Skip to main content
NCBI home page
Zoological Studies logoLink to Zoological Studies
. 2025 Oct 29;64:e47. doi: 10.6620/ZS.2025.64-47

The Phylogenetic Position of Stylochoposthia Faubel, 1983 (Polycladida, Acotylea) Provides Insights into the Evolution of the Copulatory Apparatus

Yuki Oya 1,*, Aoi Tsuyuki 2
PMCID: PMC12813612  PMID: 41561879

Abstract

Stylochoposthia Faubel, 1983 is a genus of acotylean polyclads, and its family-level assignment is controversial. In this study, we inferred the phylogenetic position of the genus represented by Stylochoposthia sp. using the combined data generated from partial sequences of nuclear 18S and 28S ribosomal DNA, mitochondrial 16S ribosomal DNA, and the cytochrome c oxidase subunit I gene. The phylogenetic analysis revealed that Stylochoposthia sp. was nested in a robust clade comprising the species of four genera within Planoceridae Stimpson, 1857 (Aquaplana Hyman, 1953, Heteroplanocera Oya & Kajihara, 2021, Paraplanocera Laidlaw, 1903, and Planocera Blainville, 1828). Specifically, Stylochoposthia sp. was closely related to Planocera pellucida (Mertens, 1833), with high support values. This study revealed that an eversible cirrus with hard structures (numerous spines, thorns, or teeth) in the male copulatory apparatus, a diagnostic characteristic of Planoceridae, has been lost in the lineage of Stylochoposthia.

Keywords: Eversible cirrus, Marine flatworm, Systematics, Taxonomy, Turbellarian

BACKGROUND

Polycladida is an order constituting approximately 1,000 free-living flatworm species (Tyler et al. 2006– 2025). Polyclad flatworms, like other turbellarians, generally exhibit morphologically diverse male/ female copulatory apparatuses. The morphological characteristics of the copulatory apparatus have been used to identify polyclads at either the family or genus levels, particularly Acotylea, a suborder of polyclads lacking a cotyle (a sucker-like structure in the ventral surface of the body). Recent molecular phylogenetic studies have revealed that not a few families and genera within Acotylea are not monophyletic (e.g., Oya and

Kajihara 2020), suggesting that the morphology of the copulatory apparatus is phylogenetically more unstable than previously assumed. As an example, the types of prostatic vesicle (free or interpolated), emphasized in a diagnosis of superfamily-level identification, are modified in several lineages of lower taxa (Aguado et al. 2017; Bahia et al. 2017; Tsunashima et al. 2017; Oya et al. 2022a).

Stylochoposthia Faubel, 1983 is an acotylean genus established by Faubel (1983). The genus is based on Pseudostylochus bellus Hyman, 1959, which was originally described from Australia (Hyman 1959). Later, Prudhoe (1989) described the second species, Stylochoposthia inhacae Prudhoe, 1989 from Africa, resulting in these two species being assigned to this genus. The systematic position of Stylochoposthia remains disputed; Faubel (1983) placed the genus in Pseudostylochidae Faubel, 1983, whereas Prudhoe (1989) assigned this genus to Callioplanidae Hyman, 1953 sensu Prudhoe (1985). To date, no molecular phylogenetic evaluations have been conducted.

During a faunal survey conducted along Japan’s coastline, we found a polyclad flatworm identified as Stylochoposthia sp. In this study, we provide the first molecular data for the genus represented by this specimen and evaluated its phylogenetic position within Acotylea.

MATERIALS AND METHODS

Specimen

A polyclad flatworm was collected from the undersurface of a stone in the intertidal zones of Otsuki, Kochi, Japan. The living specimen was anesthetized in MgCl2 solution prepared with tap water, adjusted to match the salinity of seawater. The specimen was then photographed using a digital camera. For DNA extraction, a portion of the body margin was excised from the specimen and fixed in 100% ethanol. The rest of the body was fixed in Bouin’s solution for 24 h and then preserved in 70% ethanol.

The tissue fixed in Bouin’s solution was divided into two portions: the anterior one-third and the posterior two-thirds. The anterior portion was dehydrated in an ethanol series, cleared with xylene, and mounted in Canada balsam. The posterior portion was dehydrated and cleared with the same procedure, embedded in paraffin wax, and sectioned at a thickness of 7 μm. The sections were stained with hematoxylin and eosin (HE) and then mounted using Entellan New (Merck, Germany).

The tissue sections containing components of the copulatory apparatus, which were mounted on two slides, were re-stained using the Masson-Goldner (MG) method to provide a distinct contrast between muscular and connective tissues. The cover glass was removed by soaking the preparations in xylene for one week. The tissue sections on the slide were hydrated using an ethanol series. The HE stain was then removed using deColorizing Solution 1 (FUJIFILM Wako Pure Chemical Corporation, Japan) for 24 h. After MG staining, the tissue sections were mounted in Entellan New.

Sequencing

Total DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Germany). Sequences of 16S, 18S, 28S, and cytochrome c oxidase subunit I (COI) marker fragments were generated using the following primers: 16Sar-L and 16Sbr-H (Palumbi et al. 1991) for 16S, hrms18S_F and hrms18S_R (Oya and Kajihara 2020) for 18S, fw1 and rev2 (Sonnenberg et al. 2007) for 28S, and Acotylea_ COI_F and Acotylea_COI_R (Oya and Kajihara 2017) for COI. The sequences were validated and processed using MEGA ver. 7.0 (Kumar et al. 2016); 442 bp of 16S, 1,734 bp of 18S, 1,003 bp of 28S, and 712 bp of COI were obtained.

Molecular phylogeny

Phylogenetic analyses were conducted using the maximum-likelihood (ML) method in RAxML-NG ver. 1.2.1 (Kozlov et al. 2019) and Bayesian inference (BI) using MrBayes ver. 3.2.2 (Ronquist and Huelsenbeck 2003). Additional sequences of acotyleans and outgroup species were downloaded from GenBank (Table 1). The 16S, 18S, and 28S rDNA sequences were aligned using MAFFT ver. 7 (Katoh and Standley 2013), with the L-INS-i strategy selected through the “Auto” option. Sites with ambiguous alignments were removed using Gblocks ver. 0.91b (Castresana 2000) with the “With Half” option. The COI sequences were manually aligned using MEGA ver. 7.0 (Kumar et al. 2016). Two datasets were compiled, each with four gene markers (16S, 18S, 28S, and COI) and with varying operational taxonomic units (OTUs). Dataset 1 (93 OTUs, 2,706 bp; concatenated 405 bp of 16S, 934 bp of 18S, 851 bp of 28S, and 516 bp of COI) included as many acotylean taxa as possible with sequence data available in public databases. Dataset 2 (16 OTUs, 3,547 bp; concatenated 439 bp of 16S, 1647 bp of 18S, 936 bp of 28S, and 525 bp of COI) was considered as a subset of Dataset 1 and focused on the Planoceridae clade. The optimal substitution models for the analyses were selected using PartitionFinder ver. 2.1.1 (Lanfear et al. 2016), based on the Akaike information criterion (Akaike 1974) with a greedy algorithm (Lanfear et al. 2012). The models used for Dataset 1 were GTR+I+G (16S, 18S, 28S, and all position in COI) for the ML and the BI analyses. The models used for Dataset 2 were GTR+I+G (28S), TIM+G (16S), TRN+G (third codon position in COI), TRN+I+G (first codon position in COI), and TVM+I (18S and second codon position in COI) for the ML analysis and GTR+I+G (28S and the first codon of COI), GTR+I (18S and second codon position in COI), and GTR+G (16S and third codon position of COI) for the BI analysis. Nodal support within the ML tree was evaluated by analyzing 1,000 bootstrap pseudoreplicates. For BI, the Markov chain Monte Carlo process used random starting trees and involved four chains that ran for 50,000,000 and 10,000,000 generations for Datasets 1 and 2, respectively, with the initial 25% trees discarded as burn-in. We considered standard ML bootstrap (BS) values of ≥ 85% and posterior probability (PP) values of ≥ 0.98 as indicative of strong clade support, with combined nodal support represented as BS/PP in the text.

Table 1.

List of species included in the molecular phylogenetic analysis and respective GenBank accession numbers

graphic file with name zoolstud-64-047-t001-1.jpg

graphic file with name zoolstud-64-047-t001-2.jpg

Open in a new tab

Data treatment

The voucher slides have been deposited in the Invertebrate Collection of the Hokkaido University Museum (ICHUM), Sapporo, Japan. All sequences obtained in this study were deposited in the DDBJ/ EMBL/GenBank databases under the accession numbers LC884712–LC884715.

RESULTS

Stylochoposthia sp.

(Figs. 1, 2)

Material examined: ICHUM 8981, sagittal sections of copulatory apparatus (12 slides) and whole mount of head, intertidal, Otsuki (32°46'44"N 132°43'57"E), Kochi, Japan, 10 April 2019, A. Tsuyuki, N. Hasegawa, N. Jimi leg.

Morphology: Live specimen 39 mm in length, 29 mm in maximum width. Body oval, brownish translucent (Fig. 1A, B). Intestine orangish, not anastomosed. Pharynx whitish, ruffled in shape, with five pairs of shallow folds, occupying about onefifth of body length (8.1 mm in length), located at center of body (Fig. 1C). Pair of uterine ducts lacking anterior confluence (Fig. 1C). Pair of nuchal tentacles prominent, orangish with white tip, located about onethird of body length (12 mm in length) from anterior end (Fig. 1A, B). Tentacular eye clusters congregated in bases of each nuchal tentacle. Cerebral eye clusters separated into two groups, located anterior and posterior to nuchal tentacles (Fig. 1D).

Fig. 1.

Fig. 1.

Open in a new tab

Stylochoposthia sp., ICHUM 8981, photographs taken in life (A–C) and whole mounted specimen of head (D). A, dorsal view with black back; B, dorsal view with white back; C, ventral view; D, eyespots and nuchal tentacles. Abbreviations: cec, cerebral eyespot cluster; nt, buchal tentacles; ov; oviduct, ph, pharynx, sd; sperm duct; tec, tentacular eyespot cluster; ud, uterine duct.

Male copulatory apparatus located posterior to pharynx and ventral to vagina, consisting of seminal vesicle and free prostatic vesicle (Fig. 2A, B). Testes distributed in ventral parenchyma. Pair of sperm ducts running anteriorly, turning medially at 3.6 mm posterior to posterior end of pharynx, fusing to common sperm duct (Fig. 2B). Common sperm duct 903 µm in length, entering proximal end of seminal vesicle (Fig. 2A). Seminal vesicle elliptic, having strong muscular wall (Fig. 2A, B). Prostatic vesicle oval, smaller than seminal vesicle, coated with muscular wall, lined with ridged glandular epithelium (Fig. 2B, C). Distal end of prostatic vesicle forming prostatic duct and connecting distal end of seminal vesicle (Fig. 2C). From uniting point, ejaculatory duct 528 µm in length, running posteriorly, opening to small male atrium; penis papilla lacking (Fig. 2D, E). Prostatic vesicle and ejaculatory duct surrounded sheath formed by fibrous tissue (Fig. 2A–F). Fibrous tissue dyed pale red with eosin in HE staining (Fig. 2D) but scarcely stained in MG stain (Fig. 2F); each dyed with same intensity as walls of seminal vesicle and prostatic vesicle.

Fig. 2.

Fig. 2.

Open in a new tab

Stylochoposthia sp., ICHUM 8981, photomicrographs (A–F) of sections and schematic diagram (H) of copulatory apparatuses, HE stain (A– E, G) and MG stain (F). A, male and female copulatory apparatuses; B, C, seminal vesicle and prostatic vesicle; D, prostatic vesicle and ejaculatory duct; E, male and female gonopores and vagina bulbosa; F, prostatic vesicle and sheath re-stained by MG method; G, female copulatory apparatus; H. reconstructed schematic diagram of male and female copulatory apparatuses. Abbreviations: cg, cement gland; cov, common oviduct; csd, common sperm duct; dsv, distal part of seminal vesicle; ed, ejaculatory duct; fg, female gonopore; ld, Lang’s vesicle duct; lv, Lang’s vesicle; mg, male gonopore; pd, prostatic duct; pv, prostatic vesicle; sh, sheath; sv, seminal vesicle; v, vagina; vb, vagina bulbosa.

Female copulatory apparatus consisting of vagina and Lang’s vesicle; bursa copulatrix absent (Fig. 2A, G). Ovaries distributed in dorsal parenchyma. Pair of uterine ducts running posteriorly, entering common oviduct. Common oviduct 293 µm in length, running posteriorly to enter vagina. Elongated Lang’s vesicle duct (1,013 µm in length) running posteriorly (Fig. 2G) to connect to Lang’s vesicle. Lang’s vesicle oval, located anterior to female gonopore. Vagina 2,657 µm in length, lined with smooth and ciliated epithelium, running anteriorly, then turning postero-ventrally to exit at female gonopore; about one-half of vagina surrounded cement glands. Distal one-fourth of vagina forming vagina bulbosa, lacking cuticularized structures (Fig. 2E).

Remarks: According to Prudhoe (1989), Stylochoposthia can be identified by the following characteristics: i) an oval or discoid body; ii) developed nuchal tentacles; iii) circularly arranged tentacular eyespots at the base of the tentacles; iv) cerebral eyespots forming two loose clusters between the tentacles; v) a pharynx with five or six pairs of lateral folds; vi) a thick connective tissue sheath surrounding the male copulatory apparatus; vii) a fusiform seminal vesicle; viii) a small prostatic vesicle with longitudinally folded epithelium; ix) a long vagina that forms an anteriorly-directed loop and runs dorsally to the male copulatory apparatus; x) a small, bulbous Lang’s vesicle; and xi) uterine ducts without anterior confluence. The present specimen meets the diagnostic criteria, except for criterion vi), which stipulates the presence of a sheath composed of connective tissues (Figs. 1, 2). The sheath in the specimen was composed of fibrous tissue (Fig. 2D, F), which we were reluctant to identify as connective tissue due to their histological appearance (See DISCUSSION). However, the general morphology of the external and internal structures aligns with those of Stylochoposthia; thus, we identified the specimen as a species of Stylochoposthia.

Stylochoposthia consists of two species: S. bella (Hyman, 1959) and S. inhacae Prudhoe, 1989. The current species exhibits greater similarity to S. inhacae than to S. bella, as both share the following characteristics: i) presence of a long common sperm duct, ii) absence of a penis stylet, and iii) presence of separated gonopores. However, S. inhacae possesses a small penis papilla, whereas the currently examined specimen lacks a papilla-like structure within the male copulatory apparatus (Fig. 2H). Furthermore, the common sperm duct in our specimen was longer than that in S. inhacae; the proximal end of the common sperm duct located anterior to the curve point of the vagina in our polyclad (Fig. 2H), whereas that in Prudhoe’s species positioned posterior to the curve although the actual length of the common sperm duct is unclear because the figure by Prudhoe (1989, fig. 15) does not include any scales. We could not determine whether these differences were due to interspecific variation or artifacts resulting from the fixation or sectioning of the specimen; thus, we identify this polyclad flatworm as Stylochoposthia sp.

Molecular phylogeny

The BI and ML trees exhibited nearly identical topologies; hence, we present only the ML tree in both analyses (Figs. 3, 4). In the tree involving acotylean polyclads (Dataset 1), the present species was placed within the clade of Planoceridae with high support values (95% in BS and 1.00 in PP). This species was separated from the species of Callioplanidae and Pseudostylochidae (Fig. 3). In the tree focused on planocerid polyclads (Dataset 2), the present species was placed within the clade of Planocera Blainville, 1828, with high support values (90% in BS and 1.00 in PP) (Fig. 4). This species formed a robust clade with Planocera pellucida in both datasets.

Fig. 3.

Fig. 3.

Open in a new tab

Maximum likelihood tree based on Dataset 1 (acotylean polyclads; 16S, 18S, 28S, COI) (concatenated length 2,706 bp). Numbers near nodes are bootstrap values greater than 85% or posterior probability greater than 0.98, respectively.

Fig. 4.

Fig. 4.

Open in a new tab

Maximum likelihood tree based on Dataset 2 (planocerid polyclads; 16S, 18S, 28S, COI) (concatenated length 3,547 bp). Numbers near nodes are bootstrap values greater than 85% or posterior probability greater than 0.98, respectively.

DISCUSSION

Molecular phylogenetic analyses did not support the classification of Stylochoposthia as proposed by Faubel (1983) or Prudhoe (1989). Stylochoposthia sp. did not form an exclusive clade with Callioplana marginata Stimpson, 1857, the type species of the type genus of Callioplanidae and Pseudostylochus including P. takeshitai Yeri & Kaburaki, 1918, the type species of the genus of Pseudostylochidae (Fig. 3). The morphology of Stylochoposthia, excluding the male copulatory apparatus, resembled that of planocerids, particularly Planocera species. Several diagnostic characteristics of Stylochoposthia described by Prudhoe (1989), such as the body shape, the presence of developed nuchal tentacles, the distribution of eyespots, the shape of the pharynx, and the lack of anterior confluence in the uterine ducts, were shared by all the planocerid polyclads included in this study. Moreover, Stylochoposthia shared characteristics with Planocera, including a reduced Lang’s vesicle and a developed vagina bulbosa (Fig. 2). The similarity between Stylochoposthia and planocerids suggests that the aforementioned characteristics are more stable than the type of an intromittent organ, the latter of which was emphasized by Faubel (1983).

Our study indicates that the male copulatory apparatus underwent drastic structural modifications within the Stylochoposthia sp. lineage. The planocerid polyclads in this study (Aquaplana, Heteroplanocera, Paraplanocera, and Planocera) are characterized by possessing an eversible cirrus with numerous teeth and a cavity surrounded by the cirrus bulb; the latter is alternatively referred to as “intromuscular space,” “intermuscular space,” or “spaces in the wall of the cirrus sac” (Prudhoe 1945; Ramos-Sánchez et al. 2019; Oya and Kajihara 2021). The presence of the aforementioned cirrus may represent an ancestral character state for the clade Planoceridae. The loss of numerous teeth and the cavity would have occurred following the divergence of Stylochoposthia sp. from its common ancestor with P. pellucida.

The histological characteristics of the sheath surrounding the prostatic vesicle and the ejaculatory duct in Stylochoposthia sp. are crucial for assessing the evolutionary modifications of the cirrus in planocerids. We speculate that the sheath is homologous to the cirrus bulb in planocerids; this homology is important for investigating the processes underlying the loss of these structures and their function during copulation. The sheath histologically resembled the walls of the seminal vesicle and the prostatic vesicles, which are composed of muscle tissues (Fig. 2D, F). The MG-stained sections revealed that the sheath was not composed of collagen fibers, which are typically stained light green, as seen in basement membranes (Fig. 2F). Furthermore, the sheath showed weak staining with azophloxine, which typically stains muscle fibers, similar to the bright red color of the body wall muscles observed in this section (Fig. 2F). This result; however, could be due to re-staining and/or artifacts resulting from the staining or fixation process because the walls of the seminal and prostatic vesicles were stained similarly to the sheath. Furthermore, we could not exclude the possibility that the sheath (and the walls of the seminal and prostatic vesicles) was composed of elastic fibers, a type of connective tissue. Therefore, further studies using additional specimens and alternative staining techniques (e.g., Elastica Van Gieson stain) that can differentiate tissue types are warranted. A histological comparison of the cirrus in planocerids with the sheath in Stylochoposthia could offer significant insights into the evolution of copulatory apparatuses in Polycladida.

The phylogenetic position of Stylochoposthia represented by our specimen suggests that Stylochoposthia is a junior synonym of Planocera. Despite the histological or structural similarities between the cirrus in Planocera and the sheath in Stylochoposthia, we could not provide a definite diagnosis encompassing both Planocera and Stylochoposthia. We propose deferring the synonymization of Stylochoposthia Faubel, 1983 with Planocera Blainville, 1828 until further morphological and histological assessments of these structures have been conducted.

CONCLUSIONS

Molecular phylogenetic analysis showed that Stylochoposthia, represented by Stylochoposthia sp., is nested within the Planoceridae clade and is a sister taxon to P. pellucida. The results indicate the loss of eversible cirrus with hard structures within the lineage of Stylochoposthia. Future studies involving detailed histological comparisons of the male copulatory apparatus between planocerids and Stylochoposthia are required to understand the evolution of this organ and its role in copulation. This study suggests that Stylochoposthia is a junior synonym of Planocera; however, we defer the synonymization of Stylochoposthia until a clear definition, based on a detailed morphological examination of the copulatory apparatuses, is established.

Supplementary materials

Fig. S1.

Stylochoposthia_full_BI.

(download) (7.2KB, pdf)
Fig. S2.

Stylochoposthia_full_ML.

(download) (7.3KB, pdf)

Acknowledgments

We thank Dr. Naoto Jimi (Nagoya University) and Dr. Naohiro Hasegawa (Hiroshima Shudo University) for helping sampling the specimen; Prof. Hiroshi Kajihara (Hokkaido University) for managing the voucher specimen. The authors would like to thank Enago (www.enago.jp) for the English language review. This study was partially supported by JSPS KAKENHI Grant Number JP23K14254 for YO and Kuroshio Biological Research Foundation for AT.

Footnotes

Authors’ contributions: YO designed the present study and conducted morphological observation and molecular phylogenetic analyses as well as drafted the manuscript. AT collected materials and contributed to improvement of the manuscript. All authors read and approved the final manuscript.

Competing interests: The authors declare that they have no conflict of interests.

Availability of data and materials: Voucher slides have been deposited in the Invertebrate Collection of the Hokkaido University Museum (ICHUM). Sequences determined in this study were deposited into the DNA Data Bank of Japan (DDBJ) database. The ML and BI phylogenies generated from Dataset 1 were deposited at Figshare (doi:10.6084/m9.figshare.29586680).

Consent for publication: Not applicable.

Ethics approval consent to participate: Not applicable.

References

  1. Akaike H. 1974. A new look at the statistical model identification. IEEE Trans Autom Control 19: 716–723. doi:10.1109/TAC.1974. 1100705.
  2. Aguado MT, Noreña C, Alcaraz L, Marquina D, Brusa F et al. 2017. Phylogeny of Polycladida (Platyhelminthes) based on mtDNA data. Org Divers Evol 17: 767–778. doi:10.1007/s13127-017 0344-4.
  3. Bahia J, Padula V, Schrödl M. 2017. Polycladida phylogeny and evolution: integrating evidence from 28S rDNA and morphology. Org Divers Evol 17:653–678. doi:10.1007/s13127-017-0327-5.
  4. Castresana J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552. doi:10.1093/oxfordjournals.molbev.a026334. [DOI] [PubMed]
  5. Dittmann IL, Cuadrado D, Aguado MT, Noreña C, Egger B. 2019. Polyclad phylogeny persists to be problematic. Org Divers Evol 19:585–608. doi:10.1007/s13127-019-00415-1.
  6. Faubel A. 1983. The Polycladida, Turbellaria. Proposal and establishment of a new system. Part I. The Acotylea. Mitt Hamb Zool Mus Inst 80:17–121.
  7. Gutiérrez A, Auby I, Gouillieux B, Daffe G, Massé C et al. 2023. A new polyclad flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Platyhelminthes, Polycladida) from France. Can this foreign flatworm be responsible for the deterioration of oyster and mussel farms? Zool Stud 62:15. doi:10.6620/ZS.2023.62-15. [DOI] [PMC free article] [PubMed]
  8. Hyman LH. 1959. Some Australian polyclads (Turbellaria). Rec South Aust Mus 25:1–17. doi:10.3853/j.0067-1975.25.1959.653.
  9. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. doi:10.1093/molbev/mst010. [DOI] [PMC free article] [PubMed]
  10. Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. 2019. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 35:4453– 4455. doi:10.1093/bioinformatics/btz305. [DOI] [PMC free article] [PubMed]
  11. Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. doi:10.1093/molbev/msw054. [DOI] [PMC free article] [PubMed]
  12. Lanfear R, Calcott B, Ho SYW, Guindon S. 2012. PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Mol Biol Evol 29:1695–1701. doi:10.1093/molbev/mss020. [DOI] [PubMed]
  13. Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B. 2016. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol Biol Evol 34: 772–773. doi:10.1093/molbev/ msw260. [DOI] [PubMed]
  14. Laumer CE, Giribet G. 2014. Inclusive taxon sampling suggests a single, stepwise origin of ectolecithality in Platyhelminthes. Biol J Linn Soc 111:570–588. doi:10.1111/bij.12236.
  15. Littlewood DTJ, Rohde K, Clough KA. 1999. The interrelationships of all major groups of Platyhelminthes: phylogenetic evidence from morphology and molecules. Biol J Linn Soc 66:75–114. doi:10.1111/j.1095-8312.1999.tb01918.x.
  16. Litvaitis MK, Bolaños DM, Quiroga SY. 2019. Systematic congruence in Polycladida (Platyhelminthes, Rhabditophora): are DNA and morphology telling the same story? Zool J Linn Soc 186:865– 891. doi:10.1093/zoolinnean/zlz007.
  17. Lockyer AE, Olson PD, Littlewood DTJ. 2003. Utility of complete large and small subunit rRNA genes in resolving the phylogeny of the Neodermata (Platyhelminthes): implications and a review of the cercomer theory. Biol J Linn Soc 78:155–171. doi:10.1046/j.1095-8312.2003.00141.x.
  18. Mallatt J, Winchell CJ. 2002. Testing the new animal phylogeny: first use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes. Mol Biol Evol 19:289– 301. doi:10.1093/oxfordjournals.molbev.a004082. [DOI] [PubMed]
  19. Oya Y, Kajihara H. 2017. Description of a new Notocomplana species (Platyhelminthes: Acotylea), new combination and new records of Polycladida from the northeastern Sea of Japan, with a comparison of two different barcoding markers. Zootaxa 4282:526–542. doi:10.11646/zootaxa.4282.3.6.
  20. Oya Y, Kajihara H. 2019. A new species of Phaenoplana (Platyhelminthes: Polycladida) from the Ogasawara Islands. Species Divers 24:1–6. doi:10.12782/specdiv.24.1.
  21. Oya Y, Kajihara H. 2020. Molecular phylogenetic analysis of Acotylea (Platyhelminthes: Polycladida). Zool Sci 37:271–279. doi:10.2108/zs190136. [DOI] [PubMed]
  22. Oya Y, Kajihara H. 2021. Description and phylogenetic relationships of a new genus of Planoceridae (Polycladida, Acotylea) from Shimoda, Japan. J Mar Biol Assoc UK 101: 81–88. doi:10.1017/ S0025315421000060.
  23. Oya Y, Kimura T, Kajihara H. 2019. Description of a new species of Paraplehnia (Polycladida, Stylochoidea) from Japan, with inference on the phylogenetic position of Plehniidae. ZooKeys 864:1. doi:10.3897/zookeys.864.33955. [DOI] [PMC free article] [PubMed]
  24. Oya Y, Tsuyuki A, Kajihara H. 2020. A new species of Zygantroides (Platyhelminthes: Polycladida) from Amakusa, Japan. Species Divers 25:189–196. doi:10.12782/specdiv.25.189.
  25. Oya Y, Nakajima H, Kajihara H. 2022a. A new symbiotic relationship between a polyclad flatworm and a mantis shrimp: description of a new species of Emprosthopharynx (Polycladida: Acotylea) associated with Lysiosquilla maculata (Crustacea: Stomatopoda). Mar Biodivers 52:46. doi:10.1007/s12526-022-01288-y.
  26. Oya Y, Tsuyuki A, Kajihara H. 2021. Description of a new species of Alloioplana (Polycladida: Stylochoplanidae) with an inference on its phylogenetic position in Leptoplanoidea. Proc Biol Soc Wash 134:306–317.
  27. Oya Y, Tsuyuki A, Kajihara H. 2022b. Descriptions of two new species of Armatoplana (Polycladida: Stylochoplanidae) from the coasts of Japan, with their phylogenetic positions in Leptoplanoidea. Zootaxa 5178:433–452. doi:10.11646/zootaxa.5178.5.2. [DOI] [PubMed]
  28. Palumbi S, Martin A, Romano S, McMillan WO, Stice L et al. 1991. The Simple Fools Guide to PCR, Ver. 2. Department of Zoology and Kewalo Marine Laboratory, University of Hawaii, USA.
  29. Prudhoe S. 1945. On the species of the polyclad genus Paraplanocera. Annals Mag Nat Hist 12:195–202. doi:10.1080/00222934508527505.
  30. Prudhoe S. 1985. A Monograph on Polyclad Turbellaria. Oxford University Press, Oxford, UK.
  31. Prudhoe S. 1989. Polyclad turbellarians recorded from African waters. Bull Br Mus Nat Hist 55:47–96.
  32. Ramos-Sánchez M, Bahia J, Bastida-Zavala JR. 2019. New genus, new species and new records of marine acotylean flatworms (Platyhelminthes: Polycladida: Acotylea) from Oaxaca, southern Mexican Pacific. Zootaxa 4700: 30–58. doi:10.11646/ zootaxa.4700.1.2. [DOI] [PubMed]
  33. Rawlinson KA, Gillis JA, Billings RE, Borneman EH. 2011. Taxonomy and life history of the Acropora-eating flatworm Amakusaplana acroporae nov. sp. (Polycladida: Prosthiostomidae). Coral Reefs 30:693–705. doi:10.1007/s00338-011-0745-3.
  34. Rodríguez J, Hutchings PA, Williamson JE. 2021. Biodiversity of intertidal marine flatworms (Polycladida, Platyhelminthes) in southeastern Australia. Zootaxa 5024: 1–63. doi:10.11646/ zootaxa.5024.1.1. [DOI] [PubMed]
  35. Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574. doi:10.1093/bioinformatics/btg180. [DOI] [PubMed]
  36. Sonnenberg R, Nolte AW, Tautz D. 2007. An evaluation of LSU rDNA D1–D2 sequences for their use in species identification. Front Zool 4:1–12. doi:10.1186/1742-9994-4-6. [DOI] [PMC free article] [PubMed]
  37. Tsunashima T, Hagiya M, Yamada R, Koito T, Tsuyuki N et al. 2017. A molecular framework for the taxonomy and systematics of Japanese marine turbellarian flatworms (Platyhelminthes, Polycladida). Aquat Biol 26:159–167. doi:10.3354/ab00682.
  38. Tsuyuki A, Oya Y, Jimi N, Kajihara H. 2020. Description of Pericelis flavomarginata sp. nov. (Polycladida: Cotylea) and its predatory behavior on a scaleworm. Zootaxa 4894: 403–412. doi:10.11646/ zootaxa.4894.3.6. [DOI] [PubMed]
  39. Tsuyuki A, Oya Y, Kajihara H. 2022. Two new species of the marine flatworm Pericelis (Platyhelminthes: Polycladida) from southwestern Japan with an amendment of the generic diagnosis based on phylogenetic inference. Mar Biol Res 17: 946–959. doi: 10.1080/17451000.2022.2048669.
  40. Tyler S, Schilling S, Hooge M, Bush LF. 2006–2025. Turbellarian taxonomic database. Version 2.2. Available at: http://turbellaria. umaine.edu. (Accessed 1 Jan. 2025)
  41. Ueda H, Itoi S, Sugita H. 2018. TTX-bearing planocerid flatworm (Platyhelminthes: Acotylea) in the Ryukyu Islands, Japan. Mar Drugs 16:37. doi:10.3390/md16010037. [DOI] [PMC free article] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1.

Stylochoposthia_full_BI.

(download) (7.2KB, pdf)
Fig. S2.

Stylochoposthia_full_ML.

(download) (7.3KB, pdf)

Articles from Zoological Studies are provided here courtesy of Biodiversity Research Center, Academia Sinica

RESOURCES