Species Diversity 24: 1–6 Published online 25 January 2019 DOI: 10.12782/specdiv.24.1 A New Species of Phaenoplana (Platyhelminthes: Polycladida) from the Ogasawara Islands Yuki Oya1,3 and Hiroshi Kajihara2 1 Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan E-mail: [email protected] 2 Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan 3 Corresponding author (Received 26 July 2018; Accepted 8 November 2018) http://zoobank.org/8282F788-F3C7-4918-99D3-6C672BA8DE58 We describe a new species of polyclad flatworm, Phaenoplana kopepe sp. nov., from Chichijima island in the Ogas- awara Islands, Japan. This is the first report of Phaenoplana from Japan. Phaenoplana kopepe sp. nov. is characterized by i) a vagina that curves anteriorly, ii) gonopores well-separated from each other, and iii) a Lang’s duct that is shorter than the vagina. We provide a partial sequence (610 bp) from the mitochondrial cytochrome c oxidase subunit I gene as a DNA bar- code for the species. Key Words: Acotylea, COI, cox1, DNA barcoding, marine flatworm, Pacific, Stylochoplanidae. cus, 1952 in the reproductive organs and its epithelial ultra- Introduction structure. The morphological heterogeneity and the conflict in taxonomic opinions between Faubel (1983) and Prudhoe The polyclad flatworm genus Phaenoplana Faubel, 1983, (1985) indicate that Phaenoplana requires a thorough revi- belonging to Stylochoplanidae, is distinguished from other sion. However, such a taxonomic revision should involve ex- stylochoplanid genera by possessing a penis rod and a Lang’s tensive molecular phylogeny of acotyleans, which is beyond vesicle. Faubel (1983) assigned five species to Phaenoplana; the scope of this paper. the history of taxonomic changes of each species has been In this study, we describe a new species of Phaenoplana detailed in Pérez-García et al. (2018). Additionally, Pérez- as the first representative of the genus from Japan, providing García et al. (2018) described one new species in Phaeno- a partial sequence from the cytochrome c oxidase subunit I plana from the East Atlantic. Thus, six species are currently (COI) gene as a DNA barcode for the species. classified in Phaenoplana (Tyler et al. 2006–2016; Pérez-García et al. 2018). All of them have been reported from tropical and subtropical areas (Fig. 1), but not from Japanese waters. Materials and Methods Phaenoplana is a morphologically heterogeneous as- semblage and taxonomically problematic. Of the major Two specimens were collected intertidally in Chichijima morphological characters that have been used in the lepto- island under permission from the Tokyo Metropolitan Gov- planoid taxonomy, the penis rod, seminal vesicle, and ten- ernment to collect wild animals (Permission No. 28, Special tacular and cerebral eyes are the only ones that are shared Permission No. 29). Worms were anaesthetized in a MgCl2 by all the members of Phaenoplana. Faubel (1983) stated in solution prepared with tap water so that it had the same the definition of Phaenoplana that they usually have a horse- refractive index (or ‘salinity’) as the seawater, using an IS/ shoe-like Lang’s vesicle, but actually only three—P. chal - Mill-E refractometer (AS ONE, Japan), and then photo- lengeri (Graff, 1892), P. conoceraea (Schmarda, 1859), and P. graphed with a Nikon D5200 digital camera with external taiwanica (Kato, 1943)—have such a Lang’s vesicle. Another strobe lighting provided by a pair of Morris Hikaru Koma- species, Phaenoplana peleca (Du Bois-Reymond Marcus chi Di flash units. For DNA extraction, a piece of the body and Marcus, 1968), which was transferred from Phaenocelis margin (lateral to ocelli) was cut away from each specimen Stummer-Traunfels, 1933 (Cryptocelidae) to Phaenoplana and fixed in 100% ethanol. The rest of the body was fixed in by Faubel (1983), has marginal eyes. Prudhoe (1985) placed Bouin’s solution for 24 hours and preserved in 70% ethanol. Phaenoplana peleca in Phaenocelis while classifying other It was then cut into two (anterior and posterior) pieces. The congeners (except for Phaenoplana caetaria Pérez-García, anterior piece not containing the pharynx was dehydrated Noreña and Cervera, 2018) in Stylochoplana Stimpson, in an ethanol series, cleared in xylene, and mounted on a 1857. Liana et al. (2012) argued that Phaenoplana peleca glass slide in Entellan New (Merck, Germany). The posterior should be placed in Phaenocelis rather than in Phaenoplana, piece containing the pharynx was also dehydrated in an eth- based on the similarity between Phaenocelis medvedica Mar- anol series and cleared in xylene, then embedded in paraffin © 2019 The Japanese Society of Systematic Zoology 2 Y. Oya and H. Kajihara Fig. 1. Map showing distribution of Phaenoplana species: 1, P. conoceraea (Schmarda, 1859) from east coast of Sri Lanka; 2, P. challengeri (Graff, 1892) from Humboldt Bay, New Guinea; 3, P. taiwanica (Kato, 1943) from Su’ao, Taiwan; 4, P. longipenis (Hyman, 1953) from coast of south California to Mexico and Neguange Bay, Colombia (Quiroga et al. 2004); 5, P. peleca (Du Bois-Reymond Marcus and Marcus, 1968) from Curaçao Island; 6, P. caetaria Pérez-García, Noreña and Cervera, 2018 from Cádiz, Spain; 7, P. kopepe sp. nov. from Chichijima island, Ogasawara Islands. wax, sectioned at 7 µm thickness, stained with hematoxylin and eosin, and also mounted in Entellan New (Merck, Ger- Taxonomy many). They were observed under an Olympus BX51 com- pound microscope and photographed with a Nikon D5200 Family Stylochoplanidae Meixner, 1907 digital camera. Type slides have been deposited in the In- vertebrate Collection of the Hokkaido University Museum, Stylochoplaninae Meixner, 1907: 447. Sapporo, Japan (ICHUM). Stylochoplanidae: Faubel 1983: 98. Total DNA was extracted using silica (Boom et al. 1990). A COI fragment (610 bp) was amplified by nested poly- Nomenclatural remarks. Tyler et al. (2006–2016) as- merase chain reaction (PCR) because a standard PCR am- cribed the authorship and date of Stylochoplanidae to plification with the outer primers (see below) yielded poor Faubel (1983). However, according to the Principle of Co- chromatogram data after sequencing. The first PCR ampli- ordination as stipulated in Article 36.1 of the International fication conditions were 94°C for 5 min; 35 cycles of 94°C Code of Zoological Nomenclature (International Commis- for 30 s, 50°C for 30 s, and 72°C for 1.5 min; and 72°C for sion on Zoological Nomenclature 1999), these should be 7 min, with the outer primers Acotylea_COI_F and Acoty- deemed as Meixner (1907), who established the subfamily lea_COI_R (Oya and Kajihara 2017). The second set of Stylochoplaninae before Faubel (1983) upgraded it to the conditions were 94°C for 5 min; 35 cycles of 94°C for 30 s, family level. 58°C for 30 s, and 72°C for 1 min; and 72°C for 7 min, with the nested internal primer pair, nestCOI_F1 (5′-GGT GT Phaenoplana kopepe sp. nov. A TGG TCA GGT CTA ATRGGWAC-3′) and nestCOI_R1 (Figs 2, 3) (5′-ATA CTT CCG GAT GAC CAA AGA AYCA-3′); these primers were newly designed with PerlPrimer ver. 1.1.21 Etymology. The new specific name is an indeclinable (Marshall 2003–2011) based on COI sequences determined noun, taken from the type locality, Kopepe Beach. Accord- from the first author’s personal polyclad specimens. All nu- ing to a local anecdote, Kopepe was the name of a man who cleotide sequences were determined by direct sequencing came from the Gilbert Islands all the way across the Pacific with a BigDye Terminator Kit ver. 3.1 and ABI 3730 Genetic to Chichijima island and lived near the beach. Analyzer (Life Technologies, California, USA). Sequences Material examined. Two specimens: holotype, ICHUM were checked and edited using MEGA ver. 5.2 (Tamura et 5343, 10 slides (1 slide for whole mount of the anterior body al. 2011). All sequences determined in this study have been and 9 slides for serial sagittal sections), intertidal, Kopepe deposited in DDBJ/EMBL/GenBank databases with the ac- Beach (27°03′52″N, 142°11′32″E), Chichijima island, the cession numbers LC369778 and LC369779. Ogasawara Islands, Japan, 3 September 2016, Y. Oya leg.; New species of Phaenoplana 3 Fig. 2. Phaenoplana kopepe sp. nov., ICHUM5343 (holotype), photographs taken in life and sketch of cerebral eyes. A, Dorsal view; B, ven- tral view; C, tentacles; D, distribution of cerebral eyespots. Abbreviations: fg, female gonopore; mg, male gonopore; ov; oviduct, ph, pharynx, sd; sperm duct, te; tentacle. paratype, ICHUM 5344, 5 slides (1 slide for whole mount male gonopores separate; male gonopore opening at about of the anterior body and 4 slides for serial sagittal sections), one-third of body length (5.6–7 mm, 7 mm in holotype) collected along with the holotype. from posterior end; female gonopore situated 0.5–0.7 mm Description. Live specimens 17–21 mm (21 mm in ho- (0.7 mm in holotype) posterior to male gonopore (Fig. 2B). lotype) in length, 5.5–7 mm (7 mm in holotype) in width. Male copulatory apparatus located posteriorly to pharynx Body thin, elongate oval, narrow toward posterior end. (Fig. 3A), consisting of true seminal vesicle, interpolated Ground body color translucent to whitish opaque. General prostatic vesicle, and penis rod (Fig. 3B, C). Pair of sperm appearance of body light brown (Fig. 2A, B). Dorsal body ducts running anteriorly, then turning medially at point tinged with light brown due to minute granules scattered of about one-fifth length (1.4 mm in holotype, not clearly over entire surface except around margin. Dorsal surface of visible in paratype) of pharynx from posterior end, subse- body around pharynx brown. Body margin translucent. Pair quently running posteriorly along both sides of pharynx of tentacles present at between one-fifth to one-sixth body and extending further posteriorly for short distance beyond length (3.4–3.5 mm, 3.5 mm in holotype) from anterior end level of posterior end of pharynx, before turning medially (Fig.