Description of a new species of Alloioplana (Polycladida: Stylochoplanidae) with an inference on its phylogenetic position in Leptoplanoidea

Polycladida is a group of marine free-living flatworms. Most polyclads are benthic and their vertical distribution ranges from the supralittoral zone (Newman & Cannon 1997) to the bathyal zone down to 3232 m (Quiroga et al. 2006). Generally, polyclad flatworms are predators feeding on other invertebrates; some polyclads are significant pests in commercial oyster cultures (Newman & Cannon 2003).

Stylochoplanidae Meixner, 1907 is a family in Leptoplanoidea Ehrenberg, 1831. Within this superfamily, Stylochoplanidae is characterized by species that possess a developed, interpolated, prostatic vesicle lined with a smooth inner epithelium; currently, the family contains 12 genera (Faubel 1983). However, a recent molecular phylogenetic study of Acotylea, which employed three stylochoplanid genera (i.e., ArmatoplanaFaubel, 1983, ComoplanaFaubel, 1983, and PhaenoplanaFaubel, 1983), revealed that Stylochoplanidae was not a monophyletic taxon (Oya & Kajihara 2020).

AlloioplanaPlehn, 1896 is a genus in Stylochoplanidae characterized by the possession of a penis stylet and the lack of a Lang's vesicle. Currently, four species are classified in this genus by Faubel (1983). To date, Alloioplana has not been included in any molecular phylogenetic analyses (Aguado et al. 2017, Bahia et al. 2017, Tsunashima et al. 2017, Dittmann et al. 2019, Litvaitis et al. 2019, Oya & Kajihara 2020).

In the present study, we describe a new species of Alloioplana from the coast of Japan. We also determine a partial sequence of the cytochrome c oxidase subunit I (COI) gene as a DNA barcode, as well as 18S and 28S ribosomal RNA genes (18S and 28S) for the inference of the phylogenetic position of the new species in Leptoplanoidea.

Flatworms were collected from Misaki (Kanagawa) and Usa (Kochi) on the Pacific coast of Japan (Table 1). Living specimens were anesthetized in MgCl₂ solution prepared with tap water until it had the same salinity as seawater. Flatworms were then photographed with a digital camera. For DNA extraction, a piece of the body margin was cut away from the specimen and fixed in 100% ethanol. The rest of the body was fixed in Bouin's solution for 24 h and preserved in 70% ethanol. The specimens for histological observation were dehydrated in an ethanol series and cleared in xylene, embedded in paraffin wax, and sectioned at 7 μm thickness. The sections were stained with hematoxylin and eosin and mounted in Entellan New (Merck, Germany). Morphological terminology in this study followes Hyman (1953) and Oya & Kajihara (2017).

Total DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Germany). As a reference for DNA barcoding, a partial sequence of the COI (712 bp) gene was determined from five specimens using the primer pair Acotylea_COI_F and Acotylea_COI_R (Oya & Kajihara 2017); genetic distances were calculated using MEGA ver. 7.0 (Kumar et al. 2016). The fragments of 18S (1736 bp) and 28S (1015 bp) rDNA were also amplified from one specimen using hrms18S_F and hrms18S_R (Oya & Kajihara 2020) for 18S and fw1 and rev2 (Sonnenberg et al. 2007) for 28S. Sequences were checked and edited using MEGA ver. 7.0 (Kumar et al. 2016).

Additional sequences of leptoplanoid and outgroup species were downloaded from GenBank; two acotylean species, Discocelis sp. and Stylochus cf. aomori (Kato, 1937), were chosen as outgroup taxa (Table 2). The sequences of 18S and 28S were aligned using MAFFT ver. 7 (Katoh & Standley 2013), with the L-INS-i strategy selected by the “Auto” option. Ambiguous sites were removed with Gblocks ver. 0.91b (Castresana 2002) using the option “Do not allow many contiguous nonconserved positions.” The sequences of COI were aligned and trimmed manually using MEGA ver. 7.0 (Kumar et al. 2016). The concatenated dataset from the three genes was 3163 bp long and contained 33 terminal taxa.

Phylogenetic analyses were performed with the maximum likelihood (ML) method executed in IQtree ver. 2.0 (Minh et al. 2020) under a partition model (Chernomor et al. 2016) and with Bayesian inference (BI) executed in MrBayes ver. 3.2.2 (Ronquist & Huelsenbeck 2003). The optimal substitution models for the ML analysis were 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) as follows: GTR+I (18S, second codon position in COI), GTR+I+G (28S), TIM+I+G (third codon position in COI), and TRN+G (first codon position in COI). For BI, the optimal substitution models were GTR+G (first codon position in COI), GTR+I (18S, second codon position in COI), and GTR+I+G (28S, second codon positions in COI). Nodal support within the ML tree was assessed by analyses of 1000 bootstrap pseudoreplicates. For BI, the Markov chain Monte Carlo (MCMC) process used random starting trees and involved four chains run for 5,000,000 generations with the first 25% of trees discarded as burn-in. We considered ML bootstrap values ≥ 70% and posterior probability values ≥ 0.90 to indicate clade support.

Type slides were deposited in the Invertebrate Collection of the Hokkaido University Museum (ICHUM), Sapporo, Japan. All sequences determined in this study were deposited in DDBJ/EMBL/GenBank databases with the accession numbers LC582941–LC582944, LC651420–LC651422. This work has been registered in ZooBank with the registration number urn:lsid:zoobank.org:pub:34ADD862-F10A-43C2-B452-2B95DA01B2E2.

Genus AlloioplanaPlehn, 1896 

Stylochoplanidae with tentacular and cerebral eye clusters, seminal vesicle, penis armed with a slender stylet housed in a tubular atrium, and without Lang's vesicle. Gonopores or common gonopore arranged in posterior part of body (modified from Faubel (1983: p. 101)).

We eliminated references to character states of the nuchal tentacle and gonopores from the definition by Faubel (1983). Faubel (1983: p. 101) included the absence of the tentacle as one of the diagnostic characters of Alloioplana. However, this was rather self-contradictory. As Bahia and Schrödl (2018) indicated, Alloioplana sensu Faubel (1983) contained two species with nuchal tentacles, Alloioplana aulica (Marcus, 1947) and Al. delicataPlehn, 1896; the latter is the type species of the genus. In addition, Faubel (1983, p. 101) listed the presence of separated gonopores in the genus definition. However, Al. aulica has a common gonopore (Marcus 1947: p. 114, fig. 24). Other characters included by Faubel (1983: p. 101) (the eyespot distribution, the structure of male copulatory apparatus, and the position of gonopores) are applicable to all Alloioplana species.

The above redefinition of Alloioplana was meant to avoid confusion caused by Faubel's (1983) definition. The monophyly of this genus should be tested using molecular data, as suggested by Bahia & Schrödl (2018).

Alloioplana yerii sp. nov. Figs. 1–3

The specific name is a noun in the genitive case honoring Megumi Yeri, who studied Japanese polyclads.

See Table 1.

Alloioplana without nuchal tentacles, with common sperm duct, oval prostatic vesicle smaller than seminal vesicle, and ejaculatory duct projecting into prostatic vesicle (Figs. 1–3).

Live specimens 7.8–17 mm in length (17 mm in holotype), 2.8–5.9 mm in maximum width (5.9 mm in holotype). Body elongate oval, narrow toward posterior end (Fig. 1A, B). Dorsal body tinged with light brown due to minute granules scattered over entire surface except around margin. Dorsal surface of body around pharynx brown. Body margin translucent. General appearance of body brownish (Fig. 1). Nuchal tentacles lacking. Cerebral and tentacular eyespots distributed continuously and forming cerebro-tentacular eye cluster. Each of cerebro-tentacular eye clusters containing 14–46 eyespots (27 in right cluster, 32 in left cluster in holotype) (Fig. 1C), arranged near median line. Pharynx whitish, ruffled in shape, occupying about one-fourth of body length (2.0–4.6 mm in length, 4.5 mm in holotype), located at center of body (Fig. 1B). Mouth opening at slightly posterior to center of pharyngeal cavity. Intestine not anastomosed. Male and female gonopores separate; male gonopore opening at about one-fifth to one-fourth of body length (2.0–4.6 mm, 3.8 mm in holotype) from posterior end; female gonopore situated 0.1–0.5 mm (0.3 mm in holotype) posterior to male gonopore.

Male copulatory apparatus located posterior to pharynx, consisting of seminal vesicle, interpolated prostatic vesicle, and penis stylet; spermiducal bulbs lacking (Figs. 2, 3A). Pair of sperm ducts extending anteriorly, turning medially at point about one-fourth length of pharynx from posterior end, subsequently extending posteriorly along both sides of pharynx and extending further posteriorly for short distance beyond level of posterior end of pharynx, then turning anteriorly (Fig. 1B) and fusing to common sperm duct (Figs. 2, 3A). Common sperm duct entering curved-oval seminal vesicle with strong muscular wall (Figs. 2, 3A, B). Distal end of seminal vesicle slender, long, meandering, entering proximal end of prostatic vesicle and projecting as intra-prostatic ejaculatory duct (Fig. 3B). Intra-prostatic ejaculatory duct lacking muscular wall, reaching to half length of prostatic vesicle (Fig. 3B, C). Prostatic vesicle oval, lacking tubular chambers, smaller than seminal vesicle, coated with thick muscular wall and lined with smooth glandular epithelium (Fig. 3B). Post-prostatic ejaculatory duct with thin muscular wall compared to that in prostatic vesicle connecting to penis stylet. Penis stylet straight, projecting into penis pocket (Fig. 3D). Penis pocket lined with non-ciliated epithelium, opening to male atrium. Male atrium more than 10 times larger than penis pocket and lined with ciliated epithelium (Fig. 3A, D). Shape of male atrium varying depending on fixation (Fig. 3A, E).

Pair of oviducts extending posteriorly and entering the proximal end of the vagina; Lang's vesicle and bursa copulatrix lacking (Figs. 2, 3F). Vagina lacking ampulla, lined with smooth, ciliated epithelium and surrounded by cement glands, curving antero-dorsally, extending anteriorly, then turning postero-ventrally to exit at female gonopore (Figs. 2, 3F).

Arai-hama Beach (35°09′34″N, 139°36′42″E), Misaki, Kanagawa, Japan (Table 1).

Intertidally to subtidally, on the surface of coralline algae and among kelp holdfasts (Kanagawa), or in oyster beds on a mooring rope hanging in the sea (Kochi) (Table 1).

The partial COI sequences (712 bp) from the five specimens (LC582941–LC582944, LC651422) were almost identical to each other. The uncorrected p-distance among specimens was 0.001–0.010.

The topology was almost identical between BI and ML trees (we show only the ML tree in Fig. 4). Alloioplana yerii sp. nov. was the sister to a clade comprised of Echinoplana celerrimaHaswell, 1907 and Notoplana sp. identified by Bahia et al. (2017) with relatively high bootstrap values (72%) and high posterior probability (0.99). The stylochoplanid species employed in the analyses did not form a monophyletic clade.

Faubel (1983) assigned four species to Alloioplana (Table 3). Alloioplana yerii sp. nov. is distinguished from these species as follows: from Al. aulica (Marcus, 1947) and Al. delicataPlehn, 1896 by the presence/absence of nuchal tentacles (absent in Al. yerii; present in Al. aulica and Al. delicata); from Al. stylifera (Hyman, 1953) by the length of the pharynx relative to the body length (about one-forth in Al. yerii; about three-fifths in Al. stylifera) and the shape of the prostatic vesicle (oval in Al. yerii; long and posteriorly curved in Al. stylifera); and from Al. wyona (Du Bois-Reymond Marcus & Marcus, 1968) by the presence/absence of the common sperm duct (present in Al. yerii; absent in Al. wyona) as well as the size of the prostatic vesicle compared with that of the seminal vesicle (small in Al. yerii; almost the same size in Al. wyona). In addition, among the congeners, the intra-prostatic ejaculatory duct is only observed in Al. yerii sp. nov.

This is the first report of Alloioplana from the West Pacific. Two Atlantic species, Al. aulica and Al. wyona, have been described from Brazil (Marcus 1947; Bahia & Schrödl 2018) and the Caribbean Sea (Du Bois-Reymond Marcus & Marcus 1968), respectively. Other congeners are known from the East Pacific: Al. delicata from Payta, Peru (Plehn 1896) and Al. stylifera from the Gulf of California (Hyman 1953).

Our results support previous studies by Dittmann et al. (2019), Litvaitis et al. (2019), and Oya & Kajihara (2020) in showing that Stylochoplanidae sensu Faubel (1983) is not monophyletic (Fig. 4). In the tree produced here, Al. yerii did not form a sister group relationship to any of the stylochoplanids included in our analyses. Stylochoplanidae as envisaged by Faubel (1983) should be divided into several family-level taxa as previously suggested by Dittmann et al. (2019) and Litvaitis et al. (2019).

There is a possibility that Alloioplana sensu Faubel (1983) is not monophyletic. Currently, Alloioplana contains polyclad flatworms with nuchal tentacles (Al. aulica and Al. delicata) and without nuchal tentacles (Al. stylifera, Al. wyona, and Al. yerii) (Bahia and Schrödl 2018; Table 3). In other stylochoplanid polyclads, i.e., Armatoplana, molecular phylogenies have indicated the possibility of non-monophyly in that Armatoplana divae (with nuchal tentacles) and Ar. leptalea (without tentacles) are separately positioned in the leptoplanoid clade (Fig. 4; Litvaitis et al. 2019, Oya & Kajihara 2020). Although the presence or absence of the nuchal tentacles has hardly been applied for diagnoses, especially in species-rich genera (cf. Faubel 1983; Prudhoe 1985), the characteristic may be useful for distinguishing higher-rank taxa (families or genera) in Leptoplanoidea, as was demonstrated by Oya & Kajihara (2020). To evaluate the suitability of this diagnostic characteristic in Alloioplana, the phylogenetic positions of other Alloioplana species are required.

In this study, we could not detect remarkable morphological similarities among Al. yerii, E. celerrima, and Notoplana sp. although these three species formed a relatively robust clade (Fig. 4). In the external morphology, Al. yerii and E. celerrima share two taxonomic characters: i) lacking nuchal tentacles and ii) having cerebro-tentacular eye clusters (Fig. 1C; Haswell 1907: p. 475, pl. 37, fig. 1); these traits are not observed in other gnesiocerotid polyclads included in the phylogeny. However, the character states in Notoplana sp. are unknown and these are also seen in other leptoplanoid species we employed in the analysis (e.g., Amyris hummelincki Du Bois-Reymond Marcus & Marcus, 1968). In the internal characters, the difference of anatomical features of the copulatory apparatuses between the three polyclads are so large that they are assigned in different families in Leptoplanoidea, respectively. Although there may be some similarities in histological characteristics of the copulatory apparatus, we could not evaluate this aspect because histological specimens of E. celerrima and Notoplana sp. were not available. Investigation of morphological synapomorphies or similarities among these species is a future subject.

We thank Mr. Hisanori Kohtsuka, Ms. Michiyo Kawabata, and Prof. Toru Miura (the University of Tokyo) and Dr. Kohei Oguchi (National Institute of Advanced Industrial Science and Technology) for providing living specimens in Misaki. We also thank Dr. Keiichi Kakui (Hokkaido University) for helping with sampling in Kochi. YO is thankful to Dr. Hiroshi Namikawa (National Museum of Nature and Science) for providing YO an opportunity to collect flatworms in Kochi and to the staff of Usa Marine Biological Institute for support with sampling. The authors would like to thank Enago (www.enago.jp) for the English language review. This study was funded by Research Institute of Marine Invertebrates (No. 2017 IKU-3) and the Japan Society for the Promotion of Science (JSPS) under KAKENHI grant number JP20J11958 to YO.