Description of a new species of Paraplehnia (Polycladida, Stylochoidea) from Japan, with inference on the phylogenetic position of Plehniidae

Abstract We describe a new species of polyclad flatworm, Paraplehniaseisuiaesp. nov., from 298–310 m depths in the Sea of Kumano, West Pacific, Japan. Paraplehniaseisuiaesp. nov. is characterized by i) a developed muscular wall proximally occupying about one-third of the prostatic vesicle, ii) no common duct between the spermiducal bulbs and the prostatic vesicle, and iii) a genital pit between the male and female gonopores. We provide a partial sequence (712 bp) of the mitochondrial cytochrome c oxidase subunit I gene as a DNA barcode for the species. Our phylogenetic analyses based on 603-bp 28S rDNA sequences indicate that P.seisuiaesp. nov. is nested in a clade consisting of stylochoid species along with unidentified species of Stylochus. It suggests that Plehniidae belongs to Stylochoidea, although this should be confirmed by future studies that contain Plehniaarctica (Plehn, 1896), the type species of the type genus of the family. The interfamily relationship among the superfamily Stylochoidea remains poorly resolved.


Introduction
Polyclad flatworms in the family Plehniidae Bock, 1913 are characterized by possessing sperm ducts or a common sperm duct entering the neck of a prostatic vesicle, the latter lacks an ejaculatory duct (Hyman 1953). The majority of plehniids have been reported from sublittoral zones by dredging (e.g. Bock 1913Bock , 1923Kato 1939;Hyman 1953;Hagiya 1993); some species were described from more than 200 m depths (Bock 1913;Hyman 1953).
In this paper, we describe a new species of plehniid flatworm from Japan. We provide a partial sequence of the cytochrome c oxidase subunit I (COI) gene as a DNA barcode for the new species. We estimate the phylogenetic position of Plehniidae, represented by the new species, among other acotylean polyclads by molecular analyses using partial 28S rDNA sequences.

Material and methods
A single polyclad specimen was collected by dredging during the research cruise No. 1722 by Training/Research Vessel (TRV) Seisui-maru. The worm was anesthetized in a MgCl 2 solution prepared with tap water so that it had the same refractive index (or "salinity") as the seawater, using an IS/Mill-E refractometer (AS ONE, Japan), and then photographed with a Nikon D5300 digital camera with external strobe lighting provided by a pair of Morris Hikaru Komachi Di flash units. 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. It was then cut into two (anterior and posterior) pieces. Both pieces were dehydrated in an ethanol series and cleared in xylene, then embedded in paraffin wax, sectioned at 7 µm thickness, stained with hematoxylin and eosin (HE), and embedded in Entellan New (Merck, Germany). They were observed under an Olympus BX51 compound microscope and photographed with a Nikon D5300 digital camera.
Sections containing part of copulatory apparatus, mounted on one of the slides, were re-stained by Mallory's trichrome method to yield clear contrast between the muscular and connective tissues. The cover glass was removed by steeping the prepara-tion in xylene for 24 h. The sections on the slide were hydrated in an ethanol series. HE staining was then removed by washing in 50% ethanol containing 0.5% HCl for 2 h. After Mallory's staining, the sections were likewise embedded in Entellan New.
Total DNA was extracted by using Boom et al.'s (1990) silica method. A fragment of the cytochrome c oxidase subunit I (COI) (712 bp) was amplified with primers Acotylea_COI_F and Acotylea_COI_R (Oya and Kajihara 2017) as a reference for DNA barcoding. A 1004-bp fragment of 28S rDNA was amplified with primers fw1 and rev2 (Sonnenberg et al. 2007) for molecular phylogenetic analyses; the primer pairs have been used in other phylogenetic studies of polyclads (e.g. Bahia et al. 2017;Litvaitis et al. 2019). Polymerase chain reaction (PCR) amplification conditions were 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 50 °C (COI) or 52.5 °C (28S rDNA) for 30 s, and 72 °C for 1.5 min (COI) or 2 min (28S rDNA); and 72 °C for 7 min. All nucleotide sequences were determined by direct sequencing with a BigDye Terminator Kit ver. 3.1 and 3730 Genetic Analyzer (Life Technologies, California, USA); two internal primers, fw2 and rev4 (Sonnenberg et al. 2007), were used in sequencing 28S rDNA. Sequences were checked and edited using MEGA ver. 5.2 (Tamura et al. 2011). Table 1. List of species that were used for the molecular phylogenetic analysis and respective GenBank accession numbers.
Phylogenetic analyses were performed with maximum-likelihood (ML) methods and Bayesian Inference (BI). The ML analysis was performed with RAxML ver. 8.2.3 (Stamatakis 2014). Nodal support within the ML tree was assessed by analyses of 1,000 bootstrap pseudoreplicates (Felsenstein 1985). BI was performed with MrBayes ver. 3.2.2 (Ronquist and Huelsenbeck 2003). The Markov chain Monte Carlo (MCMC) process used random starting trees and involved four chains for 1,000,000 generations. The first 25% of the trees were discarded as burn-in.
Type slides have been deposited in the Invertebrate Collection of the Hokkaido University Museum, Sapporo, Japan (ICHUM). The sequences determined in this study have been deposited in DDBJ/EMBL/GenBank databases with the accession numbers LC466999 (COI) and LC467000 (28S rDNA).
Description. Live specimen 26 mm in length, 11 mm in width. Body thick, elongate, oval, narrow toward posterior end (Fig. 1A, B). Anterior and posterior ends pointed. Body ground color translucent to whitish opaque. General appearance of body light brown. Dorsal body without any pattern. Body margin translucent. Tentacles lacking. Pharynx, ruffled in shape, 7.4 mm in length, located at center of body. Mouth opening at center of pharyngeal cavity (Fig. 1B). Intestine highly branched and not anastomosing, spreading throughout body except margin. Pair of sperm ducts and oviducts whitish, visible through ventral surface. Male and female gonopores separate; male gonopore opening at 9 mm from posterior end; female gonopore situated 2.5 mm posterior to male gonopore.
Marginal and cerebral eyespots small and embedded in parenchyma (Fig. 1C, D). At least 47 and 28 eyespots arranged in anterior body margin and from just behind brain to anterior to brain, respectively, but detailed distribution of eyespots could not be observed.
Male copulatory apparatus located posteriorly to pharynx, consisting of pair of spermiducal bulbs, prostatic vesicle, and penis papilla ( Fig. 2A-E). Distal end of each sperm duct forming oval spermiducal bulb, latter having thick muscular wall ( Fig.  2A). Distal end of each spermiducal bulb slender and separately connecting to neck of prostatic vesicle (Fig. 2B-D). Prostatic vesicle pear-shaped, having strong muscular wall occupying its proximal one-third, distally coated with connective tissue and enclosed by muscular bulb (Fig. 2F). Canals of extra-vesicular gland penetrating prostatic-vesicle wall. Glandular epithelium with numerous teardrop-shaped cells folded in prostatic vesicle. Ejaculatory duct lacking; distal end of prostatic vesicle directly forming a part of penis papilla. Penis papilla large, conical, and projecting posteriorly into male atrium. Male atrium lined with thin, non-ciliated epithelium.
Pair of oviducts forming common oviduct, which run postero-dorsally to enter vagina (Fig. 2E). From this point, elongated duct of Lang's vesicle, lined with ciliated epithelium, running posteriorly. Lang's vesicle sac-shaped, lined with squamous cells, positioned posterior to female gonopore. Vagina lined with smooth ciliated epithelium, running antero-dorsally, curving postero-ventrally as it becomes slenderer, turning postero-dorsally as it becomes wider, eventually leading ventrally to exit at female atrium (or vagina externa). Medial part of vagina surrounded by numerous cement glands (Fig. 2E). Female atrium large, folded, with thick basement membrane opening at female gonopore.
Genital pit with smooth epithelium and basement membrane similar to those in vagina (Fig. 2G), located between male and female gonopores (Fig. 2F, H).
Habitat. Judging from the nature of the dredged material, the sediment type of the species' habitat is likely to be sandy mud.
Molecular phylogeny. The resulting BI and ML trees were almost identical to each other in topology. Paraplehnia seisuiae sp. nov. was nested in a clade composed of stylochoids except Koinostylochus elongatus and Pseudostylochus obscurus (Fig. 3); the latter two appeared to be more closely related to leptoplanoids than to stylochoids, as indicated by Tsunashima et al. (2017). The majority of stylochoids except Callioplana marginata, Koinostylochus elongatus, and Pseudostylochus obscurus formed a clade which also included Stylochus sp. of Bahia et al. (2017) and was supported by 0.99 BI posterior probability and 73% ML bootstrap (Fig. 3). Given Bahia et al.'s (2017) generic identification of Stylochus sp., this clade can be regarded as representing the "true" Stylochoidea, because Stylochus is the type genus for this family-group name. While Paraplehnia seisuiae sp. nov. appeared as sister to Hoploplana spp., its supporting values were low (0.64 BI posterior probability; 27% ML bootstrap). The inter-family relation of Plehniidae among Stylochoidea was thus not fully resolved in the present study.
Remarks. In this paper, we follow the classification system by Prudhoe (1985), in which Plehniidae consists of five genera (Discocelides, Myoramyxa, Nephtheaplana, Paraplehnia, and Plehnia); for Faubel's (1983) system, see Discussion below. Hyman (1953)  characterized Paraplehnia as possessing a prostatic vesicle that has i) a strong muscular wall in its proximal end and ii) a reduced glandular part. Paraplehnia seisuiae sp. nov. is characteristic of the genus by possessing these characteristics. By these characters, our new species cannot be placed in Plehnia sensu Prudhoe (1985), because the latter is diagnosed to have a prostatic vesicle whose proximal end is not particularly thick-walled. Paraplehnia seisuiae sp. nov. differs from Discocelides and Myoramyxa in that it does not have a vaginal duct (ductus vaginalis) and from Nephtheaplana in that our specimen has a pair of spermiducal bulbs.
Paraplehnia has contained two species, P. japonica (Bock, 1923) and P. pacifica (Kato, 1939), and both were originally described from the sublittoral zone in Japan. Paraplehnia seisuiae sp. nov. can be distinguished from the two congeners by the thickness of the muscular wall of the prostatic vesicle (about one-third of the prostatic vesicle in P. seisuiae sp. nov.; about one-half in P. japonica and P. pacifica), the presence/ absence of a common duct between spermiducal bulbs and prostatic vesicle (absent in P. seisuiae sp. nov.; present in P. japonica and P. pacifica), and the presence/absence of a genital pit between the male and the female gonopores (present in P. seisuiae sp. nov.; absent in P. japonica and P. pacifica) ( Table 2). In addition, P. seisuiae sp. nov. differs from P. japonica by the length of the Lang's duct (elongated in P. seisuiae sp. nov.; short in P. japonica) and from P. pacifica by the range of developed connective tissues in the female copulatory apparatus (from the female atrium to around the female gonopore and the genital pit in P. seisuiae sp. nov.; only around the female atrium in P. pacifica).
The eyespots were invisible in the living specimen (Fig. 1A), probably because of the thickness and opaqueness of the body, as well as the small size of each eyespot. We noticed the presence of eyespots only after sectioning (Fig. 1C, D). Bock (1923: 3) also remarked for Paraplehnia japonica that eyespots were undetectable in the living specimens and became apparent only after histological sectioning. Because we failed to observe the arrangement of eyespots from dorsal view in intact body, we had to categorize each eyespot into marginal ones or cerebral ones according to the relative position from the body margin and the brain.
It is for the first time that a genital pit (or genital sucker) was found in a species of plehniid. Among Acotylea, genital pits have been known in Itannia ornata Marcus, 1947(Hoploplanidae Stummer-Traunfels, 1933, three species of Leptoplana (Leptoplanidae Stimpson, 1857) (Gammoudi et al. 2012), and Persica qeshmensis Maghsoudlou, Bulnes, & Rahimian, 2015(Pleioplanidae Faubel, 1983. Genital pits in I. ornata are present in a pair, situated on both sides of the female gonopore (Marcus 1952). On the other hand, a single genital pit is present between the male and female gonopores in three Leptoplana species and Persica qeshmensis, as well as in Paraplehnia seisuiae sp. nov. (Fig. 2H, I).

Discussion
In this paper, we adopted Prudhoe's (1985) -instead of Faubel's (1983) -classification system as to the infrafamilial classification of Plehniidae because this system was followed by some of the subsequent researchers (e.g. Hagiya 1993;Newman and Cannon 1997). Faubel (1983) did not accept Paraplehnia because he considered that "the presence (P. japonica) or the absence (P. pacifica) of Lang's vesicle demands a separation of both these species" (Faubel 1983: 54-55) and classified Paraplehnia pacifica as a Diplehnia, which was characterized by lacking a Lang's vesicle (Faubel 1983). However, Kato (1939: 68, fig. 3) clearly stated that the "Lang's vesicle is small and irregularly elongated, disposed immediately behind the vagina bulbosa in the ventral part of the body" in the original description of Paraplehnia pacifica and also included Table 2. Comparison of characters between species of Paraplehnia. P. japonica (Bock, 1923) P. pacifica (Kato, 1939 Only around the female atrium From the female atrium to the genital pit and the female gonopore Reference Bock 1923Kato 1939Hagiya 1993 This study a line drawing of the Lang's vesicle as a schematic figure of the copulatory apparatus of Paraplehnia pacifica. The validity of Diplehnia should be tested by future molecular studies along with Diplehnia caeca (Hyman, 1953), the type species of the genus. Our 28S rDNA analyses corroborate the taxonomic views by Faubel (1983) and Prudhoe (1985) in that Plehniidae, as represented by Paraplehnia seisuiae sp. nov. in this study, should be placed in Stylochoidea (Fig. 3), rather than in Cryptoceloidea as Bahia et al. (2017) suggested. Faubel (1983) and Prudhoe (1985) placed Plehniidae in Stylochoidea based primarily on the reproductive-system morphology and the arrangement of eyespots, respectively. Bahia et al. (2017) carried out a 28S-rDNA-based molecular phylogenetic analysis covering 19 families and 32 genera of polyclads. Based on the analysis, Bahia et al. (2017: 674) circumscribed Cryptoceloidea as having "oval to elongated body, without tentacles, and with cerebral, nuchal, and marginal eyespots" and Stylochoidea as having "rounded body, nuchal tentacles, and cerebral, nuchal, and sometimes marginal eyespots", among other super-familial redefinitions. Bahia et al. (2017: 675) stated that "Polyposthiidae and Plehniidae possibly belong to Cryptoceloidea", probably because Plehniidae had been defined as having no tentacles (Bock 1913); our new species, Paraplehnia seisuiae sp. nov., also lacks tentacles. Litvaitis et al. (2019) inferred the internal relationships of Polycladida using 28S rDNA sequences representing 22 families and 37 genera, and identified morphological characters for each clade recovered. Although Stylochoidea was found to be monophyletic, Litvaitis et al. (2019) concluded that this superfamily cannot be defined by any morphological or developmental synapomorphy. It was because in Litvaitis et al.'s (2019) analyses, Stylochoidea turned out to contain members that lack tentacles (e.g., Latocestidae) and have elongated body (e.g., Latocestidae and Leptostylochus), which do not fit to Bahia et al.'s (2017) circumscription for this superfamily. In our analysis, Stylochoidea was "split" into two clades (Fig. 3), and our new species Paraplehnia seisuiae sp. nov., having no tentacles, appeared in one of the two stylochoid clades along with Stylochus sp. of Bahia et al. (2017). If we suppose that Paraplehnia seisuiae sp. nov. is more closely related to Plehnia arctica (Plehn, 1896) (originally in Acelis; type species of Plehnia, which in turn is the type genus for Plehniidae) than any other type species of the type genera of all the nominal families potentially belonging to Stylochoidea, our new species should belong to Plehniidae. If so, and given that Bahia et al.'s (2017) identification of Stylochus sp. (see Molecular phylogeny above) was correct, Plehniidae should belong to Stylochoidea. Our study corroborates the opinion of Litvaitis et al. (2019) in that at least the presence/ absence of tentacles is not appropriate to circumscribe Stylochoidea.
The inter-familial relation of Plehniidae was not resolved in this study. It is probably due to the shortness of the 28S rDNA sequence (603 bp) employed in the analyses. Future studies should be done with additional molecular makers and more extensive taxon sampling.
Jimi (National Institute of Polar Research) and Mr Akito Ogawa (The University of Tokyo) for inviting YO to this cruise; Dr Juliana Bahia (Ludwig-Maximilians-Universität München) for providing the sequence data. This study was funded by Research Institute of Marine Invertebrates (No. 2017 IKU-3) for YO.