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Vol. 26: 159–167, 2017 AQUATIC BIOLOGY Published September 5§ https://doi.org/10.3354/ab00682 Aquat Biol OPENPEN ACCESSCCESS A molecular framework for the taxonomy and systematics of Japanese marine turbellarian flatworms (Platyhelminthes, Polycladida) Tadasuke Tsunashima1,**, Morio Hagiya1,**,†, Riko Yamada1, Tomoko Koito1, Nobuaki Tsuyuki1, Shin Izawa1, Keita Kosoba2, Shiro Itoi1,*, Haruo Sugita1 1Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa 252-0880, Japan 2The Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan ABSTRACT: The order Polycladida comprises a highly diverse and cosmopolitan group of marine turbellarian flatworms. Owing to the great morphological diversity and the absence of a molecular phylogeny, the classification of this group has always been controversial. Here we seek to add resolution by reporting the results of molecular phylogenetic analysis based on sequences of the 28S ribosomal RNA gene, thus providing a framework for understanding relationships and the evolution of characters within the group. The phylogeny provides strong support for 10 and 7 distinct families within the suborders Acotylea and Cotylea, respectively. In addition, an analysis based on the mitochondrial gene sequences (cytochrome c-oxidase subunit I) reveals further details of the relationships within Acotylea, which were classified by morphological analysis, but not by 28S rRNA sequence-based analyses. These analyses also showed that several species corresponded to previously described genera based on morphological features and character combinations. We conclude that a classification of genera in Acotylea and Cotylea based on molecular phylogeny reflects the morphological diversity of these polyclad flatworms. KEY WORDS: Flatworms · Molecular systematics · Morphology · Polycladida · Taxonomy INTRODUCTION mobile prey (Miyazawa et al. 1986, 1987, Tanu et al. 2004, Ritson-Williams et al. 2006, Yamada et al. Polyclad turbellarian flatworms commonly occur in 2017). There have also been several reports of dogs coastal and rocky reefs worldwide (Hyman 1951), in New Zealand having been affected after feeding and are a significant presence in marine ecosystems. on the opisthobranch Pleurobranchaea maculata which For example, Stylochus spp. are known predators had washed up on the beach after having been toxi- of commercially important oysters (Arakawa 1970, fied by the polyclad Stylochoplana sp. (McNabb et Galleni 1976, Newman et al. 1993) and the mussel al. 2010, Wood et al. 2012, Salvitti et al. 2015). Mytilus galloprovincialis (Galleni et al. 1980). In Polyclad flatworms are acoelomate bilaterians, addition, several planocerid species accumulate high usually hermaphroditic, oviparous with densely cili- concentrations of tetrodotoxin in their eggs—as a ated epithelial cells, and are characterized by a possible defense against predators—and in the phar- highly branched intestine (Rieger et al. 1991). They ynx, where it has been shown to aid in capturing also lack a circulatory system or specific respiratory *Corresponding author: [email protected] **These authors contributed equally to this work © The authors 2017. Open Access under Creative Commons by †Deceased Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. §Corrections were made after publication. For details see www.int-res. com/abstracts/ab/v26/c_p159-167/ Publisher: Inter-Research · www.int-res.com This corrected version: September 26, 2017 160 Aquat Biol 26: 159–167, 2017 or gans, relying on the epidermis for gas exchange MATERIALS AND METHODS (Rieger et al. 1991). Polycladida contains 2 suborders, Acotylea and Cotylea, which are distinguished Animals by the absence or presence of a ventral sucker, respectively (Lang 1884). Further classification Polyclad flatworms were collected from inshore below the suborder level has traditionally been reefs at Hayama (35° 15’ N, 139° 34’ E) and Man- based on morphological characteristics, such as the azuru, Kanagawa (35° 8’ N, 139° 9’ E), and Shimoda, presence and arrangement of eyespots (e.g. cere- Shizuoka (Ebisu Island: 34° 39’ N, 138° 57’ E; Tsume - bral, tentacular, clustered, and marginal eyespots), kizaki: 34° 39’ N, 138° 59’ E), Japan (from 2 locations the presence of either true tentacles or pseudotenta- at Table S1 in the Supplement at www.int-res.com/ cles (i.e. mere folds of the anterior body margin), articles/ suppl/b026p159_supp.pdf). These areas har- and the structure of the pharynx (Newman & bor a variety of flatworm species found in the Japan- Cannon 1994). ese Islands (Kato 1944). Each specimen was pho- Several authors have maintained that color pat- tographed and its external features recorded (see terns, particularly in Cotylea, represent valid system- ‘Morphological analysis’), after which a small portion atic characteristics, useful for classification, because of the body tissue was excised for nucleotide sequen- many species in this suborder are conspicuously cing. The rest of the specimen was fixed in Bouin’s colored and exhibit striking color patterns (Hyman fluid (saturated picric acid, formaldehyde, and 1954, Prudhoe 1944, 1989, Newman & Cannon 1995). glacial acetic acid in a 15:5:1 ratio) for histological In contrast, Faubel (1983, 1984) believes that un - examination. equivocal species classification is best done, espe- cially in acotyleans, based on the characteristics of the male reproductive organ, specifically the struc- Morphological analysis ture of the prostatic vesicle and its orientation and relationship to the ejaculatory duct, on serial sections External features and reproductive organ structure of preserved specimens. Thus, 2 morphology-based were characterized following Kato (1944) and Hagiya classification systems are currently being used to (1992). Briefly, the fixed flatworms were removed understand polyclad taxonomy (Faubel 1983, 1984, from Bouin’s fluid and transferred to 70% ethanol for Prudhoe 1985). preservation. They were then embedded in paraffin The existence of 2 classification methods is due and subsequently serially sectioned (10 µm thick- to the almost exclusive reliance on morphological ness) and stained with Delafield’s hematoxylin and traits rather than molecular data for the classifica- eosin. Slides were observed under a microscope to tion of polyclad flatworms, except for a few studies determine reproductive organ structure following (Katayama et al. 1996, Litvaitis & Newman 2001, the criteria of Kato (1944). Rawlinson et al. 2011, Vanhove et al. 2013). Con- sequently, molecular data for polyclad flatworms are sparse in DNA databases worldwide: only 312 DNA extraction and PCR amplification and 59 nucleotide sequences of Acotylea and Cotylea, respectively, were available in the Gen- Total genomic DNA was extracted from the excised Bank, EMBL, and DDBJ databases, including gene tissue sample for each specimen using the method of sequences and expressed sequence tags (as of 5 Sezaki et al. (1999) with some modifications. Briefly, September 2016). proteinase K-treated samples were subjected to To remedy this situation, we collected specimens of phenol-chloroform extraction with MaXtract High various marine polyclad species from the coastal Density (Qiagen) following the manufacturer’s areas of the Japanese Islands and conducted phylo- protocol. Partial fragments of the 28S rRNA gene genetic analyses based on nuclear gene sequences (approx. 1100 bp), including the D1−D2 region, were after having first used morphological criteria to clas- amplified by PCR using the universal primers HRNT- sify the specimens. Additional phylogenetic analyses F2 (5’-AGTTC AAGAG TACGT GAAAC C-3’) based on mitochondrial gene sequences were car- and HRNT-R2 (5’-AACAC CTTTT GTGGT ATCTG ried out to classify polyclad flatworms in Acotylea. ATGA-3’), which were designed based on the 28S Sub sequently, the phylogenetic classification was rRNA gene sequence of the polyclad worm Notocom- compared with the morphological character-based plana humilis, which in turn had been determined classification. using a primer set for worms of the order Tricladida Tsunashima et al.: Taxonomy of marine flatworms 161 (Alvarez-Presas et al. 2008). Partial fragments of the the respective outgroup species for the 28S and COI mitochondrial gene (approx. 750 bp), cytochrome c- trees. oxidase subunit I (COI), were amplified by PCR using The nucleotide sequences of the partial 28S rRNA the universal primers HRpra2 (5’-AATAA GTATC and COI genes for all the flatworms were aligned ATGTA RACTD ATRTC T-3’) and HRprb2-2 (5’- using the program MUSCLE in MEGA version 6.0.6 GDGGV TTTGG DAATT GAYTA ATACC TT-3’), (Tamura et al. 2013). After alignment, 28S sequences which were designed based on the COI gene se - with poorly aligned regions were trimmed using quences from the polyclad worms N. humilis, Noto- trimAL, with the following parameters: minimum plana delicata and Pseudostylochus obscurus, which conservation threshold of 60%, gap threshold of again, in turn had been designed using a primer set 80%, or a similarity score lower than 0.001 (Capella- for Tricladida (Alvarez-Presas et al. 2008). The re - Gutiérrez et al. 2009). action mixture for PCR amplification contained ge - The maximum likelihood method was used to deter- nomic DNA as a template, 0.625 units of TaKaRa mine the phylogeny, with the GTR+G model (as de- ExTaq DNA polymerase (Takara

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