TY - JOUR
AU - Quiroga, Sigmer, Y
AB - Abstract Using 28S ribosomal DNA sequences, we inferred the internal relationships of the order Polycladida. We identified morphological characters for clade support when possible. Monophyletic Acotylea and Cotylea were consistently recovered. In Acotylea, the superfamilies Stylochoidea, Cryptoceloidea and Leptoplanoidea were supported, with Stylochoidea representing the most basal acotylean lineage. In Leptoplanoidea, we united genera lacking a penis armature into the new family Notocomplanidae. Gnesiocerotidae was recovered as the most basal leptoplanoid lineage, and Stylochoplanidae and Notoplanidae were paraphyletic. Among cotyleans, Cestoplanidae, Diposthus popeae + Pericelis spp., Boniniidae, Pseudocerotidae and Prosthiostomidae formed clades. Genera in Euryleptidae were monophyletic, but the family itself was recovered with low support only. The established superfamilies Pseudocerotoidea, Euryleptoidea, Periceloidea and Chromoplanoidea are not supported. Pericelis has been moved to Diposthidae and Pericelidae has been abolished. A clade of Boniniidae + Theama spp. + Chromyella sp. was supported. In Pseudocerotidae, the number of male reproductive structures unites Pseudobiceros and Thysanozoon. Tytthosoceros has been abolished, with all currently described species now placed in Phrikoceros. Our results support several additional synonymies and taxonomic corrections. This new phylogeny provides an increased understanding of relationships in the order and offers a framework for future testing of hypotheses of character evolution and life-history strategies. Bayesian inference, maximum likelihood, molecular systematics, phylogeny, taxonomic revision INTRODUCTION The order Polycladida contains >800 recognized species (Tyler et al., 2006–2018), but traditional classifications mostly placed species with little phylogenetic inference. The major subdivision of the order is based on the presence or absence of a taxonomically distinguishing ventral adhesive structure (cotyl) posterior to the female gonopore, which divides the order into Cotylea and Acotylea, respectively (Lang, 1884). In a first attempt to place all taxonomic information available at that time into an evolutionary framework, Lang (1884) proposed a division of Acotylea into three major families (a basal Planoceridae, plus derived Leptoplanidae and Cestoplanidae). In Cotylea, Lang (1884) recognized four families, a basal Anonymidae, plus Pseudoceridae (now Pseudocerotidae), Euryleptidae and Prosthiostomidae. Since this early classification, additional information has been added by Laidlaw (1903), Bock (1913), Poche (1926), Marcus & Marcus (1966), Faubel (1983, 1984) and Prudhoe (1985), increasing our understanding of internal relationships of the order. Currently, the two most inclusive, morphology-based classifications are incongruent (Faubel, 1983, 1984, largely based on Bock, 1913; Prudhoe, 1985, largely based on Poche, 1926). This is primarily attributable to an emphasis on very different morphological characters. However, both classifications divide Acotylea into three superfamilies. Faubel (1984: 193) argued that, because of their complexity, traits associated with the reproductive systems (e.g. the prostatic vesicle) intuitively carry more phylogenetic information than the position of eyes or the position and shape of tentacles. Accordingly, he recognized Stylochoidea (prostatic vesicle free; Fig. 1A), Leptoplanoidea (prostatic vesicle interpolated; Fig. 1B) and Ilyplanoidea [true prostatic vesicle lacking (Fig. 1C) or diffuse prostatoid glands (Fig. 1D)]. Given that he considered diffuse prostatoid glands a plesiomorphic character of the entire order, Faubel (1984) rooted his phylogeny with Ilyplanoidea and reasoned that taxa with either a free (Stylochoidea) or interpolated (Leptoplanoidea) prostatic vesicle are derived Acotylea. Figure 1. View largeDownload slide Structural arrangements and orientation of male reproductive system. Arrows and shading indicate prostatic vesicle/prostatoids; no shading indicates seminal vesicle. The anterior of the animal is to the left. A, free prostatic vesicle. B, interpolated prostatic vesicle. C, without prostatic vesicle. D, numerous prostatoid organs instead of a prostatic vesicle (e.g. Adenoplana). E, male copulatory system located anterior to male gonopore; common in Acotylea. F, male copulatory system located posterior to male gonopore; common in Cotylea. Figure 1. View largeDownload slide Structural arrangements and orientation of male reproductive system. Arrows and shading indicate prostatic vesicle/prostatoids; no shading indicates seminal vesicle. The anterior of the animal is to the left. A, free prostatic vesicle. B, interpolated prostatic vesicle. C, without prostatic vesicle. D, numerous prostatoid organs instead of a prostatic vesicle (e.g. Adenoplana). E, male copulatory system located anterior to male gonopore; common in Acotylea. F, male copulatory system located posterior to male gonopore; common in Cotylea. Additional characters of importance in Faubel’s (1983, 1984) classification are based on the shape of the epithelial lining of the prostatic vesicle (e.g. smooth, ridged, fingered, chambered, glandular), the overall orientation of the male copulatory system (located anterior vs. posterior to the male pore; Fig. 1E, F), the number of male reproductive systems and any cuticular elements of the male copulatory organ (e.g. stylet, cirrus; Fig. 2). With respect to the female system, Faubel (1983, 1984) placed great importance on the presence (mostly in acotyleans) of Lang’s vesicle and its shape (Fig. 3A–C) and on uterine vesicles (present only in cotyleans; Fig. 3D). Figure 2. View largeDownload slide Armature of the male copulatory organ. Shading indicates prostatic vesicle. A, penis papilla armed with a short stylet (arrow). B, penis papilla armed with a long, thin stylet (arrow). C, D, variations of penis papilla armed with teeth or hooks (arrows), e.g. Gnesioceros (C) and Styloplanocera (D). Figure 2. View largeDownload slide Armature of the male copulatory organ. Shading indicates prostatic vesicle. A, penis papilla armed with a short stylet (arrow). B, penis papilla armed with a long, thin stylet (arrow). C, D, variations of penis papilla armed with teeth or hooks (arrows), e.g. Gnesioceros (C) and Styloplanocera (D). Figure 3. View largeDownload slide Structural arrangement of female reproductive system. Arrows and shading indicate Lang’s vesicle; patterned area indicates cement glands. A, female system without Lang’s vesicle. B, female system with Lang’s vesicle. C, female system with horseshoe-shaped Lang’s vesicle (e.g. Discocelis, Idioplana and Imogine). D, female system in Cotylea with uterine vesicles (arrow) and cement pouches (arrowheads) in terminal part of vagina. Figure 3. View largeDownload slide Structural arrangement of female reproductive system. Arrows and shading indicate Lang’s vesicle; patterned area indicates cement glands. A, female system without Lang’s vesicle. B, female system with Lang’s vesicle. C, female system with horseshoe-shaped Lang’s vesicle (e.g. Discocelis, Idioplana and Imogine). D, female system in Cotylea with uterine vesicles (arrow) and cement pouches (arrowheads) in terminal part of vagina. Based on the arrangement of eyes and different types of tentacles, Prudhoe’s (1985) system also recognized three acotylean superfamilies. With a few exceptions, his Stylochoidea is characterized by tentacular, cerebral and frontal clusters of eyes, in addition to eyes along the margins of the body. Members of Planoceroidea possess cerebral and tentacular eyes only, with no eyes distributed along the body margin. His third superfamily, Cestoplanoidea, has eyes widely distributed over the cephalic region, but they are absent from the marginal zone (Prudhoe, 1985). Cotylean classification is also disparate between the two systems. Typically, cotylean reproductive systems possess a free prostatic vesicle (male) and cement pouches (female; arrowheads in Fig. 3D), therefore offering few diagnostic traits. Other differentiating morphological characters include the shape and position of the pharynx or of the tentacles, and the arrangement of eyes. Faubel (1984) separated Euryleptoidea (Prosthiostomidae and Euryleptidae) from Pseudocerotoidea (Pseudocerotidae, Pericelidae, Boniniidae, Amyellidae and Diposthidae) based on the shape of the pharynx (tubular vs. ruffled) and on the pseudotentacles. Two additional minor superfamilies (Ditremagenioidea and Opisthogenioidea) were also included in Cotylea (Faubel, 1984). However, Ditremagenioidea has been removed to triclads (Prudhoe, 1985; A. Faubel, pers. comm.) and Opisthogenioidea is monospecific. Prudhoe (1985), in contrast, recognized familial clades only in Cotylea. Consequently, species identifications, especially in pseudocerotids and euryleptids, are reliant on colour and colour pattern (Hyman, 1954, 1955a, b, 1959a, b; Prudhoe, 1989; Newman & Cannon, 1994). Specifically, Michiels & Newman (1998) observed copulation occurring only among like-coloured pseudocerotids, and Litvaitis et al. (2010) showed congruence in molecular systematics among similar colour morphs of the Pseudoceros bicolor Verrill, 1902–Pseudoceros rawlinsonae Bolaños et al., 2007 complex. The only other morphology-based cotylean classification is the cladistic analysis by Rawlinson & Litvaitis (2008). They confirmed monophyly of four families and a basal position for Boniniidae among Cotylea. However, neither Faubel’s (1983, 1984) nor Prudhoe’s (1985) classification scheme received unequivocal support in that study. Specifically, Boniniidae and Pericelidae were not included in Pseudocerotoidae and Prosthiostomidae was not a member of Euryleptoidea, thus rendering the two superfamilies invalid (Rawlinson & Litvaitis, 2008). A handful of molecular studies have focused exclusively on polyclads (Goggin & Newman, 1996; Litvaitis & Newman, 2001; Litvaitis et al., 2010; Rawlinson et al., 2011; Aguado et al., 2017; Bahia et al., 2017; Tsunashima et al., 2017). Most recently, using partial sequences of the 28S ribosomal DNA (rDNA) gene, Bahia et al. (2017) proposed a revised phylogeny for the order, in which they recognized three superfamilies in Acotylea (Stylochoidea Poche, 1926, Cryptoceloidea and Leptoplanoidea) and five superfamilies in Cotylea (Cestoplanoidea, Periceloidea, Chromatoplanoidea, Prosthiostomoidea and Pseudocerotoidea). Focusing on polyclads from Japan, Tsunashima et al. (2017) presented another 28S rDNA-based phylogeny, whereas Aguado et al. (2017) evaluated polyclad relationships based on partial mitochondrial cytochrome oxidase c subunit I (COI) and 16S rDNA genes. In the present study, we follow the acotylean division of Bahia et al. (2017). Our results indicate a considerably different classification for Cotylea, and consequently, we do not use the superfamilial cotylean divisions of Bahia et al. (2017; see Discussion). Our objectives are threefold. First, using partial nucleotide sequences of the 28S rDNA gene, we construct an internal phylogeny of Polycladida. To this end, we include numerous polyclad species collected throughout the Caribbean, the Mediterranean and the Indo-Pacific, with multiple representatives from most major groups. We also added sequences from GenBank to our samples. Second, where possible, we identify taxonomically stable diagnostic characters that are congruent with our molecular phylogenetic hypothesis. Third, we compare our phylogeny with other recent revisions of polyclad classification (e.g. Aguado et al., 2017; Bahia et al., 2017; Tsunashima et al., 2017). By including the largest number of taxa so far and with specimens sampled from the widest geographical range, our study represents the most comprehensive phylogeny of Polycladida to date. Not only does it provide an increased understanding of relationships in the order, but also it allows us to reconcile discrepancies in existing incongruent phylogenies. MATERIAL AND METHODS During a multi-year survey of the polyclad fauna, we collected specimens at locations throughout the greater Caribbean (see Supporting Information, Appendix). Additional specimens were obtained as part of other surveys in Australia, Guam, Palau and Papua New Guinea. To improve systematic coverage, we also included specimens collected vicariously by other scientists in Israel, Madagascar, Spain, Chile and California, and two local species were collected from Massachusetts and New Hampshire. Sequences obtained from GenBank were also included, but we purposefully chose not to include available sequences of easily misidentified species. Furthermore, the sequences deposited by Bahia et al. (2017) appear to be edited (i.e. variable regions removed) and are consequently of limited value in initial alignments. Taxonomic authorities, collection localities and GenBank accession numbers for specimens used in the present study are listed in the Supporting Information (Appendix). Animals were hand collected from reefs, sea grass beds and rocky intertidal zones by lifting them off the substrate using a soft paintbrush. Specimens were measured and photographed in the laboratory and then either fixed on frozen, buffered formalin for subsequent histology or preserved in 95% ethanol for DNA analysis (for a detailed protocol, see Litvaitis et al., 2010). DNA was extracted using either Genomic Tips or DNEasy Blood and Tissue kits (Qiagen Inc., Valencia, CA, USA). DNA was amplified using primers targeting the D1–D2 expansion segment of the 28S rDNA gene (Sonnenberg et al., 2007). The PCR conditions included an initial 2 min denaturation at 96 °C, followed by 30 cycles of 96 °C for 30 s, 50 °C for 1 min and 72 °C for 2 min, with a final extension of 4 min at 72 °C. Gel-purified amplicons were sequenced in both directions by commercial sequencing laboratories (Geneway Research LLC, Hayward, CA, USA; Eurofins Genomics, Louisville, KY, USA). Trace files were analysed using Geneious v. R11 (Kearse et al., 2012). To account for the secondary structure of rRNA, sequences were aligned with the Q-INS-i algorithm in MAFFT v.7.157 (Katoh & Standley, 2013). Gaps were treated as ambiguous characters. Post-alignment editing to remove ambiguous aligned sites was done using GBlocks v.0.91b, adopting the less stringent options (Castresana, 2000). Previous morphology- and DNA-based studies have shown a sister-group relationship between Polycladida, Macrostomida and Lecithoepitheliata (Karling, 1974; Carranza et al., 1997; Litvaitis & Rohde, 1999; Baguña & Riutort, 2004; Laumer & Giribet, 2014; Egger et al., 2015; Laumer et al., 2015). Hence, we used Paromalostomum fusculum Ax, 1952 and Prorhynchus stagnalis Schultze, 1851 as outgroups in the reconstruction of maximum likelihood (ML) via RAxML v.8.2.7 (Stamatakis, 2014) and Bayesian inference (BI; MrBayes v.3.2.3; Ronquist et al., 2012) trees under the GTR+G+I model, as determined by jModelTest v.2.1.7 (Darriba et al., 2012). Node support was determined by bootstrapping the ML tree with 2000 non-parametric replicates. Bayesian posterior probabilities (BPPs) were approximated with 5 000 000 generations using four simultaneous Metropolis-coupled Monte Carlo Markov chains (MCMCs) with a sample frequency of 1000 generations. The first 25% of the 10 000 sampled trees was discarded as burn-in. Chain convergence was assessed by ensuring that the average standard deviation of split frequencies approached zero (Ronquist et al., 2012). We considered BPP values of ≥ 90% and bootstrap values of ≥ 70% as evidence of clade support. In addition to a global polyclad phylogeny, separate ML and BI trees were generated for Acotylea and Cotylea. Trees were edited in FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and Adobe Photoshop Elements v.15.0. RESULTS Both ML and BI algorithms resulted in topologically congruent trees of the global polyclad phylogeny (Fig. 4) and of two subtrees for separate Acotylea and Cotylea relationships (Figs 5 and 6, respectively); therefore, only ML trees labelled with BPP (≥ 90%) and (≥ 70%) bootstrap support values are shown. Figure 4. View largeDownload slide Global polyclad maximum likelihood phylogeny. Bayesian posterior probabilities and maximum likelihood bootstrap support (expressed as a percentage) are provided as numerical values at nodes. For support values of internal nodes, see Figures 5 and 6. Figure 4. View largeDownload slide Global polyclad maximum likelihood phylogeny. Bayesian posterior probabilities and maximum likelihood bootstrap support (expressed as a percentage) are provided as numerical values at nodes. For support values of internal nodes, see Figures 5 and 6. Figure 5. View largeDownload slide Maximum likelihood (ML) phylogeny of Acotylea. Bayesian posterior probabilities and ML bootstrap support (expressed as a percentage) are provided as numerical values at nodes. –, support ≤ 0.90 Bayesian posterior probability or ≤ 70% maximum likelihood bootstrap. Figure 5. View largeDownload slide Maximum likelihood (ML) phylogeny of Acotylea. Bayesian posterior probabilities and ML bootstrap support (expressed as a percentage) are provided as numerical values at nodes. –, support ≤ 0.90 Bayesian posterior probability or ≤ 70% maximum likelihood bootstrap. Figure 6. View largeDownload slide Maximum likelihood (ML) phylogeny of Cotylea. Bayesian posterior probabilities and ML bootstrap support (expressed as a percentage) are provided as numerical values at nodes. –, support ≤ 0.90 Bayesian posterior probability or ≤ 70% ML bootstrap. Figure 6. View largeDownload slide Maximum likelihood (ML) phylogeny of Cotylea. Bayesian posterior probabilities and ML bootstrap support (expressed as a percentage) are provided as numerical values at nodes. –, support ≤ 0.90 Bayesian posterior probability or ≤ 70% ML bootstrap. Acotylea vs. Cotylea subdivision (Fig. 4) We consistently recover monophyletic Acotylea and Cotylea (sensu Bahia et al., 2017) with strong support. In Acotylea, the three superfamilies, Stylochoidea Poche, 1926, Cryptoceloidea Bahia et al., 2017 and Leptoplanoidea Faubel, 1984, each form clades, with Stylochoidea representing the most basal lineage. Ilyplanoidea (sensu Faubel, 1983, 1984) represent the immediate sister group to a clade of Phaenocelis species. Among cotyleans, Cestoplanidae, Pseudocerotidae, Prosthiostomidae, Boniniidae + Theama spp. + Chromyella sp. and Pericelidae + Diposthus popeae Hyman, 1959 are all recovered as monophyletic groups. Euryleptidae is not supported as a family, but individual genera in the family are monophyletic. The two superfamilies Pseudocerotoidea (sensu Faubel, 1984) and Euryleptoidea (sensu Faubel, 1984) are not supported. Relationships in Acotylea (Fig. 5) In Stylochoidea (sensu Poche, 1926), our specimens include representatives of six families (Callioplanidae, Planoceridae, Latocestidae, Pseudostylochidae, Stylochidae and Hoploplanidae). Callioplana marginata Stimpson, 1857 consistently forms the most basal lineage to two clades: Planoceridae + Hoploplana spp. form a sister group to a clade containing Stylochidae, Pseudostylochidae and Latocestidae. The superfamily Cryptoceloidea (sensu Bahia et al., 2017) contains two clades. Discocelis, Ilyella and Adenoplana, corresponding to Ilyplanoidea (sensu Faubel 1983, 1984), is recovered as an immediate sister group to a clade containing several species of Phaenocelis + Phaenoplana plus Amemiyaia pacifica Kato, 1944 (see Discussion section for reassignment of Phaenoplana to Phaenocelis and of Amemiyaia pacifica to Cryptocelidae). According to Faubel (1983, 1984), Leptoplanoidea comprises 12 families, two of which have been removed to Cotylea (Bahia et al., 2017; this study), one has been moved to the superfamily Cryptoceloidea (Bahia et al., 2017; this study), and four others are monogeneric/monospecific. We included species of five of the remaining leptoplanoid families in our phylogeny (see Supporting Information, Appendix). We consistently recover Gnesiocerotidae [Styloplanocera fasciata (Schmarda, 1859) and Gnesioceros sargassicola (Mertens, 1833)] as the most basal lineage. A second clade containing Melloplana (for reassignment to Notocomplana, see Discussion) and Notocomplana plus a heterogeneous clade of Persica, Pleioplana (for reassignment to Notoplana, see Discussion) and Notoplana are also identified. Although the clade is supported by BBP of one, bootstrap support was below our significance level. The sequences of the two species of Amyris (Amyris ujara Marcus & Marcus, 1968 and Amyris hummelincki Marcus & Marcus, 1968) are identical and formed a sister group to Armatoplana leptalea (Verrill, 1900). However, Armatoplana divae (Marcus, 1947) plus Notoplana queruca Marcus & Marcus, 1968 do not cluster with other Armatoplana or Notoplana. Instead, these species form a supported, separate basal clade in Leptoplanoidea. Relationships in Cotylea (Fig. 6) Cestoplanidae and Pericelidae + Diposthus popeae are recovered as two basal monophyletic groups. A supported clade of Boniinidae, Theamatidae and Amyellidae is recovered. Although the inclusion of Chromyella sp. with these two taxa is confirmed, support for a group containing only Boniinidae + Theamatidae is weak. A monophyletic Prosthiostomidae is strongly supported and contains a distinct, monophyletic Prosthiostomum and Enchiridium. Euryleptidae and Pseudocerotidae are sister taxa. Eurylepta, Cycloporus and Maritigrella are all monophyletic in Euryleptidae, but the family itself is not supported. Prostheceraeus forms a monophyletic group, but support is not significant other than at the species level. Pseudocerotidae consists of a strongly supported clade containing a monophyletic Pseudobiceros + Thysanozoon. However, relationships in Thysanozoon proper could not be resolved. A monophyletic Tytthosoceros plus Phrikoceros represents the immediate sister group. Pseudoceros was recovered as a monophyletic group, albeit two distinct subclades are evident. DISCUSSION Recently, three molecular phylogenies focusing solely on polyclads have become available (Aguado et al., 2017; Bahia et al., 2017; Tsunashima et al., 2017). Taxon coverage varies among the three, with the study by Bahia et al. (2017) being taxonomically the most inclusive. Aguado et al. (2017) specifically focused on two mitochondrial genes, whereas Bahia et al. (2017) selected the 5′ end of the 28S rDNA gene, and Tsunashima et al. (2017) also based their conclusions mostly on partial 28S rDNA sequences, but additionally provided COI sequences for a select subset of their specimens. Although the mitochondrial COI gene is a preferred choice to determine lower taxonomic relationships in free-living platyhelminths, high third position substitution rates in COI codons and high variability in mitochondrial gene order (Sakai & Sakaizumi, 2012; Vanhove et al., 2013; Golombek et al., 2015; Solà et al., 2015) prevent the design of efficient, taxon-wide primers, as evidenced by Aguado et al. (2017). It appears that taxon coverage, rather than choice of marker, is the single most crucial factor for accurately reconstructing polyclad relationships. With 196 sequences (153 of which are new), representing 37 genera in 22 families (Supporting Information, Appendix), our phylogeny represents the most comprehensive analysis of the internal relationships of Polycladida to date. With a few exceptions, we included multiple representatives from all major families. Acotylea vs. Cotylea subdivision Even though its systematic value has been questioned (Marcus & Marcus, 1966; Faubel, 1984; Laumer & Giribet, 2014), the defining taxonomic character separating the order into two suborders is the cotyl (Lang, 1884). Our results strongly support a division of the order based on this character (for inclusion of acotylean-like polyclads in Cotylea, see Discussion on Relationships in Cotylea). Similar support has been provided by Aguado et al. (2017), Bahia et al. (2017) and Tsunashima et al. (2017). However, here we want to reconfirm Lang’s original definition that only a ventral adhesive structure (e.g. adhesive disc, glandulomuscular organ) located medially at any point posterior to the female gonopore and including the terminal end, be considered a cotyl. A cotyl can consist of merely a small depression or a patch of a few glandular cells on the ventral side and thus may be difficult to discern, especially on a preserved or damaged specimen (e.g. Diposthus spp.; see below). Furthermore, polyclad cotyls have commonly been called suckers. Such terminology is reminiscent of an easily distinguishable adhesive structure (e.g. ventral sucker of trematodes). The absence of a prominently visible sucker might therefore have contributed to the present confusion regarding the subdivision of Polycladida. Here, we advocate for using the term ‘cotyl’ (sensu Lang, 1884) rather than sucker, in reference to the adhesive structures found in polyclads. Consequently, the genital suckers of the acotyleans Leptoplana tremellaris (O.F. Müller, 1773), Itannia ornata Marcus, 1947 and Persica qeshmensis Maghsoudlou et al., 2015, which are located anterior (or adjacent) to the female gonopore, do not represent a cotyl as defined and therefore should not be used for subordinal taxonomic placement. A few cotylean species apparently lack adhesive organs. However, some of these descriptions may represent fixation artefacts (e.g. Diposthus popeae), descriptive omissions (e.g. Diposthus corallicola Woodworth, 1898) or are based on damaged specimens (e.g. Chromyella saga Correa, 1958). The absence of a cotyl in Nymphozoon bayeri Hyman, 1959a has been attributed to the multiplication of the female reproductive structures, extending posteriorly and occupying the area where a cotyl would be located (Hyman, 1959a). Since then, a definite cotyl has been described for Nymphozoon bayeri by Bolaños et al. (2016). Diplopharyngeata filiformis Plehn, 1896 has been listed as an example of a cotylean lacking a cotyl (Kato, 1938: 575; Marcus & Marcus, 1966: 320; Rawlinson et al. 2011: 703). However, Faubel (1983: 34) recognizes its acotylean affinity and correctly places Diplopharyngeata filiformis into Euplanidae. To date, it appears then that only Theama (recently moved to Cotylea by Bahia et al., 2017), Amakusaplana and Simpliciplana marginata Kaburaki, 1923 (not included in this study) exemplify cotylean genera lacking adhesive structures. Although we consider the presence/absence of a cotyl as defined by Lang (1884) to be the major taxonomic trait subdividing the order, the orientation of the male copulatory complex with respect to the male pore is also diagnostic (Faubel, 1983, 1984). In acotyleans, the male complex is positioned anterior to the male pore (Fig. 1E), whereas in most cotyleans, the male reproductive apparatus is located posterior to the male pore (Fig. 1F). Other traits exclusively uniting the Acotylea include a centrally located, ruffled pharynx, the absence of marginal tentacles (if present, tentacles are nuchal) and, although not present throughout the suborder, Lang’s vesicle also indicates acotylean affinity (except in Boniniidae and Praestheceraeus). Cotylea, in contrast, are characterized further by marginal tentacles, a short, posteriorly directed vagina surrounded by cement pouches (Fig. 3D, arrowheads) and by either indirect or intracapsular development via a Müller’s larva (aberrant Müller’s larva in Boniniidae; Bolaños, 2008). Relationships in Acotylea Previous studies have divided Acotylea into three major superfamilies (Table 1), and Faubel (1983) postulated the lack of a true prostatic vesicle (in his Ilyplanoidea) as the plesiomorphic condition. Stylochoidea (sensu Faubel, 1983) characterized by a free prostatic vesicle would then be derived. Although we recovered three superfamilies as monophyletic groups, Stylochoidea (sensu Poche, 1926), not Ilyplanoidea, rooted Acotylea. Thus, the absence of a prostatic vesicle must be considered as the derived state. This view is in accordance with Laidlaw (1903) and Bock (1913), who also regarded the lack of a distinct prostatic vesicle as the secondary state. Laidlaw (1903) assumed that the wide distribution of a free prostatic vesicle found in many acotyleans and most Cotylea is an indication of its plesiomorphy. Our results, in which Stylochoidea (sensu Poche, 1926) represents the most basal acotylean clade, strongly support this hypothesis. Table 1. Superfamily- and family-level comparison of morphology- and DNA-based classification systems Acotylea Family Faubel (1983, 1984) Bahia et al. (2017) This study Superfamily Stylochoidea Poche, 1926 Callioplanidae Callioplanidae N/A Callioplanidae Hoploplanidae Leptoplanidae Hoploplanidae Hoploplanidae Latocestidae Latocestidae N/A Latocestidae Planoceridae Planoceridae Planoceridae Planoceridae Pseudostylochidae Pseudostylochidae Pseudostylochidae Pseudostylochidae Stylochidae Stylochidae Stylochidae Stylochidae Superfamily Cryptoceloidea (sensu Bahia et al., 2017); Ilyplanoidea (sensu Faubel, 1983, 1984) is not a valid superfamily Discocelidae Discocelidae Discocelidae Discocelidae Ilyplanidae Ilyplanidae N/A Ilyplanidae Cryptocelidae In Leptoplanoidea Cryptocelidae Cryptocelidae Superfamily Leptoplanoidea (sensu Faubel, 1983) Gnesiocerotidae Gnesiocerotidae Gnesiocerotidae Gnesiocerotidae Leptoplanidae Leptoplanidae Leptoplanidae Leptoplanidae Notoplanidae Notoplanidae Notoplanidae Notoplanidae Notocomplanidae Notoplanidae Notoplanidae Notocomplanidae fam. nov. Pleioplanidae Pleioplanidae Pleioplanidae Notoplanidae Stylochoplanidae Stylochoplanidae Stylochoplanidae Stylochoplanidae Acotylea Family Faubel (1983, 1984) Bahia et al. (2017) This study Superfamily Stylochoidea Poche, 1926 Callioplanidae Callioplanidae N/A Callioplanidae Hoploplanidae Leptoplanidae Hoploplanidae Hoploplanidae Latocestidae Latocestidae N/A Latocestidae Planoceridae Planoceridae Planoceridae Planoceridae Pseudostylochidae Pseudostylochidae Pseudostylochidae Pseudostylochidae Stylochidae Stylochidae Stylochidae Stylochidae Superfamily Cryptoceloidea (sensu Bahia et al., 2017); Ilyplanoidea (sensu Faubel, 1983, 1984) is not a valid superfamily Discocelidae Discocelidae Discocelidae Discocelidae Ilyplanidae Ilyplanidae N/A Ilyplanidae Cryptocelidae In Leptoplanoidea Cryptocelidae Cryptocelidae Superfamily Leptoplanoidea (sensu Faubel, 1983) Gnesiocerotidae Gnesiocerotidae Gnesiocerotidae Gnesiocerotidae Leptoplanidae Leptoplanidae Leptoplanidae Leptoplanidae Notoplanidae Notoplanidae Notoplanidae Notoplanidae Notocomplanidae Notoplanidae Notoplanidae Notocomplanidae fam. nov. Pleioplanidae Pleioplanidae Pleioplanidae Notoplanidae Stylochoplanidae Stylochoplanidae Stylochoplanidae Stylochoplanidae Cotylea – Euryleptoidea and Pseudocerotoidea (sensu Faubel, 1984); Periceloidea and Chromoplanoidea (sensu Bahia et al., 2017) are not valid superfamilies Family Faubel (1983, 1984) Bahia et al. (2017) This study Cestoplanidae In Stylochoidea In Cestoplanoidea Cestoplanidae Theamatidae In Leptoplanoidea In Chromoplanoidea Theamatidae Amyellidae In Pseudocerotoidea In Chromoplanoidea Amyellidae Boniniidae In Pseudocerotoidea In Chromoplanoidea Boniniidae Prosthiostomidae In Euryleptoidea In Prosthiostomoidea Prosthiostomidae Euryleptidae In Euryleptoidea In Pseudocerotoidea Euryleptidae Pseudocerotidae In Pseudocerotoidea In Pseudocerotoidea Pseudocerotidae Diposthidae In Pseudocerotoidea N/A Diposthidae, amended Pericelidae In Pseudocerotoidea In Periceloidea Diposthidae, amended Cotylea – Euryleptoidea and Pseudocerotoidea (sensu Faubel, 1984); Periceloidea and Chromoplanoidea (sensu Bahia et al., 2017) are not valid superfamilies Family Faubel (1983, 1984) Bahia et al. (2017) This study Cestoplanidae In Stylochoidea In Cestoplanoidea Cestoplanidae Theamatidae In Leptoplanoidea In Chromoplanoidea Theamatidae Amyellidae In Pseudocerotoidea In Chromoplanoidea Amyellidae Boniniidae In Pseudocerotoidea In Chromoplanoidea Boniniidae Prosthiostomidae In Euryleptoidea In Prosthiostomoidea Prosthiostomidae Euryleptidae In Euryleptoidea In Pseudocerotoidea Euryleptidae Pseudocerotidae In Pseudocerotoidea In Pseudocerotoidea Pseudocerotidae Diposthidae In Pseudocerotoidea N/A Diposthidae, amended Pericelidae In Pseudocerotoidea In Periceloidea Diposthidae, amended Monogeneric families, families with very few species or families that appear only in one classification system have been omitted. Comparisons with Aguado et al. (2017) and Tsunashima et al. (2017) are found in the Discussion. Abbreviation: N/A, not assessed. View Large Table 1. Superfamily- and family-level comparison of morphology- and DNA-based classification systems Acotylea Family Faubel (1983, 1984) Bahia et al. (2017) This study Superfamily Stylochoidea Poche, 1926 Callioplanidae Callioplanidae N/A Callioplanidae Hoploplanidae Leptoplanidae Hoploplanidae Hoploplanidae Latocestidae Latocestidae N/A Latocestidae Planoceridae Planoceridae Planoceridae Planoceridae Pseudostylochidae Pseudostylochidae Pseudostylochidae Pseudostylochidae Stylochidae Stylochidae Stylochidae Stylochidae Superfamily Cryptoceloidea (sensu Bahia et al., 2017); Ilyplanoidea (sensu Faubel, 1983, 1984) is not a valid superfamily Discocelidae Discocelidae Discocelidae Discocelidae Ilyplanidae Ilyplanidae N/A Ilyplanidae Cryptocelidae In Leptoplanoidea Cryptocelidae Cryptocelidae Superfamily Leptoplanoidea (sensu Faubel, 1983) Gnesiocerotidae Gnesiocerotidae Gnesiocerotidae Gnesiocerotidae Leptoplanidae Leptoplanidae Leptoplanidae Leptoplanidae Notoplanidae Notoplanidae Notoplanidae Notoplanidae Notocomplanidae Notoplanidae Notoplanidae Notocomplanidae fam. nov. Pleioplanidae Pleioplanidae Pleioplanidae Notoplanidae Stylochoplanidae Stylochoplanidae Stylochoplanidae Stylochoplanidae Acotylea Family Faubel (1983, 1984) Bahia et al. (2017) This study Superfamily Stylochoidea Poche, 1926 Callioplanidae Callioplanidae N/A Callioplanidae Hoploplanidae Leptoplanidae Hoploplanidae Hoploplanidae Latocestidae Latocestidae N/A Latocestidae Planoceridae Planoceridae Planoceridae Planoceridae Pseudostylochidae Pseudostylochidae Pseudostylochidae Pseudostylochidae Stylochidae Stylochidae Stylochidae Stylochidae Superfamily Cryptoceloidea (sensu Bahia et al., 2017); Ilyplanoidea (sensu Faubel, 1983, 1984) is not a valid superfamily Discocelidae Discocelidae Discocelidae Discocelidae Ilyplanidae Ilyplanidae N/A Ilyplanidae Cryptocelidae In Leptoplanoidea Cryptocelidae Cryptocelidae Superfamily Leptoplanoidea (sensu Faubel, 1983) Gnesiocerotidae Gnesiocerotidae Gnesiocerotidae Gnesiocerotidae Leptoplanidae Leptoplanidae Leptoplanidae Leptoplanidae Notoplanidae Notoplanidae Notoplanidae Notoplanidae Notocomplanidae Notoplanidae Notoplanidae Notocomplanidae fam. nov. Pleioplanidae Pleioplanidae Pleioplanidae Notoplanidae Stylochoplanidae Stylochoplanidae Stylochoplanidae Stylochoplanidae Cotylea – Euryleptoidea and Pseudocerotoidea (sensu Faubel, 1984); Periceloidea and Chromoplanoidea (sensu Bahia et al., 2017) are not valid superfamilies Family Faubel (1983, 1984) Bahia et al. (2017) This study Cestoplanidae In Stylochoidea In Cestoplanoidea Cestoplanidae Theamatidae In Leptoplanoidea In Chromoplanoidea Theamatidae Amyellidae In Pseudocerotoidea In Chromoplanoidea Amyellidae Boniniidae In Pseudocerotoidea In Chromoplanoidea Boniniidae Prosthiostomidae In Euryleptoidea In Prosthiostomoidea Prosthiostomidae Euryleptidae In Euryleptoidea In Pseudocerotoidea Euryleptidae Pseudocerotidae In Pseudocerotoidea In Pseudocerotoidea Pseudocerotidae Diposthidae In Pseudocerotoidea N/A Diposthidae, amended Pericelidae In Pseudocerotoidea In Periceloidea Diposthidae, amended Cotylea – Euryleptoidea and Pseudocerotoidea (sensu Faubel, 1984); Periceloidea and Chromoplanoidea (sensu Bahia et al., 2017) are not valid superfamilies Family Faubel (1983, 1984) Bahia et al. (2017) This study Cestoplanidae In Stylochoidea In Cestoplanoidea Cestoplanidae Theamatidae In Leptoplanoidea In Chromoplanoidea Theamatidae Amyellidae In Pseudocerotoidea In Chromoplanoidea Amyellidae Boniniidae In Pseudocerotoidea In Chromoplanoidea Boniniidae Prosthiostomidae In Euryleptoidea In Prosthiostomoidea Prosthiostomidae Euryleptidae In Euryleptoidea In Pseudocerotoidea Euryleptidae Pseudocerotidae In Pseudocerotoidea In Pseudocerotoidea Pseudocerotidae Diposthidae In Pseudocerotoidea N/A Diposthidae, amended Pericelidae In Pseudocerotoidea In Periceloidea Diposthidae, amended Monogeneric families, families with very few species or families that appear only in one classification system have been omitted. Comparisons with Aguado et al. (2017) and Tsunashima et al. (2017) are found in the Discussion. Abbreviation: N/A, not assessed. View Large Superfamily Stylochoidea Poche, 1926 Historically, the taxonomic composition of Stylochoidea has been controversial, because the placement of Hoploplanidae has been equivocal. Laidlaw (1903), Bock (1913), Marcus & Marcus (1966) and Faubel (1983) assigned the family to Leptoplanoidea, whereas Prudhoe (1985) included it in Planoceroidea and Poche (1926) placed it in Stylochoidea. The currently accepted classification follows Poche’s (1926) placement (Tyler et al., 2006–2018). Our results, and those of Aguado et al. (2017), Bahia et al. (2017) and Tsunashima et al. (2017), support the inclusion of Hoploplana species in Stylochoidea (Table 1). In an attempt to define Stylochoidea (sensu Poche, 1926), Bahia et al. (2017) proposed ‘a rounded body shape, presence of nuchal tentacles, presence of cerebral and nuchal (and sometimes marginal) eyespots’ as synapomorphies. However, these synapomorphies do not define the superfamily as a whole. Specifically, some families (e.g. Latocestidae) do not possess nuchal tentacles, and the trait may be variable even in individual genera (e.g. Leptostylochus). Furthermore, the elongate shape of Latocestus defies the proposed rounded body shape. Among Acotylea, larvae are found only in Stylochoidea [known exceptions are Notoplana australis (Schmarda, 1859) and Stylochoplana maculata Quatrefage, 1845 (Leptoplanoidea)] (Rawlinson et al., 2008; Lapraz et al., 2013). Hence, it might be tempting to use their presence to unite species in this superfamily inclusive of Hoploplana inquilina (Wheeler, 1894). However, because we identified Stylochoidea (sensu Poche, 1926) as the most basal lineage in Acotylea, it is likely that larvae are a symplesiomorphy retained from the polyclad ancestor, rather than a synapomorphy of the superfamily. Consequently, we cannot identify a valid morphological or developmental synapomorphy for the entire superfamily, despite the molecular support of the clade. Superfamily Cryptoceloidea Bahia et al., 2017 Faubel (1983, 1984) erected the superfamily Ilyplanoidea to contain acotylean species that lack a true prostatic vesicle (Fig. 1C; Table 1). Instead, their male tract is covered with a glandular lining or with prostatic-like glands called prostatoids (Fig. 1D). Our results confirm the finding of Bahia et al. (2017) that Ilyplanoidea is not a valid taxon. We recovered monophyly of three species [Adenoplana evelinae Marcus, 1950, Discocelis tigrina (Blanchard, 1847) and Ilyella gigas (Schmarda, 1859)] representing two families (Discocelidae and Ilyplanidae) in Ilyplanoidea, but our results show that a monophyletic group containing four species of Phaenocelis plus Phaenoplana peleca (Marcus & Marcus, 1968) form the immediate sister clade to these three ilyplanoid species. Using specimens of Adenoplana evelinae and Phaenocelis medvedica Marcus, 1952, Bahia et al. (2017) also showed a paraphyletic Ilyplanoidea (Table 1). Phaenocelis belongs to Cryptocelidae, prompting the authors to create the superfamily Cryptoceloidea, which they characterize by oval to elongate body shapes, a lack of tentacles and the presence of cerebral, nuchal and marginal eyespots. Given that body shapes and eyes can be unreliable taxonomic traits, we add the presence of unarmed, rod- or cylindrical-shaped penis papillae (e.g. found in Cryptocelis, Phaenocelis) as a diagnostic character. Thus, our revised definition of Cryptoceloidea relies on the ‘absence of a true prostatic vesicle or, if an interpolated prostatic vesicle is present, an unarmed conical penis papilla’. The phylogeny of Tsunashima et al. (2017), which included Ilyella gigas, also does not resolve a monophyletic Ilyplanoidea. Instead, Ilyella gigas clustered with Amemiyaia pacifica. Amemiyaia, a monospecific genus, was initially placed in Phaenocelidae by Kato (1944) and subsequently moved to Cryptocelidae by Marcus & Marcus (1966). Currently, it belongs to Stylochoplanidae, a leptoplanoid family that was established by Faubel (1983) and that also includes Phaenoplana, among others. Based on its clustering with ilyplanoids (Tsunashima et al. 2017), and when adding it to our phylogeny where it groups with Phaenocelis spp., its position in the family Stylochoplanidae (superfamily Leptoplanoidea) can no longer be supported. It clearly belongs to the superfamily Cryptoceloidea (sensu Bahia et al., 2017). Hence, we here return it to Cryptocelidae. Likewise, the original description of Phaenoplana peleca by Marcus & Marcus (1968) placed the species in Phaenocelis. However, Faubel (1983) moved it to Phaenoplana, a genus he erected in Stylochoplanidae. Based on strong molecular support, we here return Phaenoplana peleca to Phaenocelis, thus Phaenoplana peleca becomes a junior synonym of Phaenocelis peleca. The superfamily then contains, at minimum, Ilyplanidae, Discocelidae and Cryptocelidae. The inclusion of Euplanidae awaits testing. Currently, several species of Phaenocelis are found in the greater Caribbean and the tropical western Atlantic. The type specimen of Phaenocelis purpurea (Schmarda, 1859) is from Jamaica (Schmarda, 1859), and the species is also known from the Florida Keys (Hyman, 1954) and from Curaçao (Marcus & Marcus, 1968). During our own surveys, we found Phaenocelis purpurea in Panama and Belize. Phaenocelis medvedica was originally described from the Island of São Sebastião in Brazil (Marcus, 1952) and more recently it also has been found at Ponta Verde, Brazil (Bahia et al., 2015). The type locality of Phaenocelis peleca is the same place in Curaçao that also contains Phaenocelis purpurea (Marcus & Marcus, 1968) and our surveys extends its range to Belize and Jamaica. Additionally, we found two other species of Phaenocelis. With regard to their external morphology, species in Phaenocelis are generally of elongate, slender form, with few distinguishing traits, and their colour may range from pale cream to pink. Consequently, species are difficult to identify, and conclusive identifications require sections of the internal reproductive system or DNA sequences. The D1–D2 expansion segment allows for species distinctions in the genus, because little intraspecific sequence variation exists, but distinct gaps can be recognized among species. Given that only Phaenocelis medvedica has been described from Brazil, it may be likely that the remaining four Phaenocelis species do not occur along that coast, thus facilitating the identification of Phaenocelis medvedica. With regard to the internal morphology, all five species (Phaenocelis medvedica, Phaenocelis purpurea and Phaenocelis peleca, plus the two undescribed species) have distinctly shaped Lang’s vesicles and species-specific insertion points of the duct into Lang’s vesicles (S. Quiroga and M. Bolaños, unpublished data). Superfamily Leptoplanoidea Faubel, 1984 Leptoplanoidea is a heterogenous assemblage of 12 families. Several of these families (Cestoplanidae, Cryptocelidae and Theamatidae) and individual genera (e.g. Phaenoplana and Amemiyaia) have already been transferred to other groups (Bahia et al., 2017; this study). Using two species representing Gnesiocerotidae, we recovered the family as the first branching lineage in the superfamily. This is in contrast to Bahia et al. (2017), who found the gnesiocerotid Echinoplana celerrima Haswell, 1907 embedded in a clade with Notoplana australis and an unidentified species of Notocomplana. We support the validity of Gnesiocerotidae by including two separate genera (Styloplanocera and Gnesioceros). The family forms a basal lineage with Leptoplanoidea and is morphologically united by a highly cuticularized cirrus that connects directly to the prostatic vesicle and is surrounded by a strongly developed, muscular bulb (Fig. 2C). Notoplanidae is one of the most species-rich families in the superfamily and has been the focus of several classifications and revisions (Bock, 1913; Marcus & Marcus, 1968; Faubel, 1983; Prudhoe, 1985). Given that Notoplana represents the largest genus among acotyleans (60+ species), Bock (1913) subdivided Notoplana into group A (Notoplana evansi type), group B (Notoplana atomata type) and group C (Notoplana alcinoi type). In both group A and B, the penis is armed with a stylet, whereas some members of group C do not possess a stylet. Separation between groups A and B is based on the size of the male antrum and Lang’s vesicle: group A species are characterized by a large male antrum and a small, rudimentary Lang’s vesicle, and those in group B have a small male antrum and a large Lang’s vesicle (Bock, 1913). This grouping was largely followed by Marcus & Marcus (1968), with the creation of additional subdivisions, some of which most probably represent unnatural groups by the authors’ own admission (Marcus & Marcus, 1968). In an extensive attempt at reorganizing Notoplanidae, Faubel (1983) recognized ten genera, four of which were new. Half of these genera are monospecific and three others contain two species only (Faubel, 1983). The two remaining species-rich genera are morphologically separated by the lack or presence of a penis stylet. In Notocomplana, the penis is represented by a blunt or conical papilla, whereas in Notoplana the penis is armed with a short stylet (Faubel, 1983). Finally, Prudhoe’s (1985) attempt recognized four groups in Notoplana, based on the presence or absence of a penis stylet and the presence or absence of a penis-pocket sheath. Prudhoe’s (1985) groups C and D (i.e. penis papilla without stylet or cuticular covering) corresponds to Faubel’s (1983) Notocomplana and to Bock’s (1913) group C in Notoplana. Faubel (1983) also erected the new family Pleioplanidae, using the number of tubular chambers formed by the lining of the prostatic vesicle as a distinguishing character (‘few’ in Notoplanidae vs. ‘greater’ in Pleioplanidae; Faubel, 1983: 115). Such distinctions are subjective and are most likely to be heavily influenced by the angle of the plane of sectioning and the quality of fixation. He included the new genera Melloplana and Pleioplana in the family, both containing species formerly assigned to Notoplana. According to Faubel (1983), Melloplana possess an unarmed papillate penis, whereas species with a penis armed with a stylet are placed in Pleioplana. Hence, the separation of the two new genera is based on the same trait used in his earlier separation of Notocomplana (species without an armed penis) and Notoplana (species with an armed penis) with the addition that Pleioplanidae have a ‘greater number of tubular chambers’ in their prostatic vesicle. Since Faubel’s (1983) revision, three additional genera have been placed into Pleioplanidae: Izmira (Bulnes, 2010), a monospecific Persica (Maghsoudlou et al., 2015) and Laqueusplana (Rodríguez et al., 2017). Of these, Izmira has a penis papilla, whereas the other two genera have a penis armed with a stylet. Our results revealed a paraphyletic Pleioplanidae, in which Melloplana ferruginea (Schmarda, 1859) groups with Notocomplana lapunda (Marcus & Marcus, 1968), which is characterized by an unarmed penis. Based on its clustering with other Notocomplana species in a phylogeny built on COI sequences, the only other species in Melloplana, Melloplana japonica (Kato, 1937), has been transferred to Notocomplana by Oya & Kajihara (2017). In addition to an unarmed penis papilla, our molecular results support the transfer of Melloplana ferruginea to Notocomplana to form Notocomplana ferruginea (Schmarda, 1859) comb. nov. Thus, all currently recognized species of Melloplana are now included in Notocomplana, in which the genus Melloplana is synonymized. To separate notoplanids with an unarmed penis papilla from those armed with a stylet, we erect the new family Notocomplanidae fam. nov. and formally designate Notocomplana as its type genus. Following the precedent of Faubel (1983) when he erected the genus, Notocomplana humilis (Stimpson, 1857) remains the type species. Notocomplanidae generally corresponds to Notoplanidae in Bock’s (1913) group C, Prudhoe’s (1985) groups C and D and Faubel’s (1983) Notocomplana. Morphologically, the family is defined as having the characters of Notoplanidae but possess an unarmed penis papilla. Thus, we place more importance on the armature of the penis than on more subjective traits associated with the lining of the prostatic vesicle. Combining our molecular results with the character ‘armed penis’ also led us to return Pleioplana atomata (O.F. Müller, 1776) to its original genus, Notoplana. Clearly, in Notoplanidae, ‘presence or absence of a penis stylet’ must be considered of greater phylogenetic value than more plastic traits (e.g. degree of folding of prostatic vesicle lining, distance to which the ejaculatory duct enters the prostatic vesicle). Likewise, another pleioplanid, Persica qeshmensis Maghsoudlou et al., 2015, grouped with Notoplana, all possessing a stylet. Consequently, we move Persica qeshmensis to Notoplana to form Notoplana qeshmensis comb. nov. We concur with Oya & Kajihara (2017) that the support for Pleioplanidae (even after removing Melloplana) is questionable and that it most probably is synonymous with Notoplanidae. However, this hypothesis awaits testing by including sequences of Izmira and Laqueusplana in a future molecular phylogeny. Similar to Notoplanidae, Stylochoplanidae is also a species-rich leptoplanoid family whose intrafamilial relationships are not resolved, despite several attempts at revisions (Bock, 1913; Marcus & Marcus, 1968; Faubel, 1983; Prudhoe, 1985). Bock (1913), Marcus & Marcus (1968) and Prudhoe (1985) subdivided Stylochoplana into different groups, whereas Faubel (1983) removed species possessing a long, pointed stylet, a voluminous prostatic vesicle and a Lang’s vesicle into the new genus Armatoplana to distinguish them from other species that either lack a stylet, have a short stylet only or in which the stylet is only a thickened basement membrane. We included specimens of Armatoplana divae (Marcus, 1947) and Armatoplana leptalea (Verrill, 1900) in our phylogeny. Armatoplana leptalea was originally described from Brazil (Marcus, 1947) and its range has been extended to include Curaçao, Antigua, Barbuda and the Florida Keys (Marcus & Marcus, 1968). We can add Panama, Colombia and Belize as additional localities (see Supporting Information, Appendix). In their description of Armatoplana leptalea from Curaçao, Marcus & Marcus (1968: 25) mention that the specimens differ from the original ones collected in Brazil. These differences might explain why our Caribbean specimens cluster together, whereas the specimens from Brazil (Bahia et al., 2017) form a distinctly separate group, albeit still within the species. However, the original species description of Armatoplana lactoalba (Verrill, 1900) included only a short account of the external morphology and lacked any details regarding the reproductive systems (Verrill, 1902). Therefore, we cannot exclude the possibility that our specimens are Armatoplana lactoalba rather than Armatoplana leptalea, and species equivalence of Caribbean and Brazilian samples will require additional sampling, including careful reconstruction of the reproductive systems and molecular data. Armatoplana divae has a distinct dorsal coloration, allowing for easy species identification based on external morphology alone. However, rather than clustering with Armatoplana leptalea, Armatoplana divae appears closer to Notoplana queruca. This is reflective of the unresolved classification of Stylochoplanidae and requires further study. Finally, Amyris is characterized by a muscular penis papilla, called a cirrus. Marcus & Marcus (1968) considered the cirrus unarmed, because the cuticular lining of the ejaculatory duct appears smooth. However, we observed that the lining consists of numerous small, spine-like structures that overlap to give the appearance of a smooth lining. Upon eversion, the spines unfold, resulting in an armed penis papilla. Hence, our results showing the clustering of Amyris with other notoplanids possessing an armed penis are supported by morphology. Marcus & Marcus (1968) recognized two species of Amyris, differentiated only by the size of the seminal vesicle, the degree of muscle development of the male organs, the depth of the male antrum and a wider and more folded vagina in Amyris ujara. All these traits are plastic and vary depending on fixation and age of the specimens. Based on identical sequences, our results indicate that Amyris ujara is a junior synonym of Amyris hummelincki. Relationships in Cotylea Six major clades were identified in Cotylea. The base of the tree is formed by taxa exhibiting acotylean- and cotylean-like traits; specifically, Cestoplanidae, Pericelidae + Diposthus popeae and a clade containing Boniniidae, Theamatidae and Amyellidae. Prosthiostomidae, Euryleptidae and Pseudocerotidae established the remaining three groups. Given that the male reproductive systems of cotyleans are rather uniform, with few distinguishing characters (most have a free prostatic vesicle), characters associated with the digestive (pharynx) and sensory systems (pseudotentacles, eyes) and colour patterns are more commonly used for taxonomic identifications (Newman & Cannon, 2003 and references therein). Faubel (1984) recognized three superfamilies in Cotylea. Opisthogenioidea is monospecific and was not included in our analysis. To classify the remaining two superfamilies, he placed a high taxonomic value on the shape of the pharynx. All families with a ruffled pharynx are placed into Pseudocerotoidea, whereas families with a cylindrical pharynx are placed into Euryleptoidea. The morphology-based cladistic analysis of Rawlinson & Litvaitis (2008) had already invalidated Faubel’s (1984) superfamilies, and our molecular analysis now reconfirmed that Boniniidae, Amyellidae, Diposthidae and Pericelidae are not members of Pseudocerotoidae (sensu Faubel, 1984) and that Prosthiostomidae should not be included in Euryleptoidea. Thus, the two superfamilies are not valid, and we urge the discontinuation of their use. Cestoplanidae Lang, 1884 Traditionally, the family has been included in Acotylea, despite several cotylean-like characters (e.g. male complex located posterior to the male pore and directed forward, male atrium wall folded to form a penis sheath, absence of Lang’s vesicle, posterior adhesive cells and cement/shell glands). Applying the cotylean diagnostic traits outlined above to Cestoplana rubrocincta (Grube, 1840) allows for reassignation of the family to Cotylea. This placement is strongly supported by our molecular analysis, which clearly reveals its cotylean status. This reclassification is further backed by earlier calls for a more careful examination of the posterior adhesive cells described for Cestoplana rubrocincta (Lang, 1884), by the description of cotylean-type male and female reproductive systems (Laidlaw, 1903; Faubel, 1983) and by the findings of Bahia et al. (2017). Furthermore, because our 28S rDNA sequences of Cestoplana rubrocincta and Cestoplana australis Haswell, 1907 were largely identical, we confirm that Cestoplana australis is indeed a junior synonym of Cestoplana rubrocincta. Cestoplanoidea was erected by Prudhoe (1985) to include the monospecific Diplopharyngeatidae (Diplopharyngeata filiformis) and the monogeneric Emprosthopharyngidae (six species). However, as previously mentioned, Diplopharyngeata filiformis belongs to Euplanidae and Emprosthopharynx has been removed to Stylochoplanidae (Faubel, 1983). Cestoplanidae then is the sole remaining family in Cestoplanoidea and, unlike Bahia et al. (2017), we argue that until a close sister family is described, elevating a single family to the level of a superfamily is not warranted. Boniniidae Bock, 1923, Theamatidae Marcus, 1949 and Amyellidae Faubel, 1984 Laumer & Giribet (2014) included eight polyclad species in their global phylogeny of rhabditophoran platyhelminths. Although their resulting polyclad subtree revealed two distinct clusters in Polycladida, the selected species do not separate into Acotylea and Cotylea (sensu Lang, 1884; sensu Faubel, 1983, 1984). Specifically, their cotylean clade included Theama sp. that was traditionally placed into Acotylea (Marcus, 1949). This led the authors to question the usefulness of the subordinal division of polyclads. Faubel (1983) had already doubted the acotylean placement of Theamatidae owing to the cotylean-like features of the taxon (e.g. lack of Lang’s vesicle and presence of cement gland pouches), and recently, Bahia et al. (2017) transferred Theama sp. to Cotylea to be included in their new superfamily Chromoplanoidea. According to these authors, Chromoplanoidea contained the families Boniniidae, Theamatidae, Chromoplanidae and Amyellidae represented by Boninia divae Marcus & Marcus, 1968, Theama sp., Chromoplana sp. and Chromyella sp., respectively. However, in their analysis, the authors specifically excluded Chromoplana sp. from their phylogeny on the basis of ‘discrepant sequences’ (Bahia et al., 2017: 657). As a result, Chromoplanoidea (sensu Bahia et al., 2017) no longer included its type family Chromoplanidae, invalidating the superfamily. Adding a new Theama sp. sequence plus several new Boninia sequences (Boninia antillarum (Hyman 1955) and Boninia neotethydis Curini-Galletti & Campus, 2007), we also recover a monophyletic clade of Boniniidae + Theamatidae + Chromyella sp. Currently, these species appear to be defined by a small, elongate body size and a combination of acotylean and cotylean traits associated with the reproductive structures. However, the slender body size is most probably attributable to ecological convergence rather than being of taxonomic importance, and other polyclads (e.g. cestoplanids) also possess acotylean and cotylean reproductive features. Even though Boniniidae + Theamatidae + Amyellidae cluster together, in the absence of morphological synapomorphies we contend that it is premature to unite these species into a superfamilial taxon. Since Bock’s (1923) description of Boninia mirabilis Bock, 1923, three additional species have been described in the genus, two of which have type localities in the Caribbean. However, even Bock (1923) already noted unique, and for cotyleans atypical, traits of Boninia. Unlike cotyleans, they possess Lang’s vesicle, which led Hyman (1955a) to place B. antillarum (Adenoplana antillarum Hyman, 1955) in Acotylea. Additionally, their sperm morphology resembles that of acotylean sperm (Liana & Litvaitis, 2007, 2010) and they develop via an aberrant Müller’s larva reminiscent of an acotylean Götte’s larva (Bolaños, 2008). Despite these acotylean-like traits, our molecular analysis confirms the placement of Boniniidae into Cotylea. Morphologically, placement of Boniniidae into Cotylea is supported by characters associated with the nervous system (Quiroga et al., 2015). In Cotylea, globuli cell masses, which are aggregates of sensory ganglions, tend to be little defined or absent. The globuli cell masses of B. antillarum are poorly differentiated, similar to those found in some species of euryleptids, prosthiostomids and pseudocerotids (Quiroga et al., 2015). Furthermore, the unique organization of the central nervous system, submerged in the longitudinal musculature, is an apomorphy for Boninia (Quiroga et al., 2015). Further analyses of nervous system anatomy in Theamatidae and Amyellidae might lend additional support to the monophyly of the clade. Currently, two species of Boninia are recognized in the Caribbean. Boninia antillarum was described by Hyman (1955a), with the type locality in the US Virgin Islands. Marcus & Marcus (1968) extended the distribution to Curaçao and Bonaire and added B. divae (type locality in Curaçao), which is syntopic with B. antillarum. The authors justify a second species based on the arrangement of the cerebral eyes and the number of prostatoid organs. Specifically, B. antillarum is distinguished from B. divae by three anterior groups of cerebral eyes plus a fourth group further posterior, whereas the arrangement in B. divae is as two elongated longitudinal bands of cerebral eyes (Marcus & Marcus, 1968: figs 61 and 67, respectively). Additionally, B. antillarum has < 30 prostatoid organs, whereas B. divae has > 50 (Fig. 7A). However, both traits co-vary with the age of the specimens, which can be gleaned by reported lengths of B. antillarum as 16 mm and of B. divae as 30–50 mm (Marcus & Marcus, 1968). Figure 7. View largeDownload slide Male reproductive organs in Boninia (A) and Diposthus (B), indicating unique arrangement of prostatoids or prostatoid-like organ. A, schematic diagram and histological section of Boninia, showing prostatic vesicle (shading) and multiple prostatoids (shading plus arrows) armed with stylets, emptying into the male atrium. Scale bar: 500 µm. B, schematic diagram and histological section of accessory prostatoid organ (shading, ap) in Diposthus, opening into the male atrium independent of the penis papilla (pp). Cement pouches (cp) of female system and the cotyl (co) are also visible in the histological section. Scale bar: 250 µm. Figure 7. View largeDownload slide Male reproductive organs in Boninia (A) and Diposthus (B), indicating unique arrangement of prostatoids or prostatoid-like organ. A, schematic diagram and histological section of Boninia, showing prostatic vesicle (shading) and multiple prostatoids (shading plus arrows) armed with stylets, emptying into the male atrium. Scale bar: 500 µm. B, schematic diagram and histological section of accessory prostatoid organ (shading, ap) in Diposthus, opening into the male atrium independent of the penis papilla (pp). Cement pouches (cp) of female system and the cotyl (co) are also visible in the histological section. Scale bar: 250 µm. We collected > 50 specimens of Boninia (including specimens from the type localities). Attempting to follow Marcus & Marcus (1968), we divided our specimens into B. divae and B. antillarum based on cerebral eye arrangement. However, nucleotide sequences of all our specimens were largely identical, regardless of initial species assignment or collection locality. Hence, we conclude that B. divae is not a separate species but instead a variant of B. antillarum and that the diagnostic characters proposed by Marcus & Marcus (1968) are not sufficient for species separation in the genus. Boninia divae then becomes a junior synonym of B. antillarum. We established monophyly of the genus Boninia with the inclusion of B. neotethydis in our phylogeny. Diposthidae (Woodworth, 1898), amended A third, well-supported cotylean clade is formed by representatives of all currently known Pericelis species plus Diposthus popeae. Earlier studies reported that the genus Diposthus lacks a cotyl (Hyman, 1959b; Woodworth, 1898). Our examination of the type material is equivocal owing to the poor quality of fixation and sectioning. However, we collected 14 specimens of Diposthus from locations in Panama, Curaçao, Honduras and the US Virgin Islands, and all of them had a cotyl (Fig. 7B). Furthermore, Faubel (1984: 201) claimed that Diposthidae lack cement pouches (what he calls mucous gland chambers). Histological sections of our newly collected material unambiguously revealed the presence of cement pouches (Fig. 7B). The genus (and most probably the family) clearly belongs to Cotylea. Diposthus is unique in possessing an accessory prostatoid organ that empties independently of the penis papilla into the male atrium (Fig. 7B). Pericelidae is a monogeneric family with four species. Their cotylean classification is warranted because of a cotyl, cement pouches and uterine vesicles. However, like Cestoplanidae and Boniniidae, pericelids also exhibit acotylean affinities, such as a centrally located ruffled pharynx, anteriorly directed uteri and marginal eyes encircling the entire body. Furthermore, the brain of Pericelis cata Marcus & Marcus, 1968 connects to four conspicuous globuli cell masses (an acotylean trait), forming mushroom bodies (Quiroga et al., 2015). Given that our results reveal a well-supported relationship between Pericelis spp. and Diposthus popeae, we here formally subsume all pericelids in Diposthidae and abolish the family Pericelidae. Because Pericelidae is currently monogeneric, we argue that amending Diposthidae to include Pericelis is more appropriate than to create a new superfamily. Thus, Diposthidae now contains a monospecific Asthenoceros, two species of Diposthus and four species of Pericelis. The type genus continues to be Diposthus due to chronological precedence and the genera remain distinct by the absence of an accessory prostatoid organ and the presence of a true seminal vesicle in Pericelis. We here formally amend Diposthidae to include cotyleans of an oval to slightly elongate body shape, with short, well-separated marginal pseudotentacles and uterine vesicles. Additionally, the prostatic vesicle of Diposthus and Pericelis is atypical, because it consists of prostatic tissue lining the ejaculatory duct within a thick, well-developed penis papilla (Fig. 8). Possible similarities with respect to the nervous system (e.g. globuli cell masses, mushroom bodies) await an examination in Diposthus. Figure 8. View largeDownload slide Histological sections of prostatic elements of the penis papillae (pp) of Pericelis (A) and Diposthus (B). Abbreviations: ap, accessory prostatic organ; cg, cement glands; pt, prostatic tissue lining ejaculatory duct in penis papilla (pp); sb, spermiducal bulb; sv, seminal vesicle. Scale bars: 250 µm. Figure 8. View largeDownload slide Histological sections of prostatic elements of the penis papillae (pp) of Pericelis (A) and Diposthus (B). Abbreviations: ap, accessory prostatic organ; cg, cement glands; pt, prostatic tissue lining ejaculatory duct in penis papilla (pp); sb, spermiducal bulb; sv, seminal vesicle. Scale bars: 250 µm. Recently, Bahia et al. (2017) created Periceloidea based solely on two species of Pericelis. With our addition of Diposthus to the phylogeny, we showed that Periceloidea (sensu Bahia et al., 2017) is not supported, and therefore, we abolish this superfamily. Prosthiostomidae Lang, 1884 Prosthiostomidae are characterized by an absence of tentacles, a tubular, anteriorly located pharynx and a male complex characterized by a pair of spherical, free prostatic vesicles. Generic differentiation is based on the presence or absence of a main frontal intestinal branch, varying amounts (complete, partial or none) of a muscular enclosure of the prostatic vesicles, the arrangement of cerebral and marginal eyes and body shape (oval, elongate) (Kato, 1938; Marcus & Marcus, 1968; Faubel, 1984). However, eye arrangement and body shape are highly plastic and thus, their taxonomic use is questionable at best (Jokiel & Townsley, 1974; Poulter, 1975). Using representatives of five currently recognized genera [i.e. Enchiridium, Prosthiostomum, Euprosthiostomum, Lurymare and Amakusaplana (a sixth genus, Enterogonimus, is monospecific)], we recovered a strongly supported monophyletic Prosthiostomidae, in which Enchiridium and Prosthiostomum are well-supported taxa. According to Faubel (1984), a muscular sheath completely enclosing the prostatic vesicles and the seminal vesicle of Enchiridium distinguishes the genus morphologically from Prosthiostomum, in which neither the prostatic nor the seminal vesicles are enclosed by a muscular bulb. Including Prosthiostomum siphunculus (Della Chiaje, 1822), Amakusaplana acroporae Rawlinson et al., 2011, Enchiridium evelinae Marcus, 1949 plus two unidentified species of Enchiridium, Bahia et al. (2017) also found a monophyletic Prosthiostomidae. Despite the fact that currently all prosthiostomids are placed in a single family (Faubel, 1984; Prudhoe, 1985), Bahia et al. (2017) elevated the family to superfamily status (Prosthiostomoidea). We contend that such a taxonomic addition is not warranted until at least another prosthiostomid family is recognized. The genus Amakusaplana was erected for a single species (Amakusaplana oshimai Kato, 1938), separating it from Prosthiostomum based on the lack of a cotyl, a more oval rather than elongated body shape and cerebral eyes that are scattered fan-like over the brain region rather than arranged into two distinct clusters (Kato, 1938). However, the latter two characteristics are highly variable and co-vary with the age of the specimen (Jokiel & Townsley, 1974; Poulter, 1975). Since then, a second species, Amakusaplana acroporae, has been described, its species epithet reflecting the fact that its main prey items are several species of the stony coral Acropora (Rawlinson et al., 2011). The authors based their specific separation mostly on the number and arrangement of cerebral and marginal eyes and on other plastic traits associated with the reproductive system (e.g. size of the seminal vesicle, the female atrium and the cement gland pouch). Recognizing the variability of these traits, Rawlinson & Stella (2012) accepted the possibility of synonymy between Amakusaplana oshimai and Amakusaplana acroporae. Regardless of it being a separate species, our analysis places Amakusaplana acroporae firmly into Prosthiostomum with strong support. Hence, rather than recognizing a separate genus based on highly variable traits, we here support Hyman (1959a) and Faubel (1984), who proposed that the genus should be eliminated and synonymized with Prosthiostomum. By synonymizing the two genera, Amakusaplana acroporae becomes Prosthiostomum acroporae comb. nov. The absence of a cotyl then represents a secondary loss, probably attributable to a highly specific lifestyle on madreporarian corals. In addition to established generic prosthiostomid traits (e.g. elongate body shape, absence of marginal tentacles, cylindrical pharynx and a pair of muscular accessory prostatic vesicles), synonymy of Amakusaplana with Prosthiostomum is further supported by: (1) the presence of a ventral eye in each cerebral eye cluster of both genera; (2) an anteriorly extending median intestinal branch; and (3) a cleft pharynx, which has also been described in another corallivorous species, Prosthiostomum montiporae Poulter, 1975. According to Poulter (1974, 1987), Prosthiostomum montiporae is also an obligate symbiont of stony coral (Montipora spp.), and thus, a cleft pharynx might be an adaptation of the feeding mode in this genus. The taxonomic affinity of Prosthiostomum utarum Marcus, 1952 has been equivocal (Marcus, 1952; Marcus & Marcus, 1968; Faubel, 1984; Bahia et al., 2014). Originally described as Prosthiostomum utarum by Marcus (1952), it was moved to the newly erected genus Lurymare by Marcus & Marcus (1968). The amount of muscles surrounding the prostatic vesicles has been used to separate Lurymare from Prosthiostomum. However, the trait is highly variable and depends on the age of the specimen. Molecular data now support the original placement of this species in Prosthiostomum. The placement of the remaining species currently in Lurymare awaits further testing. Euryleptidae Lang, 1884 In Euryleptidae, our samples covered the genera Eurylepta, Cycloporus, Prostheceraeus and Maritigrella. Unlike Bahia et al. (2017), we recovered Cycloporus as a monophyletic genus. Morphologically, Cycloporus is distinguished from other euryleptids by the shape of the tentacles (small protuberances on the anterior margin) and by numerous small, peripheral vesicles surrounding the body margin, from which it derives its name. These vesicles are lateral branches of the digestive system that open to the exterior via pores (Newman & Cannon, 2002). This morphological distinctness might be the reason for the low molecular support of Euryleptidae as a family, and the relationship of Cycloporus with euryleptids warrants further investigation. Three species of Maritigrella formed a discrete clade. However, the two Caribbean species of Maritigrella cluster with Prostheceraeus. This apparent paraphyly led us to re-classify Maritigrella crozieri (Hyman, 1939) and Maritigrella newmanae Bolaños et al., 2007. Based on a distinct colour pattern of transverse stripes and the absence of uterine vesicles, Newman & Cannon (2000) erect the genus Maritigrella for a group of euryleptid polyclads from the Indo-Pacific. Relying on similar striped colour patterns and the absence of uterine vesicles, Newman et al. (2000) then synonymized and reclassified Pseudoceros crozieri Hyman, 1939, Prostheceraeus zebra Hyman, 1955 and Cryptoceros crozieri Faubel, 1984 into Maritigrella crozieri (Hyman, 1939). Although the reclassification into Euryleptidae is warranted (Pseudoceros belongs to Pseudocerotidae; Cryptoceros has been abolished; Litvaitis et al., 2010), Hyman’s (1955b) description of Prostheceraeus zebra clearly states that the species is lacking uterine vesicles. Hence, the fact that uterine vesicles may be absent in some species of Prostheceraeus indicates that they are of little phylogenetic value in this group. Consequently, we here move Maritigrella crozieri to Prostheceraeus, to form Prostheceraeus crozieri comb. nov. Additionally, after a re-examination of our specimens, a comparison with Hyman’s (1955a) original description and holotype material and the results of our molecular analysis, we recognize Maritigrella newmanae as a junior synonym of Prostheceraeus floridanus. Traditionally, morphological traits for the identification of euryleptids have focused on tentacle shape (pointed, slender vs. small anterior bumps), the presence or absence of marginal pores, the number of uterine vesicles (none, two or multiple) and diverse colour patterns. Characters of the male reproductive system are of little taxonomic value, because of their uniformity and resemblance to pseudocerotid systems. Although some euryleptids may be identified by unique colour patterns (e.g. some species of Prostheceraeus and Maritigrella), the inadequacy of species-specific characters compels a reliance on molecular data for positive species identification, especially in Eurylepta. Pseudocerotidae Lang, 1884 A monophyletic Pseudocerotidae containing three discrete clades (Pseudobiceros + Thysanozoon; Phrikoceros + Tytthosoceros; Pseudoceros) is recovered, and in each clade we are able to resolve existing taxonomic uncertainties and junior synonyms (see the three subsections on Pseudoceros splendidus, Phrikoceros and Tytthosoceros and Pseudoceros below). Species characterized by two male reproductive systems (Pseudobiceros and Thysanozoon) cluster together, with each genus forming a monophyletic group. This is in contrast to the findings of Bahia et al. (2017), who recovered paraphyletic Pseudobiceros and Thysanozoon. Based on a grouping of their specimen of Pseudobiceros hancockanus (Newman & Cannon, 1994) in Pseudoceros, Tsunashima et al. (2017) also questioned the monophyly of Pseudoceros and Pseudobiceros. However, this might be attributable to a misidentification (Tsunashima et al. 2017: fig. 2H on p. 163 is not Pseudobiceros hancockanus). Our specimen of Pseudobiceros hancockanus clusters with Pseudobiceros. Our analysis also includes two specimens of an undescribed species of Pseudobiceros. The colour pattern of this species (black dorsal surface, with inner wide, bright orange and outer narrower, white marginal bands encircling the entire body) led Newman & Cannon (1994: fig. 51C; 2003: 33, 39, 52, 82) to identify it as Pseudobiceros hancockanus. Accordingly, this description is widely circulated in online databases. However, the original description of Pseudobiceros hancockanus by Collingwood (1876) differs considerably. Recently, Bolaños et al. (2016) have addressed the confusion surrounding Pseudobiceros hancockanus and have designated a neotype for the species. Our results support the duplication of the male reproductive system as a valid morphological trait (Rawlinson & Litvaitis, 2008), separating the genera Pseudobiceros and Thysanozoon from the remaining pseudocerotids. However, species resolution in Thysanozoon was not possible using the D1–D2 expansion segments. In fact, morphologically distinct species [e.g. Thysanozoon brocchii (Risso, 1818) and Thysanozoon raphaeli Bolaños et al., 2007] reveal almost complete sequence concordance. Consequently, specific separations in this genus await analyses that use more rapidly evolving genetic markers (possibly COI, internal transcribed spacers). Pseudobiceros splendidus (Lang, 1884) (Fig. 9; Table 2): Pseudobiceros splendidus, initially described as Pseudoceros superbus by Lang (1884), was renamed Pseudobiceros splendidus by Stummer-Traunfels (1933) to avoid confusion with another species with the same name. Eventually, it was moved to Pseudobiceros by Faubel (1984), who recognized its double male copulatory system. Pseudobiceros splendidus is part of a group of pseudocerotids exhibiting colour patterns with a uniform dorsal colour plus one or more marginal bands of a different colour (Newman & Cannon, 1994, 1997). In most Pseudobiceros splendidus, the dorsal colour is black, and the animals possess an orange submarginal band and a black rim (Fig. 9A; Table 2). Lang’s (1884: 541) original description mentioned tiny white dots on the dorsal surface that can be discerned only with a magnifying glass, but Hyman (1955a) and Prudhoe (1989) made no mention of these minute white dots. Table 2. Comparison of synonyms, colour morphs and geographical distribution of Pseudobiceros splendidus Species Dorsal coloration Submarginal band Rim of margin Geographical distribution References Pseudoceros superbus = Pseudobiceros splendidus (Lang, 1884) Bluish black, with minute white dots not visible to the naked eye; or dark chocolate brown Orange–yellow Bluish black or dark chocolate brown Island of Nisida, Naples, Italy; Bermuda; Puerto Rico; Vietnam; Mid Turtle Shoal, Hawk Channel, Florida Keys and Atlantic coast of Florida, USA Lang, 1884; Hyman, 1939, 1955a; Prudhoe 1989; this study Pseudoceros evelinae = Pseudobiceros evelinae (Marcus, 1950) Dark reddish brown Orange Black Forte de Itaipú, Santos, São Paulo, Brazil; Extremoz, Rio Grande de Norte, Brazil; Cabo Frio, Rio de Janeiro, Brazil Marcus, 1950; Bahia et al., 2012, 2014 Pseudobiceros periculosus Newman & Cannon, 1994 Black Orange Very narrow, transparent Heron Island and One Tree Island, Great Barrier Reef, Australia; Hawaii, USA Newman & Cannon, 1994; this study Pseudobiceros hymanae Newman & Cannon, 1997 Black Orange Black Heron Island, Great Barrier Reef, Australia; Madang, Papua New Guinea; Makapu’u Point, Hawaii, USA; Rottnest Island, Western Australia; Andaman and Nicobar Islands, India; Indonesia; Maldives; South Africa; Singapore Newman & Cannon, 1997, 2003; Sreeraj & Raghunathan, 2013; Bolaños et al., 2016 Species Dorsal coloration Submarginal band Rim of margin Geographical distribution References Pseudoceros superbus = Pseudobiceros splendidus (Lang, 1884) Bluish black, with minute white dots not visible to the naked eye; or dark chocolate brown Orange–yellow Bluish black or dark chocolate brown Island of Nisida, Naples, Italy; Bermuda; Puerto Rico; Vietnam; Mid Turtle Shoal, Hawk Channel, Florida Keys and Atlantic coast of Florida, USA Lang, 1884; Hyman, 1939, 1955a; Prudhoe 1989; this study Pseudoceros evelinae = Pseudobiceros evelinae (Marcus, 1950) Dark reddish brown Orange Black Forte de Itaipú, Santos, São Paulo, Brazil; Extremoz, Rio Grande de Norte, Brazil; Cabo Frio, Rio de Janeiro, Brazil Marcus, 1950; Bahia et al., 2012, 2014 Pseudobiceros periculosus Newman & Cannon, 1994 Black Orange Very narrow, transparent Heron Island and One Tree Island, Great Barrier Reef, Australia; Hawaii, USA Newman & Cannon, 1994; this study Pseudobiceros hymanae Newman & Cannon, 1997 Black Orange Black Heron Island, Great Barrier Reef, Australia; Madang, Papua New Guinea; Makapu’u Point, Hawaii, USA; Rottnest Island, Western Australia; Andaman and Nicobar Islands, India; Indonesia; Maldives; South Africa; Singapore Newman & Cannon, 1997, 2003; Sreeraj & Raghunathan, 2013; Bolaños et al., 2016 View Large Table 2. Comparison of synonyms, colour morphs and geographical distribution of Pseudobiceros splendidus Species Dorsal coloration Submarginal band Rim of margin Geographical distribution References Pseudoceros superbus = Pseudobiceros splendidus (Lang, 1884) Bluish black, with minute white dots not visible to the naked eye; or dark chocolate brown Orange–yellow Bluish black or dark chocolate brown Island of Nisida, Naples, Italy; Bermuda; Puerto Rico; Vietnam; Mid Turtle Shoal, Hawk Channel, Florida Keys and Atlantic coast of Florida, USA Lang, 1884; Hyman, 1939, 1955a; Prudhoe 1989; this study Pseudoceros evelinae = Pseudobiceros evelinae (Marcus, 1950) Dark reddish brown Orange Black Forte de Itaipú, Santos, São Paulo, Brazil; Extremoz, Rio Grande de Norte, Brazil; Cabo Frio, Rio de Janeiro, Brazil Marcus, 1950; Bahia et al., 2012, 2014 Pseudobiceros periculosus Newman & Cannon, 1994 Black Orange Very narrow, transparent Heron Island and One Tree Island, Great Barrier Reef, Australia; Hawaii, USA Newman & Cannon, 1994; this study Pseudobiceros hymanae Newman & Cannon, 1997 Black Orange Black Heron Island, Great Barrier Reef, Australia; Madang, Papua New Guinea; Makapu’u Point, Hawaii, USA; Rottnest Island, Western Australia; Andaman and Nicobar Islands, India; Indonesia; Maldives; South Africa; Singapore Newman & Cannon, 1997, 2003; Sreeraj & Raghunathan, 2013; Bolaños et al., 2016 Species Dorsal coloration Submarginal band Rim of margin Geographical distribution References Pseudoceros superbus = Pseudobiceros splendidus (Lang, 1884) Bluish black, with minute white dots not visible to the naked eye; or dark chocolate brown Orange–yellow Bluish black or dark chocolate brown Island of Nisida, Naples, Italy; Bermuda; Puerto Rico; Vietnam; Mid Turtle Shoal, Hawk Channel, Florida Keys and Atlantic coast of Florida, USA Lang, 1884; Hyman, 1939, 1955a; Prudhoe 1989; this study Pseudoceros evelinae = Pseudobiceros evelinae (Marcus, 1950) Dark reddish brown Orange Black Forte de Itaipú, Santos, São Paulo, Brazil; Extremoz, Rio Grande de Norte, Brazil; Cabo Frio, Rio de Janeiro, Brazil Marcus, 1950; Bahia et al., 2012, 2014 Pseudobiceros periculosus Newman & Cannon, 1994 Black Orange Very narrow, transparent Heron Island and One Tree Island, Great Barrier Reef, Australia; Hawaii, USA Newman & Cannon, 1994; this study Pseudobiceros hymanae Newman & Cannon, 1997 Black Orange Black Heron Island, Great Barrier Reef, Australia; Madang, Papua New Guinea; Makapu’u Point, Hawaii, USA; Rottnest Island, Western Australia; Andaman and Nicobar Islands, India; Indonesia; Maldives; South Africa; Singapore Newman & Cannon, 1997, 2003; Sreeraj & Raghunathan, 2013; Bolaños et al., 2016 View Large Figure 9. View largeDownload slide Photographic records of look-alike Pseudobiceros splendidus. A, Pseudobiceros splendidus, Florida Keys, USA. B, Pseudobiceros hymanae. C, Pseudobiceros periculosus. D, Pseudoceros periaurantius. Specimens in B–D were collected at North Heron Island, Great Barrier Reef, Australia; photograph credit for B–D: Leslie Newman and Andrew Flowers. Figure 9. View largeDownload slide Photographic records of look-alike Pseudobiceros splendidus. A, Pseudobiceros splendidus, Florida Keys, USA. B, Pseudobiceros hymanae. C, Pseudobiceros periculosus. D, Pseudoceros periaurantius. Specimens in B–D were collected at North Heron Island, Great Barrier Reef, Australia; photograph credit for B–D: Leslie Newman and Andrew Flowers. Pseudobiceros hymanae Newman & Cannon, 1997 also has a dorsal black coloration, a submarginal orange band and a black margin (Newman & Cannon, 1997). The authors justified a new species designation because the orange band tends towards a rusty colour (Fig. 9B; Table 2), and they placed greater importance on the microscopic white dots found on Pseudobiceros splendidus (Table 2) than on the marginal band. Depending on the nutritional state or the life-history stage of a specimen, Pseudobiceros hymanae with a reddish-brown dorsal colour have also been described (Bolaños et al., 2016: 150). Furthermore, reddish-brown specimens with a submarginal orange band surrounded by a black rim are reminiscent of Pseudobiceros evelinae (Marcus, 1950) (Table 2; Bahia et al., 2012: 38; Bahia et al., 2014: 507), yet another colour morph similar to Pseudobiceros splendidus. Newman & Cannon (1994) described two additional, similarly coloured species. They defined Pseudobiceros periculosus Newman & Cannon, 1994 as distinct from Pseudobiceros splendidus because of the lack of a black rim (Fig. 9C; Table 2). Instead, an extremely thin transparent margin surrounds the animal. However, this might be attributable to the nutritional status of the specimen or might represent an artefact of light refraction when taking photographic records through the air–water interface. The second species, Pseudoceros periaurantius Newman & Cannon, 1994, has a wide orange marginal band extending all the way to the rim (Fig. 9D), and because of its single male copulatory complex, clearly is placed in Pseudoceros not Pseudobiceros. Mitochondrial sequences showing Pseudobiceros periaurantius clustering in Pseudoceros add further support to its taxonomic placement (Aguado et al., 2017). A comparison of D1–D2 expansion segment sequences of the two specimens of Pseudobiceros splendidus we collected in Florida with Pseudobiceros periculosus from the Great Barrier Reef reveals that they are identical. Furthermore, Pseudobiceros evelinae sequences available in GenBank are identical to our Pseudobiceros periculosus and Pseudobiceros splendidus sequences. Therefore, based on colour patterns and nucleotide sequences of the 28S D1–D2 expansion segments, these species can no longer be maintained as distinct entities. We conclude that Pseudobiceros periculosus, Pseudobiceros hymanae and Pseudobiceros evelinae are all junior synonyms of Pseudobiceros splendidus. A review of the geographical distribution of Pseudobiceros splendidus reveals that it might be one of the few truly cosmopolitan polyclad species (Table 2) and a testament to strong intraspecific cohesion. Phrikoceros Newman & Cannon, 1996 and Tytthosoceros Newman & Cannon, 1996: The second pseudocerotid clade forms an immediate sister group to Pseudobiceros + Thysanozoon and includes Phrikoceros and Tytthosoceros. Like species of Pseudoceros, both genera possess single male reproductive systems. However, their overall gross morphologies (e.g. highly ruffled body margin, shape of pseudotentacles, ruffled pharynx with simple folds) resemble Pseudobiceros (Newman & Cannon, 1996a). These differences led Newman & Cannon (1996a) to establish the new genus Phrikoceros. A further separation places species with ear-like pseudotentacles into Tytthosoceros, while species with square pseudotentacles are retained in Phrikoceros (Newman & Cannon, 1996b). Based on our results, this separation is artificial. Given that the establishment of Phrikoceros (Newman & Cannon, 1996a) pre-dated Tytthosoceros (Newman & Cannon, 1996b), we here abolish Tytthosoceros as a genus and move all three valid species to Phrikoceros to establish Phrikoceros nocturnus comb. nov., Phrikoceros lizardensis comb. nov. and Phrikoceros inca comb. nov. Given that Bahia et al. (2017) found that Phrikoceros clustered with other genera characterized by double male reproductive systems, they invoked the possibility that the genus has two male systems opening into a single male gonopore. However, not only does the original description of Phrikoceros show a single male system (Newman & Cannon 1996a), but our own histological examinations of Phrikoceros mopsus (Marcus, 1952) also reveal only one male system. Pseudoceros Lang, 1884: The third clade in Pseudocerotidae is formed by Pseudoceros. Although the genus is monophyletic, we find substructure in the group. With only a few exceptions, most of our samples were collected from Australia, especially the Great Barrier Reef, where they represent a conspicuous component of the reef fauna (Newman & Cannon, 2003 and references therein). The type locality for Pseudoceros prudhoei Newman & Cannon, 1994 is Heron Island, Great Barrier Reef, Australia (Newman & Cannon, 1994). The species has been recorded throughout the Indo-Pacific, including Japan, the Persian Gulf and Kenya. The original species description notes a brown-orange dorsal background colour with a wide, inner light mauve to light purple marginal band and a narrow, outer yellow band (Newman & Cannon, 1994). Recently, Velasquez et al. (2018) recorded a much darker colour variant (even black dorsal colour, inner marginal band almost white) from the Israeli coast of the Mediterranean. Based on comparisons with the original description of Pseudoceros duplicinctus Prudhoe, 1989, the authors synonymized Pseudoceros prudhoei with Pseudoceros duplicinctus. By comparing the sequence of a specimen identified as Pseudoceros prudhoei with the existing sequence of Pseudoceros duplicinctus, we here lend molecular support to this synonymy. Additionally, Velasquez et al. (2018) entertained the possibility that Pseudoceros depiliktabub Newman & Cannon, 1994 is also a junior synonym of Pseudoceros duplicinctus. Our comparison of sequences for a specimen initially identified as Pseudoceros depiliktabub with that of Pseudoceros duplicinctus confirmed this hypothesis, despite different colour variations and different collection localities of the specimens. We here designate Pseudoceros depiliktabub a junior synonym of Pseudoceros duplicinctus. Only a handful of Pseudoceros species have been reported from the Caribbean (Pseudoceros pardalis, Pseudoceros aurolineatus Verrill, 1901; Pseudoceros splendidus, Pseudoceros bicolor and Pseudoceros rawlinsonae). In our survey, we found Pseudoceros splendidus (see above) and a species complex of Pseudoceros bicolor exhibiting several colour morphs (Litvaitis et al., 2010). We also collected several specimens of Pseudoceros pardalis, a species that is characterized by a distinct colour pattern. After careful examination of its reproductive system, we had reassigned the species to Pseudobiceros (Bolaños et al. 2007). This reassignment now has been confirmed based on 28S rDNA sequences. Taxonomic value of morphological traits and summary of taxonomic revisions Our results confirm that, with few exceptions, the cotyl (sensu Lang, 1884) is a taxonomically useful character in the subordinal division of Polycladida. We encourage its use for the placement of species into Cotylea vs. Acotylea. Other traits that are valid for separating most species into Acotylea and Cotylea include the orientation of the male reproductive system and the presence of Lang’s vesicle (Faubel, 1983, 1984). Exceptions to the taxonomic usefulness of these characters are found in basal cotylean lineages (e.g. Cestoplanidae, Boniniidae and Theamatidae). Based on these characters, the basic acotylean body plan then lacks a cotyl, has a male system located anterior to the male gonopore and possesses Lang’s vesicle. Additionally, all Acotylea have a ruffled pharynx. In earlier, morphology-based classification systems, the most reliable taxonomic characters used had been associated with the reproductive systems (Faubel, 1983, 1984), the arrangement of eyes and the position and shape of tentacles (Prudhoe, 1985). Despite Faubel’s (1983, 1984) attempt to distinguish acotylean superfamilies based on one of three types of prostatic vesicles, such separation is no longer supported (Table 1) and requires the inclusion of additional characters. Faubel (1983) united Stylochoidea by the ‘presence of a free prostatic vesicle’. However, because Hoploplanidae with their atypical, interpolated prostatic vesicles are included in the superfamily, a free prostatic vesicle can no longer be considered a synapomorphy for the superfamily. Alternatively, four-lobed Götte’s larvae are taxonomically widespread in Stylochoidea (inclusive of Hoploplanidae). However, rather than use their presence as a stylochoid synapomorphy, we propose that the ancestral polyclad had a biphasic life cycle. Stylochoidea (sensu Poche, 1926) lacks a morphology-based synapomorphy at this time and is supported only by molecular data. In Cryptoceloidea (sensu Bahia et al., 2017), Ilyplanidae, Discocelidae and Euplanidae lack a true prostatic vesicle. However, Cryptocelidae possess an interpolated prostatic vesicle. Consequently, using the type of prostatic vesicle alone is taxonomically not informative. Three of the five recognized cryptocelid genera are monospecific (Faubel, 1983) and poorly defined. The two species-rich genera Cryptocelis and Phaenocelis possess unarmed, rod- or cylindrical-shaped penis papillae. We contend that Cryptoceloidea is united by the ‘absence of a true prostatic vesicle or, if an interpolated prostatic vesicle is present, an unarmed conical penis papilla’. Finally, a penis armed with cuticular elements (e.g. stylet, cirrus) is a synapomorphy of Leptoplanoidea (sensu Faubel, 1983, 1984), and the absence of armature in Notocomplanidae represents most probably a secondary loss (Table 1). Previous studies have also used the shape/amount of folding of the epithelial lining of the prostatic vesicle and the degree to which the ejaculatory duct extends into the vesicle as diagnostic characters (Faubel, 1983; Bulnes et al., 2005). We argue that such characters can be subjective and are mostly dependent on the quality of fixation and the angle of the plane of sectioning. Hence, we advise caution as to their taxonomic use. The shape of marginal tentacles provides taxonomic information in Cotylea (Faubel, 1984; Prudhoe, 1985; Rawlinson & Litvaitis, 2008). Pseudotentacles are formed by a ruffling of the anterior margin and are a characteristic of all Pseudocerotidae. Their shape may range from simple folds to more intricate structures (Newman & Cannon, 1994). The presence or absence of pseudotentacles is a useful trait, whereas the degree and complexity of folding (e.g. simple folds, ear-like or square) are dependent on the size of the animal, may be species specific and are affected by the quality of fixation. Consequently, only their presence should be noted, establishing an apomorphy for Pseudocerotidae (Table 1). Cotylean tentacles can also extend as well-separated, pointed structures from the anterior margin (Diposthus and Pericelis), arise as fine lateral tentacles (Boniniidae), form small marginal bumps (Cycloporus) or form pointed, V-shaped extensions (Maritigrella and Eurylepta). Thus, marginal tentacles can be useful for generic separation in Cotylea. Given that the position and arrangement of marginal eyes are highly variable in polyclads and can vary over the lifetime of an individual (Faubel, 1983, 1984; Prudhoe, 1985), the trait, at best, should be considered only in mature adult animals. The arrangement of pseudotentacular eyes has been used for generic separation in Pseudoceros vs. Pseudobiceros (Newman & Cannon, 1994, 1996a). However, they are often difficult to discern in animals of dark background colour. The number of male gonopores (i.e. Pseudoceros, one; Pseudobiceros, two) and the extent of folding of the pharynx are more reliable characters. Pharynx shape (ruffled vs. cylindrical) led Faubel (1984) to establish Pseudocerotoidea and Euryleptoidea, two superfamilies that can no longer be supported with the present data available (Rawlinson & Litvaitis, 2008; this study). Cotylean pharynx shape may be used to identify families in conjunction with additional traits. Our results supported many portions of earlier classifications based on morphology and molecular data, but we also identified several taxonomic corrections and additions. The following is a summary of those changes. In Acotylea, we returned Phaenoplana peleca to its original genus Phaenocelis (Phaenoplana peleca is now a junior synonym of Phaenocelis peleca), we established the new family Notocomplanidae to unite notoplanids lacking penis armature and, at the same time, transferred Melloplana ferruginea to Notocomplana ferruginea, resulting in the elimination of the genus Melloplana. Furthermore, we also returned Pleioplana atomata to its original Notoplana atomata (Pleioplana atomata is now a junior synonym of Notoplana atomata) and transferred Persica qeshmensis to Notoplana qeshmensis, resulting in the abolishment of the monospecific genus Persica. Finally, our results indicated that Amyris ujara is a junior synonym of Amyris hummelincki. Changes in Cotylea also included rearrangements of taxa and the identification of junior synonyms. We designated Chromoplanoidea (sensu Bahia et al., 2017) as invalid because the type family is not contained in the superfamily. As a consequence, Boniniidae, Theamatidae and Amyellidae remain their own distinct families. We amended Diposthidae to include all species of Pericelis, resulting in the elimination of the family Pericelidae and the invalidation of Periceloidea (sensu Bahia et al., 2017). Furthermore, we identified the following synonyms and new combinations: Boninia divae is a junior synonym of Boninia antillarum; Cestoplana australis is a junior synonym of Cestoplana rubrocincta; Maritigrella crozieri has been moved to Prostheceraeus crozieri; Maritigrella newmanae is a junior synonym of Prostheceraeus floridanus; Amakusaplana acroporae has been moved to Prosthiostomum acroporae; and Lurymare utarum has been returned to its original genus, Prosthiostomum, as Prosthiostomum utarum. In addition, the genus Tytthosoceros has been abolished, and all three valid species have been moved to Phrikoceros to establish Phrikoceros nocturnus, Phrikoceros lizardensis and Phrikoceros inca. Finally, Pseudoceros prudhoei and Pseudoceros depliktabub have been identified as junior synonyms of Pseudoceros duplicinctus, and Pseudobiceros evelinae, Pseudobiceros periculosus and Pseudobiceros hymanae are all junior synonyms of Pseudobiceros splendidus. With the application of innovative methodological approaches (e.g. DNA sequencing, phylogenomics, fluorescence microscopy and immunocytochemistry), a more stable system for Polycladida is emerging. Although multiple lines of evidence are supporting three acotylean superfamilies and a cotylean classification based on families, the relationships in some clades require additional research. Specifically, the families Notoplanidae and Stylochoplanidae (both in Leptoplanoidea) need revision. Likewise, relationships in Euryleptidae, and the monophyly of this family itself, need further resolution. Finally, the relationships of basal cotylean lineages (i.e. Boniniidae, Theamatidae and Amyellidae) deserve additional attention. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher's web-site: Appendix. Collection localities and GenBank accession numbers (D1–D2 segment of the 28S rDNA gene) of species (with taxonomic authorities) used in this study. Accession numbers in italics indicate new sequences generated during this study. [Version of Record, published online 28 March 2019; http://zoobank.org urn:lsid:zoobank.org:pub:2F256142-BA B4-4FBC-A768-B8D5D70090DD] ACKNOWLEDGEMENTS This work is dedicated to the late Dr Leslie Newman, who inspired countless students, scientists, underwater photographers and amateur taxonomists with her enthusiasm and passion for the wonderful world of polyclads. Without her encouragement, insights and contributions, this work would not have been possible. Thank you, Leslie! We also thank Dr Larry Harris, Dr Marcin Liana, Dr Kate Rawlinson and our volunteers Anne DuPont, Joseph Dunn and Andrew Allan for help with specimen collection. 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Bulletin of the Museum of Comparative Zoology Harvard Collection 32 : 61 – 67 . © 2019 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Systematic congruence in Polycladida (Platyhelminthes, Rhabditophora): are DNA and morphology telling the same story?
JF - Zoological Journal of the Linnean Society
DO - 10.1093/zoolinnean/zlz007
DA - 2019-03-28
UR - https://www.deepdyve.com/lp/oxford-university-press/systematic-congruence-in-polycladida-platyhelminthes-rhabditophora-are-71dJBL0POH
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -