr. Jorn von Dohren (University of Bonn) for putting us in contact with Prof. Aguado Molina. The authors would like to thank Enago (www.enago.jp) for the English-language review. We thank four reviewers for giving us insightful comments to improve our man- uscript. This study was funded by the Research Institute of Marine Invertebrates under Grant FY2019 No. 15 for AT and by the Japan Society for the Promotion of Science (JSPS) under KAKENHI grant number 20J11958 to YO. References Akaike H (1974) A new look at the statistical model identification. IEEE Transactions on Automatic Control 19(6): 716-723. https://doi. org/10.1109/TAC.1974.1100705 Altekar G, Dwarkadas S, Huelsenbeck JP, Ronquist F (2004) Parallel Metropolis coupled Markov chain Monte Carlo for Bayesian phy- logenetic inference. Bioinformatics 20(3): 407-415. https://doi. org/10.1093/bioinformatics/btg427 Bahia J, Padula V, Schrodl M (2017) Polycladida phylogeny and evo- lution: Integrating evidence from 28S rDNA and morphology. Organisms, Diversity & Evolution 17(3): 653-678. https://doi. org/10.1007/s13127-017-0327-5 Curini-Galletti M, Campus P, Delogu V (2008) Theama mediterranea sp. nov. (Platyhelminthes, Polycladida), the first interstitial poly- clad from the Mediterranean. The Italian Journal of Zoology 75(1): 77-83. https://doi.org/10.1080/11250000701690525 Delle Chiaje S (1828) Memorie sulla storia e notomia degli animali sen- za vertebre del regno di Napoli. Vol. II. Fratelli Fernandes, Napoli, 232 pp. https://doi.org/10.5962/bh1.title. 10021 Dittmann IL, Cuadrado D, Aguado MT, Norefia C, Egger B (2019) Polyclad phylogeny persists to be problematic. Organisms, Diver- sity & Evolution 19(4): 585-608. https://doi.org/10.1007/s13127- 019-00415-1 Du Bois-Reymond Marcus E (1957) On Turbellaria. Anais da Academia Brasileira de Ciéncias 29(1): 153-191. zse.pensoft.net Faubel A (1983) The Polycladida, Turbellaria. Proposal and establish- ment of a new system. Part I. The Acotylea. Mitteilungen aus dem Hamburgischen Zoologischen Museum und Institut 80: 17-121. Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution; International Journal of Organic Evolution 39(4): 783-791. https://doi.org/10.2307/2408678 Grube E (1840) Actinien, Echinodermen und Wtrmer des adriatischen- und Mittelmeers, nach eigenen Sammlungen beschrieben. Verlag von J.H. Bon, Konigsberg, 1-106. https://doi.org/10.5962/bh1.ti- tle.23025 Katoh K, Rozewicki J, Yamada KD (2017) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visu- alization. Briefings in Bioinformatics 20(4): 1160-1166. https://doi. org/10.1093/bib/bbx108 Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolution- ary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870-1874. https://doi.org/10.1093/ molbev/msw054 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular evolutionary genetics analysis across computing plat- forms. Molecular Biology and Evolution 35(6): 1547-1549. https:// doi.org/10.1093/molbev/msy096 Lanfear R, Calcott B, Ho SYW, Guindon S (2012) PartitionFinder: Combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29(6): 1695-1701. https://doi.org/10.1093/molbev/mss020 Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2016) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic anal- yses. Molecular Biology and Evolution 34: 772-773. https://doi. org/10.1093/molbev/msw260 Lang A (1884) Die Polycladen (Seeplanarien) des Golfes von Neapel und der angrenzenden Meeresabschnitte. Eine Monographie. Wilhelm Engelmann, Leipzig, 1-172. https://doi.org/10.5962/bhl. title. 10545 Marcus E (1949) Turbellaria Brasileiros (7). Boletim da Faculdade de Filosofia, Ciéncias e Letras, Universidade de Sao Paulo. Zoologia 14: 7-155. https://doi.org/10.11606/issn.2526-4877.bsficlzoolo- gia. 1949.129106 Oya Y, Kajihara H (2017) Description of a new Notocomplana species (Platyhelminthes: Acotylea), new combination and new records of Polycladida from the northeastern Sea of Japan, with a comparison of two different barcoding markers. Zootaxa 4282(3): 526-542. https://do1.org/10.11646/zootaxa.4282.3.6 Oya Y, Kajihara H (2019) A new bathyal species of Cestoplana (Poly- cladida: Cotylea) from the West Pacific Ocean. Marine Biodiversity 49(2): 905-911. https://doi.org/10.1007/s12526-018-0875-8 Oya Y, Kajihara H (2020) Molecular phylogenetic analysis of Acotylea (Platyhelminthes: Polycladida). Zoological Science 37(3): 271-279. https://doi.org/10.2108/zs190136 Palumbi S, Martin A, Romano S, McMillan WO, Stice L, Grabows- ki G (1991) The Simple Fools Guide to PCR. Ver. 2. Department of Zoology and Kewalo Marine Laboratory, University of Hawaii, Honolulu, 45 pp. Prudhoe S (1982a) Polyclad flatworms. In: Shepherd SA, Thomas IM (Eds) Marine Invertebrates of Southern Australia. Handbook of the Flora and Fauna of South Australia. Part I: South Australian Government, Adelaide, 220-227. Zoosyst. Evol. 99 (2) 2023, 363-373 Prudhoe S (1982b) Polyclad turbellarians from the southern coasts of Australia. Records of the South Australian Museum (Adelaide) 18: 361-384. Prudhoe S (1985) A Monograph on Polyclad Turbellaria. Oxford University Press, Oxford, 259 pp. Rodriguez J, Hutchings PA, Williamson JE (2021) Biodiversity of intertidal marine flatworms (Polycladida, Platyhelminthes) in southeastern A ustra- lia. Zootaxa 5024(1): 1-63. https://do1.org/10.11646/zootaxa.5024.1.1 Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19(12): 1572-1574. https://do1.org/10.1093/bioinformatics/btg 180 Sonnenberg R, Nolte AW, Tautz D (2007) An evaluation of LSU rDNA D1-D2 sequences for their use in species identification. Frontiers in Zoology 4(1): 1-12. https://doi.org/10.1186/1742-9994-4-6 Sopott-Ehlers B, Schmidt P (1975) Interstitielle Fauna von Galapagos XIV. Polycladida (Turbellaria). Mikrofauna des Meeresbodens 54: 193-222. Stamatakis A (2014) RAxML version 8: A tool for phylogenetic anal- ysis and post-analysis of large phylogenies. Bioinformatics 30(9): 1312-1313. https://doi.org/10.1093/bioinformatics/btu033 Steenwyk JL, Buida TJ, Li Y, Shen X-X, Rokas A (2020) ClipKIT: A multiple sequence alignment trimming software for accurate phy- logenomic inference. PLoS Biology 18(12): e3001007. https://doi. org/10.1371/journal.pbio.3001007 373 Tajika K-I, Raj U, Horiuchi S, Koshida Y (1991) Polyclad turbellar- ians collected on the Osaka University Expedition to Viti Levu, Fiji, in 1985, with remarks on distribution and phylogeny of the genus Discoplana. Hydrobiologia 227(1): 333-339. https://doi. org/10.1007/BF00027619 Tsuyuki A, Oya Y, Jimi N, Kajihara H (2020) Description of Perice- lis flavomarginata sp. nov. (Polycladida: Cotylea) and its predatory behavior on a scaleworm. Zootaxa 4894(3): 403-412. https://doi. org/10.11646/zootaxa.4894.3.6 Tsuyuki A, Oya Y, Kajihara H (2021) Two new species of the marine flatworm Pericelis (Platyhelminthes: Polycladida) from southwest- ern Japan with an amendment of the generic diagnosis based on phy- logenetic inference. Marine Biology Research 17(9-10): 946-959. https://doi.org/10.1080/17451000.2022.2048669 Tsuyuki A, Oya Y, Kajihara H (2022) Reversible shifts between inter- stitial and epibenthic habitats in evolutionary history: molecular phylogeny of the marine flatworm family Boniniidae (Platyhel- minthes: Polycladida: Cotylea) with descriptions of two new spe- cies. PLoS ONE 17(11): e0276847. https://doi.org/10.1371/journal. pone.0276847 Tsuyuki A, Oya Y, Jimi N, Hookabe N, Fujimoto S, Kajihara H (2023) Theama japonica sp. nov., an interstitial polyclad flatworm showing a wide distribution along Japanese coasts. Zoological Science 40(3): 262-272. https://doi.org/10.2108/zs220105 zse.pensoft.net