Introduction
Pericelis Laidlaw, 1902 is a genus in a cotylean polyclad family of Diposthidae Woodworth, 1898 (Litvaitis et al., Reference Litvaitis, Bolaños and Quiroga2019). The genus is characterized by possessing (i) an elongated oval or circular body; (ii) a pair of marginal tentacles; (iii) cerebral, tentacular, and marginal eyespots; (iv) a pharynx located at the centre of the body; (v) a seminal vesicle and an unarmed penis papilla in the male copulatory apparatus but lacking a prostatic vesicle and (vi) uterine vesicles and no Lang's vesicle in the female copulatory apparatus (Tsuyuki et al., Reference Tsuyuki, Oya and Kajihara2022a). Eleven of 12 known Pericelis polyclads have been reported from shallow waters (intertidal to 20 m depths), mainly in tropical and subtropical areas (figure 1 in Tsuyuki et al., Reference Tsuyuki, Oya and Kajihara2022a). A single species, Pericelis tectivorum Dittmann et al., Reference Dittmann, Dibiasi, Noreña and Egger2019a, has been described from an aquarium and its habitat and distribution in nature are uncertain (Dittmann et al., Reference Dittmann, Dibiasi, Noreña and Egger2019a). In recent years, new species of Pericelis have been successively described (Dittmann et al., Reference Dittmann, Dibiasi, Noreña and Egger2019a; Ramos-Sánchez et al., Reference Ramos-Sánchez, Bahia and Bastida-Zavala2020; Tsuyuki et al., Reference Tsuyuki, Oya, Jimi and Kajihara2020, Reference Tsuyuki, Oya and Kajihara2022a).
We found two individuals of polyclads that could be identified as Pericelis on sunken wood collected from a depth of 330 m and brought to Toba Aquarium (Mie, Japan; Figure 1). One individual was successfully captured for detailed observation. The polyclad flatworm lacks eyespots; however, its copulatory apparatuses have a typical morphology of Pericelis polyclads. In this study, we describe a new species of eye-less Pericelis based on the specimen and determine cytochrome c oxidase subunit I (COI) sequences for DNA barcoding and 18S and 28S ribosomal RNA genes for inferring the phylogenetic positions of the new species within Pericelis.
Collection site and photographs of living individuals of Pericelis nivea sp. nov. on a block of sunken wood: (A) locality of specimens, red circle indicates the collection site; (B) uncaptured individual; (C) ICHUM 8562 (holotype).
Materials and methods
Sampling and fixation
Two polyclads were found on sunken wood obtained from 330 m depths by bottom trawling off the coast of Owase, Mie, Japan (Figure 1A). One individual was captured and photographed with a digital camera; another was photographed but not collected (Figure 1B, C). Fixation was performed according to the method of Tsuyuki et al. (Reference Tsuyuki, Oya and Kajihara2022a). The captured worm was anaesthetized in an MgCl2 solution prepared with tap water to have the same salinity as seawater. The ventral view of the worm was photographed with a digital camera under an anaesthetized state. 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.
Histological observation
The whole body of the specimen was dehydrated in an ethanol series and cleared in xylene. The cleared specimen was embedded in paraffin wax and sagittally sectioned at 7 μm thickness. The sections were stained with haematoxylin and eosin and mounted in Entellan New (Merck, Germany).
Measurements of the specimens were carried out using ImageJ. The body size and pharynx length were measured from photographs of the anaesthetized specimens. The size of copulatory apparatuses was measured from photographs of the histological sections obtained by a digital camera (DP20, OLYMPUS) mounted on a microscope (Olympus BX41).
DNA extraction and sequencing
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) was determined from the specimen using the primer pair Acotylea_COI_F and Acotylea_COI_R (Oya and Kajihara, Reference Oya and Kajihara2017). For molecular phylogenetic analyses, 18S (1736 bp) and 28S (1007 bp) fragments were sequenced using hrms18S_F and hrms18S_R (Oya and Kajihara, Reference Oya and Kajihara2020) for 18S and fw1 and rev2 (Sonnenberg et al., Reference Sonnenberg, Nolte and Tautz2007) for 28S, respectively. The procedures of PCR amplification were as follows: 94°C for 1 min; 35 cycles of 94°C for 30 s, 50°C (COI and 18S) or 52.5°C (28S) for 30 s, and 72°C for 1 min (COI), 2 min (18S), or 1.5 min (28S); and 72°C for 7 min. Sequences were checked and edited using MEGA version 7.0 (Kumar et al., Reference Kumar, Stecher and Tamura2016).
Molecular phylogenetic analysis
Additional sequences of Pericelis and four cotylean species were downloaded from GenBank (Table 1). The 18S and 28S sequences were aligned using MAFFT version 7 (Katoh and Standley, Reference Katoh and Standley2013) with the L-INS-i strategy. Ambiguous sites were removed with Gblocks (Castresana, Reference Castresana2000) using the option ‘With Half’. The concatenated dataset from the four genes was 2652 bp long and contained 16 terminal taxa.
List of species included in the molecular phylogenetic analysis and their respective GenBank accession numbers
Phylogenetic analyses were performed using the maximum likelihood (ML) method executed in IQtree version 2.0 (Minh et al., Reference Minh, Schmidt, Chernomor, Schrempf, Woodhams, von Haeseler and Lanfear2020) under a partition model (Chernomor et al., Reference Chernomor, von Haeseler and Minh2016) and Bayesian inference (BI) executed in MrBayes version 3.2.2 (Ronquist and Huelsenbeck, Reference Ronquist and Huelsenbeck2003). The optimal substitution models for ML analysis selected with PartitionFinder version 2.1.1 (Lanfear et al., Reference Lanfear, Frandsen, Wright, Senfeld and Calcott2016) under the Akaike information criterion (Akaike, Reference Akaike1974) using the greedy algorithm (Lanfear et al., Reference Lanfear, Calcott, Ho and Guindon2012) were TRN + I (18S) and GTR + I + G (28S). For BI, optimal substitution models were GTR + I (18S) and GTR + I + G (28S). Nodal support within the ML tree was assessed by analyses of 1000 bootstrap pseudoreplicates. For BI, the Markov chain Monte Carlo process used random starting trees and involved four chains run for 10,000,000 generations, with the first 25% of trees discarded as burn-in. Convergence was confirmed using an average standard deviation of split frequencies of 0.003556, potential scale reduction factors for all parameters of 1.000–1.001, and effective sample sizes for all parameters of >5052.
Data treatment
Type slides have been deposited in the Invertebrate Collection of the Hokkaido University Museum (ICHUM), Sapporo, Japan. All sequences determined in this study have been deposited in DDBJ/EMBL/GenBank databases with accession numbers LC794541–LC794543.
Results
Order Polycladida Lang, 1881
Suborder Cotylea Lang, 1884
Family Diposthidae Woodworth, 1898
Genus Pericelis Laidlaw, 1902
Pericelis nivea sp. nov.
[New Japanese name: shiromuku-perikerisu]
urn:lsid:zoobank.org:act: 8A91AC26-52FE-4BC2-A885-DEFFB910694E
(Figures 1B–4)
Material examined
Holotype, ICHUM 8562, sagittal sections (15 slides), found on sunken wood collected from 330 m deep, off the coast of Owase (34°01′N, 136°22′E), Mie, Japan, 3 February 2019, T. Moritaki leg.
Etymology
The new specific name nivea (-us, -a, -um) is a Latin adjective meaning ‘snow white’. It was named after the appearance of the living worm. The new Japanese name for the new species is derived from shiromuku (a pure white kimono dress) and perikerisu (Pericelis polyclad) in the Japanese language.
Type locality
Off the coast of Owase, Mie, Japan (Figure 1A).
Diagnosis
Pericelis without eyespots and colour pattern, with glandular epithelium in penis papilla and separated gonopores (Figures 2–4).
Pericelis nivea sp. nov. (ICHUM 8562, holotype), photographs taken in life: (A) dorsal view without anaesthetization, scale unknown, anterior to the left; (B) enlarged view of anterior margin (C) dorsal view with anaesthetization, anterior to the top; and (D) ventral view with anaesthetization, anterior to the top: Abbreviations: mt, marginal tentacles; ph, pharynx; su, sucker.
Photomicrographs of sagittal sections of Pericelis nivea sp. nov. (ICHUM 8562, holotype), anterior to the left: (A) whole body; (B) male and female copulatory apparatuses; (C and D) uterine vesicle. Abbreviations: br, brain; cg, cement gland; cp, cement pouch; ed, ejaculatory duct; fa, female atrium; fg, female gonopore; luv, large uterine vesicle; ma, male atrium; mg, male gonopore; ph, pharynx; pp, penis papilla; sv, seminal vesicle; su, sucker; suv, small uterine vesicle; v, vagina.
Schematic diagram of copulatory apparatuses in Pericelis nivea sp. nov, anterior to the left. Abbreviations: cg, cement gland; cp, cement pouch; ed, ejaculatory duct; fa, female atrium; fg, female gonopore; ma, male atrium; mg, male gonopore; pp, penis papilla; sd, sperm duct; sv, seminal vesicle; su, sucker; v, vagina.
Description
Live specimen about 10 mm, elongated oval (Figure 2A). Anaesthetized specimen 9.3 mm long, 8.5 mm wide maximum (Figure 2C, D). Body translucent. Intestine visible whitish, highly branched, and not anastomosing, spreading throughout body, not reaching body margin. Dorsal and ventral surfaces without any colour pattern. General appearance of body white (Figures 1B, C, 2). Pair of marginal tentacles inconspicuous, not folded, slightly pointed (Figure 2B). Eyespots absent. Pharynx whitish, ruffled in shape, occupying about one-third of body length, 3.1 mm in anaesthetized state, located at almost centre of body (Figure 2A, D). Mouth opening at centre of pharyngeal cavity. Gonopores separate; female gonopore situated 281 μm posterior to male gonopore (Figures 3A, B, 4).
Male copulatory apparatus located immediately posterior to pharynx, consisting of seminal vesicle and unarmed penis papilla (Figures 3B, 4). Pair of sperm ducts entering laterally into seminal vesicle. Seminal vesicle oval, 292 μm on short axis and 446 μm on long axis, with thin (8.8–12 μm in thickness) muscular wall (Figure 3B). Distal end of seminal vesicle opening almost directly into penis papilla. Penis papilla cylindrical, 274 μm on short axis and 288 μm on long axis, with developed internal glandular epithelium, directing ventrally, occupying almost whole male atrium (Figures 3B, 4).
Female copulatory apparatus lacking Lang's vesicle (Figures 3B, 4). Pair of oviducts, each with 7–8 small uterine vesicles (Figure 3C) and single large uterine vesicle (Figure 3D) arranged from anterior to posterior, running posteriorly lateral to pharynx, leading to proximal end of vagina. Vagina 728 μm long, running posterodorsally and turning anteroventrally, opening into cement pouch. Cement glands opening cement pouch. Female atrium 166 μm long, opening to exterior through female gonopore. Sucker situated posterior to female copulatory apparatus (Figures 3A, B, 4).
Phylogenetic position
The topology was almost identical between BI and ML trees (only the ML tree is shown in Figure 5). Pericelis nivea sp. nov. was encompassed in the clade of Pericelis with high support values (87/0.99). Within the Pericelis species, P. nivea sp. nov was sister to the clade formed by other Pericelis except P. lactea; however, the nodal support was low (50/0.65).
Maximum likelihood (ML) phylogenetic tree based on sequences from two genes (18S and 28S; concatenated length: 2652 bp). The numbers near nodes are ML bootstrap values/posterior probability.
Habitat
Sunken wood in the bathyal zone (Figure 1B, C).
Distribution
Only from the type locality.
Remarks
We assign the P. nivea sp. nov. to Pericelis although it lacks eyespots. The presence of eyespots in the body margin is a diagnostic character of the genus (cf. Tsuyuki et al., Reference Tsuyuki, Oya and Kajihara2022a). However, other morphological characteristics, such as body shape, presence of marginal tentacles, position of the pharynx, and structures of male and female reproductive organs in the present polyclad flatworm, fit the definition of the genus. The new species is also nested in the clade of Pericelis with high support values in phylogenetic analyses (Figure 5). Here, we avoid modifying the definition of Pericelis and classify the present species as an exception of the genus. The absence of eyespots in P. nivea sp. nov. may be related to its habitat (cf. Oya and Kajihara, Reference Oya and Kajihara2019).
This is the first record of Pericelis from the bathyal zone. Among 12 species of Pericelis, P. nivea sp. nov. can be readily distinguished from other congeners by lacking eyespots and colour patterns in the dorsal surface (cf. table 3 in Tsuyuki et al., Reference Tsuyuki, Oya and Kajihara2022a). In addition, the present species differs from five species (P. flavomarginata, P. hymanae, P. lactea, P. maculosa, and P. orbicularis) by possessing glandular epithelium in the penis papilla. Moreover, our species is distinguished from four species (P. alba, P. ernesti, P. nazahui, and P. sigmeri) of the rest congeners by having separated gonopores. Furthermore, P. nivea sp. nov. is also differentiated from P. byerleyana by the penis-papilla shape (length/width: about 1 in P. nivea sp. nov.; 4–5 in P. byerleyana). In addition to the morphology, the present polyclad is well separated from nine Pericelis species by the molecular information (Figure 5). Here, we judged the worm to be a new species of Pericelis.
Discussion
This polyclad is the fourth polyclad species described from the bathyal zone around Japan (Oya and Kajihara, Reference Oya and Kajihara2019, Reference Oya and Kajihara2021; Oya et al., Reference Oya, Kimura and Kajihara2019, this study). In Japan, approximately 150 species of Polycladida have been reported from the coast of Japan (Kato, Reference Kato1944), representing 15% of the described polyclads in the world. In addition, despite easily accessible sites such as the intertidal zone, new polyclad flatworms have been successively described from Japan (e.g. Oya et al., Reference Oya, Tsuyuki and Kajihara2021, Reference Oya, Tsuyuki and Kajihara2022); this fact suggests that Japanese waters have a rich polyclad fauna. Although knowledge of the polyclad fauna in deep areas is scarce, it is natural that many species will be discovered on the deep sea bottom around Japan as the faunal survey progresses.
Unintentionally captured specimens are important for investigating the diversity of deep-sea polyclads. Deep-sea polyclads are rarely collected; for example, Paraplehnia seisuiae Oya et al., Reference Oya, Kimura and Kajihara2019, which was described from the bathyal zone of the Kumano Sea, has not been collected except for a single specimen of the holotype although the area has been continuously surveyed since 2017 (Kimura et al., Reference Kimura, Kimura, Jimi, Kakui, Tomioka, Oya, Matsumoto, Tanabe, Hasegawa, Hookabe, Homma, Hosoda Y, Fujimoto, Kuramochi, Fujita, Ogawa, Kobayashi, Ishida, Tanaka, Onishi, Shimetsugu, Yoshikawa, Tanaka, Kushida, Maekawa, Nakamura, Okumura and Tanaka2018, Reference Kimura, Kimura, Jimi, Kuramochi, Fujita, Komai, Yoshida, Tanaka, Okanishi, Ogawa, Kobayashi, Kodama, Saito, Kiyono, Katahira, Nakano, Yoshikawa, Uyeno, Tanaka, Oya, Maekawa, Nakamura, Okumura and Tanaka2019a, Reference Kimura, Kimura, Kakui, Hookabe, Kuramochi, Fujita, Ogawa, Kobayashi, Jimi, Okanishi, Yamaguchi, Hirose, Yoshikawa, Fukuchi, Shimomura, Kashio, Uyeno, Fujiwara, Naruse, Kushida, Kise, Maekawa, Nakamura, Okumura and Tanaka2019b; Jimi et al., Reference Jimi, Kimura, Ogawa and Kimura2020). As Quiroga et al. (Reference Quiroga, Bolanos and Litvaitis2006) pointed out, polyclads in deep waters may be broken or wafted away during dredging in many cases even though many species inhabit the seafloor; Quiroga et al. (Reference Quiroga, Bolanos and Litvaitis2006) stated that sampling by research submersibles or remotely operated vehicles is the only way to collect intact polyclads in the deep sea. These machines are indeed effective; however, it is not considered suitable for surveying large areas of the seafloor. In terms of covering the limitations of the methods, bycatch in other research and commercial fisheries would be an effective way to collect bathyal polyclads.
Pericelis nivea sp. nov. is expected to be a predator on wood falls. In Pericelis, several observations about feeding habits have been reported (Bahia et al., Reference Bahia, Padula, Lavrado and Quiroga2014; Dittmann et al., Reference Dittmann, Dibiasi, Noreña and Egger2019a; Tsuyuki et al., Reference Tsuyuki, Oya, Jimi and Kajihara2020). Bahia et al. (Reference Bahia, Padula, Lavrado and Quiroga2014) described that P. cata fed on a sea slug, Felimare lajensis (Troncoso et al., 1998) when they were placed in the same container and Dittmann et al. (Reference Dittmann, Dibiasi, Noreña and Egger2019a) observed that P. tectivorum preyed on a marine snail, Tectus fenestratus (Gmelin, 1791). In another study, Tsuyuki et al. (Reference Tsuyuki, Oya, Jimi and Kajihara2020) reported that P. flavomarginata fed on a scaleworm, Iphione muricata (Lamarck, 1818). Like these congeners, P. nivea sp. nov. may feed on other invertebrates, such as annelids and molluscs, on sunken wood. As Quiroga et al. (Reference Quiroga, Bolanos and Litvaitis2008) pointed out, taxonomic studies of polyclads on wood falls would be important not only for revealing polyclad fauna but also for understanding a community in deep-sea environments.
Polyclad flatworms may have independently colonized deep-sea wood falls in several lineages. Four species of polyclads from two acotylean (Anocellidus profundus Quiroga et al., Reference Quiroga, Bolanos and Litvaitis2006 in Anocellidae Quiroga et al., Reference Quiroga, Bolanos and Litvaitis2006 and Didangia carneyi Quiroga et al., Reference Quiroga, Bolanos and Litvaitis2008 in Didangiidae Faubel, 1983) and one cotylean families (Oligocladus bathymodiensis Quiroga et al., Reference Quiroga, Bolanos and Litvaitis2008 and O. voightae Quiroga et al., Reference Quiroga, Bolanos and Litvaitis2006 in Euryleptidae Stimpson, 1857) have been described from sunken wood in the deep sea (Quiroga et al., Reference Quiroga, Bolanos and Litvaitis2006, Reference Quiroga, Bolanos and Litvaitis2008). In the group known in the wood falls, Oligocladus Lang, 1884 is expected to provide some insights into the colonization of deep-sea substrates because it contains species inhabiting shallow waters (e.g. Noreña et al., Reference Noreña, Marquina, Perez and Almon2014) as well as bathyal zones (Quiroga et al., Reference Quiroga, Bolanos and Litvaitis2008) to abyssal zones (Quiroga et al., Reference Quiroga, Bolanos and Litvaitis2006). Pericelis may be another candidate of polyclad flatworms to study the colonization process from shallow waters to bathyal wood falls.
Data availability
The data that support the findings of this study are available from the corresponding author, Y. O., upon reasonable request.
Acknowledgements
The authors thank the crew of the trawler Jinsho-maru XVIII (Jinsho Co., Ltd.) for allowing us to study polyclads on wood falls. Y. O. thanks Hiroshi Kajihara (Hokkaido University) for managing the voucher specimen. The authors thank Enago (www.enago.jp) for the English language review.
Author contributions
Y. O. prepared the histological sections, conducted morphological observations, performed molecular analyses, and wrote the manuscript. T. M. collected the specimens and photographed the living polyclads. A. T. improved the description and the figures. All authors read and approved the manuscript.
Financial support
This study was funded by the Japan Society for the Promotion of Science (JSPS) under KAKENHI grant number 20J11958.
Competing interest
None.
