Organisms Diversity & Evolution (2019) 19:585–608 https://doi.org/10.1007/s13127-019-00415-1

ORIGINAL ARTICLE

Polyclad phylogeny persists to be problematic

Isabel L. Dittmann1 & Daniel Cuadrado2 & Maria Teresa Aguado3,4 & Carolina Noreña2 & Bernhard Egger1

Received: 12 April 2019 /Accepted: 14 August 2019 /Published online: 16 September 2019 # The Author(s) 2019

Abstract Two conflicting morphological approaches to polyclad systematics highlight the relevance of molecular data for resolving the interrelationships of Polycladida. In the present study, phylogenetic trees were reconstructed based on a short alignment of the 28S rDNA marker gene with 118 polyclad terminals (24 new) including 100 different polyclad species from 44 genera and 22 families, as well as on a combined dataset using 18S and 28S rDNA genes with 27 polyclad terminals (19 new) covering 26 different polyclad species. In both approaches, Theamatidae and Cestoplanidae were included, two families that have previously been shown to switch from Acotylea to Cotylea. Three different alignment methods were used, both with and without alignment curation by Gblocks, and all alignments were subjected to Bayesian inference and maximum likelihood tree calculations. Over all trees of the combined dataset, an extended majority-rule consensus tree had weak support for Theamatidae and Cestoplanidae as acotyleans, and also the cotylean genera Boninia, Chromyella and Pericelis appeared as acotyleans. With the most inclusive short 28S dataset, on the other hand, there is good support for the aforementioned taxa as cotyleans. Especially with the short 28S matrix, taxon sampling, outgroup selection, alignment method and curation, as well as model choice were all decisive for tree topology. Well-supported parts of the phylogeny over all trees include Pseudocerotoidea, Prosthiostomoidea, Stylochoidea, Leptoplanoidea and Cryptoceloidea, the latter three with new definitions. Unstable positions in the tree were found not only for Theamatidae, Cestoplanidae, Boninia, Chromyella and Pericelis, but also for Anonymus, Chromoplana and Cycloporus.

Keywords Platyhelminthes . Polycladida . Cotylea . Acotylea . Molecular phylogenetics . Systematics

Introduction studied (Bahia et al. 2017). Usually, polyclads occur in diverse marine habitats, such as under coastal stones, on reefs and in Due to their colourful appearance, polyclad flatworms are interstitial spaces (Hyman 1951; Prudhoe 1985; Curini- among the most conspicuous members of the phylum Galletti et al. 2008). About 800 to 1000 species of polyclads Platyhelminthes, yet these animals are relatively poorly are currently recognised (Rawlinson 2008; Martín-Durán and Egger 2012). The phylogenetic position of Polycladida within Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13127-019-00415-1) contains supplementary Platyhelminthes used to be very controversial (Bahia et al. material, which is available to authorized users. 2017). Only recently, Polycladida have been consistently re- covered as sister group to Prorhynchida (a group harbouring * Bernhard Egger only freshwater dwellers), forming the Amplimatricata, which [email protected] is the sister group of all other Trepaxonemata (Egger et al. 2015;Laumeretal.2015; Laumer and Giribet 2017). 1 Institute of Zoology, University of Innsbruck, Technikerstr. 25, Lang (1884) was the first to distinguish between two 6020 Innsbruck, Austria groups of ‘marine planarians’, the Tricladida and the 2 Museo Nacional de Ciencias Naturales (CSIC), José Gutiérrez Polycladida. He further grouped the Polycladida into forms Abascal 2, 28006 Madrid, Spain with a ventral sucker behind the genital openings (Cotylea), 3 Animal Evolution and Biodiversity, Johann-Friedrich-Blumenbach and those without (Acotylea). This classification system per- Institute for Zoology & Anthropology, Georg-August-Universität Göttingen, Göttingen, Germany sists after some modifications (e.g. Laidlaw 1903;Bock1913; Hyman 1953; Marcus and Marcus 1966) until today, and in 4 Centro de Investigación en Biodiversidad y Cambio Global (CIBC-UAM). Departamento de Biología, Facultad de Ciencias, the 1980s, Faubel (1983, 1984)andPrudhoe(1985)separately Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain published monographs attempting to further clarify the 586 Dittmann I.L. et al. interrelationships of polyclads on morphological grounds, 28S sequences of a wide systematic range of polyclads. Most using genital organs, especially the organisation of the pros- importantly, we have applied three different, widely used tatic vesicle (Faubel 1983, 1984), the position of eyes and alignment algorithms and two different statistical approaches tentacles (Prudhoe 1985), or the pharynx organisation for tree reconstruction to test the stability and reliability of (Faubel 1983, 1984;Prudhoe1985) as main systematic char- molecular phylogenies using one or two genes, and also, when acters—however, the resulting classifications were largely in- possible, to infer relationships between groups based on a congruent. Interestingly, Faubel (1984) considered both bigger data set. Cotylea and Acotylea as not being monophyletic, but retained the names for taxonomic consistency. Prudhoe (1985) was also aware of problems with the classification Material and methods and he cited several cases, where some families, such as Enantiidae and Boniniidae, have features fitting both to Animal collection, identification of species Cotylea and Acotylea. and transcriptome data For more than 30 years, these two conflicting systems have been in use by polycladologists (a term coined by J. Bahia, An overview of newly generated and published sequences is personal communication), stressing the need of a unifying provided in Table 1. For most collected material, tissue was system, based on morphology, on molecules, or both. The first stored in 99% ethanol, and histological sections were made as molecular phylogenetic reconstruction of polyclad interrela- described by Aguado et al. (2017) and Dittmann et al. (2019). tionships was using a partial sequence of about 350 nucleo- Several published polyclad transcriptomes (Egger et al. 2015, tides of the marker molecule 28S (large nuclear ribosomal Laumer et al. 2015) were searched for 18S and 28S sequences subunit) and was focussed on the family Pseudocerotidae, (see Table 1) using BLAST (Altschul et al. 1990). with Pericelis as the cotylean sister group of Pseudocerotidae (Litvaitis and Newman 2001). Another mo- lecular phylogenetic analysis of Polycladida based on partial DNA extraction, PCR amplification and sequencing 28S sequences (about 900 nt long) included just eight cotylean and six acotyleans—Cotylea was not recovered as monophy- For all specimens, DNA was extracted from a small piece of letic, since the cotylean species Pericelis cata appeared out- ethanol-preserved marginal tissue. DNA extraction was per- side the other Cotylea as sister group of Acotylea, while formed following phenol-chloroform protocols (Sambrook Cestoplana rubrocincta emerged as an acotylean as in et al. 1989; Chen et al. 2010). Concentration and possible Faubel’s and Prudhoe’s systems (Rawlinson et al. 2011). contamination of extracted DNA were checked using With a very similar dataset, Rawlinson and Stella (2012)re- NanoDrop (NanoDrop Fluorospectrometer Thermo Fisher covered both, Pericelis and Cestoplana, as basally branching Scientific, USA). PCRs were performed in a total volume of cotyleans, thereby stressing the problematic position of these 25 μlor50μl. 18S rDNA was amplified either in two over- taxa. In a flatworm-wide phylogenetic study based on four lapping fragments using the published primer combinations genes, the acotylean Theama was grouped with the remaining 4fb + 1806R (ca. 1200 bp) and 5fk + S30 (ca. 900 bp) Cotylea, not with the Acotylea (Laumer and Giribet 2014), (Larsson and Jondelius 2008) or in one approach using 18S- which was corrobarated in a transcriptomic study in the fol- 1F + 18S9R (ca. 1800 bp) (Álvarez-Presas et al. 2008). 28S lowing year (Laumer et al. 2015). rDNA was amplified with the primers 28_LSU5_fw + In 2017, three large molecular phylogenies of polyclads L1642R (ca.1450 bp) or 28S_1F + 28S_6R (ca. 1600 bp) were published, two with different stretches of the 28S marker (Larsson and Jondelius 2008). PCR was performed using a gene (Bahia et al. 2017; Tsunashima et al. 2017), and one with ‘Touch Down’ protocol using the following protocol: 5 min mitochondrial genes (Aguado et al. 2017). Of these studies, of initial denaturation at 94 °C; 30 s of denaturation at 94 °C, only Bahia et al. dealt with the aforementioned problematic annealing at 68–45 °C for 30 s, extension at 72 °C for 2 min; taxa, namely Pericelis, Cestoplana and Theama—all of them 12 cycles; 30 s of denaturation at 94 °C, annealing at 45 °C for showing up as cotyleans in their tree (Bahia et al. 2017). 30 s, extension at 72 °C for 2 min; 23 cycles; final extension at However, this study only used a single alignment method 72 °C for 10 min, hold at 4 °C. Successful products were and a single model with relatively low bootstrap support purified using ExoSAP-IT (Affymetrix, USA), following levels, so the reliability of the provided reconstruction manufacturer’s protocol, or with the Wizard® SV gel and remained unclear. During the review phase of this manuscript, PCR clean-up system (Promega, USA) according to the man- another publication using the 28S marker gene was published ufacturer’s quick protocol. PCR products were sequenced by (Litvaitis et al. 2019). CBMSO (Spain) or by Microsynth Austria GmbH, respective- In the present study, we also have used partial 28S rDNA ly. Sequences were assembled and edited by hand or using the sequences, as well as a combined dataset of longer 18S and software CLC Main Workbench 7 (Qiagen, Germany). Polyclad phylogeny persists to be problematic 587

Table 1 List of all species used in this study, including authorities, sample locations and accession or SRA numbers. In trees using only a single sequence of the same species, the first listed sequence was included, with the number omitted

Species Authority Location 28Sshort6 18S28Slong SRA

Ilyella gigas (Schmarda 1859) Japan LC100080.1 Discocelis tigrina (Blanchard 1847) Valencia, Spain MN384690 MN334200, MN384690 Adenoplana evelinae Marcus 1950 Brazil KY263647.2 Cestoplana rubrocincta 1(Grube1840) Naples, Italy MN384689 MN334198, MN384689 rubrocincta 2 Australia HQ659009.1 salar Marcus 1949 Brazil KY263653.2 techa Du Bois-Reymond Marcus Brazil KY263652.2 1957 Phaenocelis medvedica Marcus 1952 Brazil KY263701.2 Echinoplana celerrima 1Haswell1907 Tunis, Tunisia MN421930 MN421936, MN421930 SRS842092 celerrima 2 Australia HQ659020.1 Hoploplana californica Hyman 1953 California KC869850.1 KC869797.1, KC869850.1 divae Marcus 1950 Brazil KY263692.2 villosa (Lang 1884) Japan LC100076.1 Leptoplana tremellaris 1(Müller1773) Cornwall, UK MN421931 MN421937, MN421931 SRS842637 tremellaris 2 Spain KY263695.2 sp. Lizard Island MN384693 (Australia) Notoplana australis 1(Laidlaw1904) Australia AY157153.1 AJ228786.1, AY157153.1 australis 2 Australia HQ659015.1 delicata (Jacubowa 1906) Japan LC100088.1 sp. Brazil KY263651.2 Notocomplana humilis (Stimpson 1857) Japan LC100085.1 japonica (Kato 1937a) Japan LC100087.1 koreana (Kato 1937b) Japan LC100086.1 sp. Japan LC100089.1 Melloplana ferruginea (Schmarda 1859) Florida HQ659014.1 Comoplana agilis (Lang 1884) Galicia, Spain MN384685 MN334199, MN384685 Armatoplana leptalea (Marcus 1947) Brazil KY263648.2 Amemiyaia pacifica Kato 1944 Japan LC100077.1 Theama mediterranea Curini-Galletti et al. 2008 Rovinj, Croatia MN384705 MN384707, MN384705 sp. Panama KC869845.1 KC869792.1, KC869845.1 Callioplana marginata Stimpson1857 Japan LC100082.1 Planocera multitentaculata Kato 1944 Japan LC100081.1 pellucida (Mertens 1833) Canary Island, Spain MN384696 MN334203, MN384696 Paraplanocera oligoglena (Schmarda 1859) Hawaii KC869849.1 KC869796.1, KC869849.1 sp. Greece KY263699.2 Idioplana australiensis Woodworth 1898 Australia HQ659008.1 Pseudostylochus obscurus (Stimpson 1857) Japan LC100084.1 sp. Japan LC100083.1 Stylochus ellipticus (Girard 1850) Woods Hole, USA Suppl. File 1 Suppl. File 1 SRS913554 ijimai Yeri and Kaburaki 1918 Japan LC100079.1 oculiferus (Girard 1853) Florida HQ659007.1 zebra (Verrill 1882) US Atlantic coast AF342800.1 AF342801.1, AF342800.1 sp. Peru KY263743.2 Imogine refertus Brazil KY263694.2 588 Dittmann I.L. et al.

Table 1 (continued)

Species Authority Location 28Sshort6 18S28Slong SRA

Du Bois-Reymond Marcus 1965 stellae Marquina et al. 2014 Valencia, Spain MN384692 MN334201, MN384692 Leptostylochus gracilis Kato 1934 Japan LC100078.1 Cycloporus gabriellae 1Marcus1950 Brazil KY263656.2 gabriellae 2 KY263658.2 variegatus 1Kato1934 Brazil KY263657.2 variegatus 2 Spain KY263659.2 variegatus 3 Brazil KY263660.2 variegatus 4 Brazil KY263661.2 japonicus Kato 1944 Japan LC100092.1 Maritigrella crozieri 1(Hyman1939) Florida Keys, USA MN421933 MN421939, MN421933 SRS844631 crozieri 2 Aquaria in Virginia, HQ659013.1 USA crozieri 3 Florida KY263686.2 fuscopunctata (Prudhoe 1978) Maltese coast KU674837.1 newmanae Bolaños et al. 2007 Belize EF514798.1 Prostheceraeus roseus Lang 1884 Tenerifa KY263688.2 vittatus (Montagu 1815) unknown Suppl. File 1 Suppl. File 1 SRS913668 Stylostomum ellipse (Dalyell 1853) Punat, Croatia MN384704 MN334208, MN384704 Euryleptodes galikias Noreña et al. 2014 Galicia, Spain MN384691 Prosthiostomum grande Stimpson 1857 Japan LC100090.1 siphunculus 1(DelleChiaje1822) Barcelona, Spain MN421934 MN421940, MN421934 SRS842699 siphunculus 2 Asturias, Spain MN384697 MN334204, MN384697 siphunculus 3 Spain HQ659012.1 vulgaris Kato 1938 Japan LC100091.1 Amakusaplana acroporae Rawlinson et al. 2011 Aquaria US East Coast HQ659010.1 Lurymare katoi Poulter 1975 Lizard Island MN384694 (Australia) Enchiridium evelinae Marcus 1949 Brazil KY263662.2 sp. 1 Lizard Island MN384686 (Australia) sp. 2 Santa Helena Island KY263665.2 Chromyella sp. Panama KC869848.1 KC869795.1, KC869848.1 Anonymus ruber Cuadrado et al. 2017 Canary Island, Spain MN384687 MN334197, MN384687 virilis Lang 1884 Canary Island, Spain MN384688 Boninia divae Marcus and Marcus 1968 Panama KC869846.1 KC869793.1, KC869846.1 Chromoplana sp. Panama KC869847.1 KC869794.1, KC869847.1 Pericelis byerleyana (Collingwood 1876) Red Sea MH047291.1 cata 1 Marcus and Marcus 1968 unknown EU679114.1 cata 2 Brazil KY263700.2 orbicularis (Schmarda 1859) unknown EU679116.1 tectivorum Dittmann et al. 2019 Aquaria Innbruck, MK181524 MN334202, MK181524 Austria Pseudoceros astorum Bulnes and Torres 2014 Brazil KY263737.2 bicolor 1 Verrill 1902 Belize GQ398095.1 bicolor 2 Brazil KY263732.2 bicolor Litvaitis et al. 2010 Belize GQ398098.1 marcusorum Polyclad phylogeny persists to be problematic 589

Table 1 (continued)

Species Authority Location 28Sshort6 18S28Slong SRA

cf bicolor Brazil KY263729.2 bimarginatus Meixner 1907 Lizard Island MN384700 MN334207, MN384700 (Australia) contrarius Newman and Cannon 1995 Papua New Guinea KY263728.2 harrisi Bolaños et al. 2007 Panama EF514802.1 jebborum Newman and Cannon 1994 Lizard Island MN384701 (Australia) cf maximus Lang 1884 Spain KY263708.2 nipponicus Kato 1944 Japan LC100096.1 periaurantius Newman and Cannon 1994 Lizard Island MN384702 (Australia) rawlinsonae 1 Bolaños et al. 2007 Bahamas GQ398101.1 rawlinsonae 2 Brazil KY263733.2 stimpsoni Newman and Cannon 1998 Lizard Island MN384703 (Australia) velutinus 1(Blanchard1847) Spain KY263726.2 velutinus 2 Japan LC100095.1 Pseudobiceros bedfordi (Laidlaw 1903) Papua New Guinea KY263715.2 caribbensis Bolaños et al. 2007 Curaçao EF514804.1 evelinae (Marcus 1950) Brazil KY263716.2 flowersi Newman and Cannon 1997 Lizard Island MN384698 MN334205, MN384698 (Australia) hancockanus (Collingwood 1876) Lizard Island MN384699 MN384706, MN384699 (Australia) nigromarginatus (Yeri & Kaburaki 1918) Japan LC100097.1 pardalis 1 (Verrill 1900) Panama EF514807.1 pardalis 2 Brazil KY263723.2 splendidus (Lang 1884) Florida HQ659016.1 wirtzi Bahia and Schroedl 2016 Senegal KY263725.2 sp. Santa Helena Island KY263724.2 Maiazoon orsaki Newman and Cannon 1996 Papau New Guinea KY263697.2 Thysanozoon alagoensis Bahia et al. 2015 Brazil KY263747.2 brocchii 1(Risso1818) Philip Island, Australia HQ659017.1 brocchii 2 Brazil KY263744.2 raphaeli Bolaños et al. 2007 Panama EF514809.1 Yungia sp. Florida HQ659018.1 Phrikoceros mopsus (Marcus 1952) Brazil KY263707.2 Monobiceros langi Faubel 1984 Spain KY263710.2 Macrostomum lignano Ladurner et al. 2005 MN421932 MN421938, MN421932 SRS842645 Xenoprorhynchus sp. KC869852.1 KC869813.1, KC869852.1

Datasets for phylogenetic analyses Bahia et al. in 2019 with a non-gappy version), 20 of which were generated by us, and Macrostomum lignano as an outgroup We made eight different single gene sequence collections of (‘28Sshort1’), while all subsequent ‘short’ 28S sequence collec- ‘short’ 28S sequences (see Table 1 for accession numbers of all tions worked with the updated second sequence versions of Bahia newly generated and used published data). In general, we only et al. (2017): ‘28Sshort2’ added Cycloporus japonicus, two used one sequence per species from the same authors. Pericelis and four pseudocerotoid sequences, while ‘28Sshort3’ The first sequence collection used 108 polyclad terminals (in- only included all (updated) sequences of ‘28Sshort1’. cluding the first, gappy version of sequences published by Bahia Variations of ‘28Sshort2’ included only Xenoprorhynchus et al. 2017 on NCBI, which was corrected and reuploaded by sp. (‘28Sshort2X’) or both Xenoprorhynchus sp. and 590 Dittmann I.L. et al.

Macrostomum lignano (‘28Sshort2XM’) as outgroups. reconstructions supported Cestoplanoidea, Chromoplanoidea, ‘28Sshort4’ is identical to ‘28Sshort3’, except the removal Periceloidea, Anonymidae and Chromoplana as cotyleans of Chromoplana sp., whereas in ‘28Sshort5’,wealsore- (Suppl. Figs. S5, S10, S11), while three trees rendered most moved Cycloporus variegatus. Finally, for ‘28Sshort6’,we of these taxa as acotyleans or polytomic (Suppl. Figs. S4, S6, used ‘28Sshort2’ sequences and included all available se- S12). We have visualised these changes caused by model quences of Cycloporus variegatus (four sequences) and choice in Fig. 1. Using Gblocks, only the two trees based on Cycloporus gabriellae (two sequences). Most of the shown a Q-INS-i alignment recovered Cestoplanoidea, trees deal with the last sequence collection, which includes Chromoplanoidea, Periceloidea, Anonymidae and 118 polyclad terminals (24 sequences provided by us), cover- Chromoplana as cotyleans (Suppl. Figs. S2,8). In both E- ing 100 polyclad species. INS-i trees and the ML MUSCLE tree, Anonymidae and Additionally, we made a combined dataset of ‘long’ 18S Chromoplana are sister group of all other Polycladida, while and 28S sequences (‘18S28Slong’), including 27 polyclad in the BI MUSCLE tree, they are polytomic with Cotylea and terminals (19 of which were newly generated) and Acotylea (see Table 2). Macrostomum lignano as an outgroup. We continued our analyses with the first 28S-only dataset Sequences for each gene were separately aligned using (28Sshort1) with many more taxa than available in the three methods: MUSCLE v3.8.31 (Edgar 2004), MAFFT Q- 18S28Slong dataset, including the first version of sequences INS-i and MAFFT E-INS-i v7.310 (Katoh and Standley published by Bahia et al. (2017). With this dataset, we calcu- 2013). They were manually trimmed, and in the case of the lated BI and ML trees based on three different alignments, and combined dataset, concatenated. For several alignments, we consistently (100%) recovered Cestoplanoidea, also used Gblocks with the least stringent settings (Castresana Chromoplanoidea, Periceloidea, Anonymidae and 2000). Conversion of fasta alignments to Nexus and Phylip Chromoplana as Cotylea. The corresponding MRE tree formats was done using ALTER (Glez-Peña et al. 2010). exhibited an identical topology as the BI MUSCLE tree Two different approaches for phylogenetic reconstructions shown in Fig. 2a. After obtaining the new sequence versions were pursued: maximum likelihood (ML) reconstructions of Bahia et al. (2017) in January 2019, we recalculated all using RAxML (Stamatakis 2014), and Bayesian inference trees with the new sequences (and adding additional se- (BI) with MrBayes (Ronquist et al. 2012). The best models quences, see 28Sshort2) and consistently (100%) recovered (GTR + I + G) were determined with jModelTest v2.1.10 the aforementioned groups as Acotylea, regardless of using the Akaike Information Criterion AIC(c) (Posada 2008). outgroup selection or alignment curation (Fig. 3). For ML trees, between 500 and 10,000 tree searches were We tested different parameters, always using a short 28S performed, and between 500 and 1000 separate bootstrap rep- dataset with BI MUSCLE with and without Gblocks for tree licates. At least 5–10 million generations were calculated for reconstruction. Using Gblocks, outgroup selection markedly BI trees, or more until convergence (average standard devia- changed other parts of the topology, such as tion of split frequences < 0.01) was reached. For extended Prosthiostomoidea alternating between Acotylea and Cotylea majority-rule consensus trees, we used RAxML with the (Fig. 3). With only Xenoprorhynchus as outgroup, concatenated trees of BI and ML analyses of the 28Sshort6 Chromoplana is the sister group of all other Polycladida dataset (see Table 2). Phylogenetic trees were visualised in (Fig. 3b), while with only Macrostomum as outgroup, Figtree (http://tree.bio.ed.ac.uk/software/figtree/) and adapted Cycloporus variegatus takes the place of sister group of all in Inkscape (https://inkscape.org/)andAdobeIllustratorCS4. other Polycladida (Fig. 3c). Using the same ingroup and The sequences generated during and/or analysed during the outgroup taxa as in Fig. 3c, but without Gblocks, we recovered current study are available in the GenBank repository, under a topology with many basal polytomies (Fig. 3d). With both the accession numbers listed in Table 1. The datasets gener- non-polyclad outgroups, a basal polytomy between Cycloporus ated during and/or analysed during the current study are avail- variegatus, Euryleptidae and all other Polycladida was able from the corresponding author on reasonable request. recovered (Fig. 3a). Prosthiostomidae are basally branching Acotylea with Macrostomum + Xenoprorhynchus, and only Macrostomum as outgroups. Xenoprorhynchus alone as Results outgroup provides a basal polytomy of Anonymus,Cotylea and Acotylea, except Chromoplana (Fig. 3b). Effects of model choice, alignment, outgroup Consequently, we tested if the newly added sequences were selection and taxon sampling on tree topology responsible for the change in tree topology, especially of Cestoplanoidea, Chromoplanoidea, Periceloidea, We recovered varied results with our combined 18S28Slong Anonymidae and Chromoplana. We therefore removed all matrix (see Table 2, Suppl. Figs. S1–12): without using additional sequences compared to our first dataset leading to Gblocks for alignment curation, three of the six phylogenetic the 28Sshort3 alignment, and with the same alignment and Polyclad phylogeny persists to be problematic 591

Table 2 Summary and overview of all trees calculated with the with Gblocks, No GB without Gblocks alignment curation, x yes, − no, p 18S28Slong and the 28Sshort6 datasets. ML maximum likelihood, BI polytomic, ? no data Bayesian inference, M MUSCLE, Q Q-INS-i, E E-INS-i alignment, GB

Suppl. Fig. S 18S28Slong 28Sshort6 Support

ML BI ML BI

GB No GB GB No GB GB No GB GB No GB

MQ E MQ E MQ E MQ E MQ E MQ E MQ E MQ E 123456789101112131415161718192021222324

1. Cotylea and Acotylea sensu – x ––x ––x – xx– xxxx– xxxxx– x15/24 Bahia et al. 2017 are monophyletic 2. Cestoplanidae appear ????????????xxxxxxxxxxxx12/12 monophyletic 3. Cestoplanidae appear within – x ––x – px– xx– xxxx– xxxxx– x15/24 Cotylea 4. Cestoplanoidea is sister group to – x ––x ––x – xx– xxxx– xxxxx– x15/24 all other Cotylea 5.Pericelidaeismonophyletic ????????????xxxxxxxxxxxx12/12 6. Pericelidae is sister group to all ––––x ––––xx––x – x –––––x ––6/24 Cotylea except Cestoplanoidea 7. Chromoplana and Anonymus xxxxxxxxxxxxxxxxxxxxpxxx23/24 recover as clade 1 8. Clade 1 appears as sister group to – x ––x – px– xxxxxxx– xx––x – x14/24 a clade including Prosthiostomoidea and Pseudocerotoidea 9. Pseudocerotoidea and xxxxxxxxxxxxxxxx– xxxxx– x22/24 Pseudocerotidae sensu Bahia et al. 2017 are monophyletic 10. The species Pseudoceros, ????????????xxxxxxxxxxxx12/12 Pseudobiceros and Thysanozoon are not monophyletic 12. Euryleptidae sensu Faubel 1984 xxxxxxxxxxxxx– xx– xxxxx– x21/24 is split into two clades 12.Clade2ismonophyletic ????????????xxxxxxxxxxxx12/12 13. The clade still called –––––––––––––x ––x –––––x – 21/24 Euryleptidae is recovered as paraphyletic 14. The genera Cycloporus and ????????????––––––––––p – 0/12 Prostheceraeus are recovered as monophyletic 15. Maritigrella is recovered as ????????????– x ––x ––x ––x – 4/12 monophyletic 16. Prosthiostomoidea appears as xxxxxxxxxxxxxxxxxxxxxxxx24/24 monophyletic 17. Prosthiostomoidea is sister xxxxxxxxxxxxxxxx– xxxxx– x22/24 group to Pseudocerotoidea 18. Within Prosthiostomidae, ????????????xxxxxxxxxxxx12/12 Enchiridium appears as sister group to a clade consisting of Prosthiostomum, Lurymare and Amakusaplana 19. Prosthiostomum appears ????????????xxxx– xxxxx– x10/12 paraphyletic, as Amakusaplana and Lurymare cluster within 20. Chromoplanoidea (including – x ––x ––x – xx– xxxx– xxxxx– x15/24 Theama, Chromyella and Boninia) clusters within Cotylea ––––––––––––x – x ––xxxx––x7/24 592 Dittmann I.L. et al.

Table 2 (continued)

Suppl. Fig. S 18S28Slong 28Sshort6 Support

ML BI ML BI

GB No GB GB No GB GB No GB GB No GB

MQ E MQ E MQ E MQ E MQ E MQ E MQ E MQ E 123456789101112131415161718192021222324

21. Chromoplanoidea as sister group to all other Cotylea, except Cestoplanoidea 22. Theamatidae is sister group to a xxxxxxxxxxxx– x – xxxxx– xxx21/24 clade consisting of Boninia and Chromyella 23. Leptoplanoidea sensu Faubel ––––––––––––––––––––––––0/24 1983 (in whose definition Hoploplana and Theama are included) is supported 24. Clade 3 can be subdivided into xxxxxxxxxxxxx– xx– xx– xxpx21/24 two clades (clades 5 and 6) 25. Clade 5 is synonymous with xxxxxxxxxxxx––––––––––––12/24 Leptoplanoidea sensu Bahia et al. 2017 26. Pseudostylochus is part of clade ????????????xxxxxxxxxxxx12/12 5 27. Leptoplana ismonophyletic????????????xxxxxxxxxxxx12/12 28. Notoplanidae as a whole, as ????????????––––––––––––0/12 well as Notoplana are monophyletic, while Notocomplana is not monophyletic 29.Clade6appearsmonophyletic????????????x– xx– xx– xxpx8/12 30. Clade 6 appears as sister group xxxxxxxxxxxxx– xx– xx– xxpx20/24 to clade 5 31. Clade 4 can be subdivided into ????????????–––x – xp––p – p2/12 two clades, clades 7, 8 and Callioplana, where the latter is sister group to clades 7 + 8 32. A polytomy exists between ????????????––––––x – xxxx5/12 Callioplana, clade 3 and clade 4 33. Clade 7 appears not ????????????– xx– x ––xx– x – 6/12 monophyletic 34. Hoploplana clustering as sister ????????????x––x – xx––x – x6/12 group to Idioplana, as clade 7 35. Hoploplana is sister group to xxxxx– xxxxp––x ––x ––xp– x – 14/24 Planoceridae/Planocera pellucida 36. Clade 8 is monophyletic –––––x ––––px–––x – xpppppp4/24 37. Planocera ismonophyletic????????????– x ––x ––x ––x – 4/12 38. Paraplanocera ismonophyletic????????????––––––––––––0/12 39. Planoceridae sensu Faubel 1983 ––––––––––p –––––––––––––0/24 are recovered as monophyletic 40. Stylochus is monophyletic – x ––x ––x – x ––––––x –––––x – 6/24 41. Imogine ismonophyletic ????????????––––––––––––0/24 Total score 17 21 17 17 20 15 17 21 17 20 18 16 34 32 34 33 23 33 32 29 30 32 21 32 Total number of points possible 22 (all lines except lines with ?) 38 (all lines except lines #14, 33, and 34) model choice, recovered a tree topology very different (Fig. 2a)—again with Cycloporus variegatus as sister group to (Fig. 2b) from the one obtained with the 28Sshort1 alignment the remaining Polycladida (Fig. 2b). Polyclad phylogeny persists to be problematic 593

Now we also removed Chromoplana from the dataset Also 63% of our trees, and the 28Sshort6 MRE tree, sup- (28Sshort4) and once more had C. variegatus as sister group port the phylogenetic position of Chromoplanoidea (Bahia to all other Polycladida. Also, Cestoplanoidea, et al. 2017, including Theama, Chromyella and Boninia)with- Chromoplanoidea, Periceloidea and Anonymidae emerged in Cotylea. Only 29% of our trees (all of them 28Sshort6 as Acotylea (Fig. 4a). With the additional removal of trees), as well as the 28Sshort6 MRE tree, place C. variegatus from the sequence collection (28Sshort5), we Chromoplanoidea as sister group to all other Cotylea, except recovered Cestoplanoidea, Chromoplanoidea, Periceloidea Cestoplanoidea. In 88% of our trees, and in both MRE trees, and Anonymidae as Cotylea once more—but only after align- Theamatidae is sister group to a clade consisting of Boninia ment curation with Gblocks (Fig. 4b). and Chromyella. In a last change, we returned to the full dataset with updated Periceloidea (Bahia et al. 2017) and thereby its only family, sequences, but also used all available sequence variations for Pericelidae, is also monophyletic in all of our phylogenetic Cycloporus variegatus and Cycloporus gabriellae,insteadof reconstructions and both MRE trees. They are most often only using one sequence per species from the same authors either sister group to all Cotylea except Cestoplanoidea and (28Sshort6, Suppl. Figs. S13–24). We now recovered Chromoplanoidea (25% and the 28Sshort6 MRE tree), or sis- Cycloporus again within Cotylea, and present the detailed ter group to Chromoplanoidea within Acotylea (25% of all results using this dataset in the following section. trees, but 100% of the 18S28Slong Gblocks trees, and the 18S28Slong MRE tree). However, in 21% of the trees, Periceloidea is placed as sister group to all Cotylea except Comparative tree topology using 28Sshort6 Cestoplanoidea, or, also in 21% of the trees, Periceloidea is and 18S28Slong matrices sister group to all Acotylea and Cestoplanoidea. All but one of our trees, and both MRE trees recover All 12 trees using the 28Sshort6 matrix (Suppl. Figs. S13–24), Chromoplana and Anonymus as clade 1 and this clade mostly and most of the 12 trees using the 18S28Slong matrix (Suppl. (58% and both MRE trees) appears as sister group to a clade Figs. S1–12) are different from each other. We have analysed including Prosthiostomoidea (with the single family the tree topologies to identify stable and unstable taxa Prosthiostomidae) and Pseudocerotoidea (consisting of (Table 2). This table also gives an overview of which tree Pseudocerotidae, Euryleptidae and clade 2, see paragraph supports which topology. Additionally, we computed extend- below). ed majority-rule consensus (MRE) trees from all 12 trees of Pseudocerotoidea and Pseudocerotidae sensu Bahia et al. the 18S28Slong matrix (Fig. 5), and all 12 trees of the 2017 are monophyletic in all but two trees, and in both MRE 28Sshort6 matrix (Fig. 6). We also calculated separate trees. Within Pseudocerotidae, all of our 28Sshort6 trees show 28Sshort6 and 18S28Slong matrix-based MREs for all align- that neither Pseudoceros,norPseudobiceros,norThysanozoon ments treated with or without Gblocks, respectively (Suppl. are monophyletic. The traditional family Euryleptidae sensu Figs. S25–28). If not otherwise stated, the MRE tree always Faubel 1984 does not appear monophyletic in any of our trees, refers to the MRE calculated from all twelve trees of each including the MRE trees. It is split into two clades (21 trees) or matrix. ‘Trees’ refers to both BI and ML trees, unless it is three clades (3 trees). In this work, we termed one of these clades preceded by ‘MRE’. Instead of citing all trees supporting a ‘clade 2’ (while retaining the name Euryleptidae for the larger particular placement of a taxonomic group, we provide this clade). The larger clade includes Cycloporus japonicus, information in Table 2 for better accessibility and overview. Cycloporus variegatus, Prostheceraeus and Maritigrella in the In the following, we focus our comparisons on already 28Sshort6 trees, while Cycloporus is lacking in the 18S28Slong defined families and superfamilies, mainly of the systems trees. Clade 2 consists of Euryleptodes galikias, Cycloporus established by Faubel (1983, 1984) and Bahia et al. (2017). gabriellae and Stylostomum ellipse in the 28Sshort6 trees, and The majority (63%) of our trees, and the 28Sshort6 MRE only Stylostomum ellipse in the 18S28Slong trees. In the three tree support Cotylea and Acotylea sensu Bahiaetal.(2017) trees, where the Euryleptidae sensu Faubel 1984 are split into and in the following we use these terms according to their three clades, even the clade still called Euryleptidae is recovered definition: in brief, Theama and Cestoplana are cotyleans in- as paraphyletic. The genera Cycloporus and Prostheceraeus are stead of acotyleans. never recovered as monophyletic in any of the 28Sshort6 trees, Cestoplanoidea (Bahia et al. 2017) and thereby its only and also Maritigrella is only recovered as monophyletic in one family, Cestoplanidae, appear monophyletic in all our trees, third of the 28Sshort6 trees. even if its position within the trees differs widely. The majority Prosthiostomoidea (Bahia et al. 2017) appears monophy- (63%) of our trees, and the 28Sshort6 MRE tree, support letic in all trees, and in all but two trees as sister group to Cestoplanoidea within (and as sister group to all other) Pseudocerotoidea. Within Prosthiostomoidea, Enchiridium Cotylea, but in the remaining trees, it is sister group to appears as sister group to a clade consisting of Acotylea (33%) or, in one case, polytomic. Prosthiostomum, Lurymare and Amakusaplana in all 594 Dittmann I.L. et al.

0.2 0.08 Chromoplana sp. Hoploplana californica 47 0.71 Anonymus ruber Clade 1 Planocera pellucida a d Stylochoidea ACOTYLEA 0.98 ML BI sensu Bahia et al. 2017 Chromoplanoidea Stylochoidea MUSCLE MUSCLE sensu Faubel 1983 without Gblocks ACOTYLEA 37 Pericelis tectivorum without Gblocks 0.99 18S28Slong Cestoplana rubrocincta 18S28Slong 19 Leptoplanoidea sensu Faubel 1983 Leptoplanoidea 26 sensu Faubel 1983

84 Clade 1 Planocera pellucida 52 Hoploplana californica 34 63 0.76 Stylochoidea 1 sensu Faubel 1983 Pseudocerotoidea Stylochoidea sensu Bahia et al. 2017 0.74 0.98 Prosthiostomoidea Pseudocerotoidea 0.74 COTYLEA 81 Chromoplanoidea Prosthiostomoidea COTYLEA 0.72 Macrostomum lignano Pericelis tectivorum Cestoplana rubrocincta 0.2 Macrostomum lignano Leptoplanoidea 0.1 b sensu Faubel 1983 Leptoplanoidea 52 ML ACOTYLEA e sensu Faubel 1983 MAFFT Q-INS-i Stylochoidea sensu Faubel 1983 BI ACOTYLEA without Gblocks 0.85 Stylochoidea MAFFT Q-INS-i Hoploplana californica 18S28Slong 67 sensu Bahia et al. 2017 without Gblocks Stylochoidea Planocera pellucida 37 18S28Slong 0.95 sensu Faubel 1983 Hoploplana californica Stylochoidea Cestoplana rubrocincta 1 sensu Bahia et al. 2017 Pericelis tectivorum Planocera pellucida COTYLEA 83 Cestoplana rubrocincta Pericelis tectivorum 56 Chromoplanoidea COTYLEA 0.99 Chromoplanoidea 56 Clade 1 0.93

80 Prosthiostomoidea 0.99 Pseudocerotoidea 93 1 Pseudocerotoidea 1 Prosthiostomoidea

Macrostomum lignano Clade 1 Macrostomum lignano Chromoplanoidea 0.2 0.09 Leptoplanoidea Chromoplanoidea c sensu Faubel 1983 f 32 Cestoplana rubrocincta ML ACOTYLEA 92 BI MAFFT E-INS-i Hoploplana californica MAFFT E-INS-i Stylochoidea without Gblocks without Gblocks 0.60 Leptoplanoidea 79 sensu Bahia et al. 2017 18S28Slong 33 18S28Slong sensu Faubel 1983 Stylochoidea sensu Faubel 1983 1 31 31 Hoploplana californica ACOTYLEA Cestoplana rubrocincta 0.62 Stylochoidea Pericelis tectivorum 0.99 1 sensu Bahia et al. 2017 Stylochoidea sensu Faubel 1983 Prosthiostomoidea COTYLEA 86 Pericelis tectivorum

Pseudocerotoidea Pseudocerotoidea

0.98

COTYLEA 0.52 Prosthiostomoidea

Anonymus ruber Clade 1 41 Clade 1 Chromoplana sp. Macrostomum lignano Macrostomum lignano Polyclad phylogeny persists to be problematic 595

ƒFig. 1 Effect of model choice and alignment on tree topology. Tree In all 18S28Slong trees, clade 5 is synonymous with reconstructions based on the 18S28Slong dataset, without using Leptoplanoidea sensu Bahia et al. (2017), as the genus Gblocks. a–c Maximum likelihood. d–f Bayesian inference used for phylogenetic reconstructions. a, d MUSCLE alignments. b, e MAFFT Pseudostylochus is not available in these datasets. In all Q-INS-i alignments. c, f MAFFT E-INS-i alignments. Node numbers 28Sshort6 trees, Pseudostylochus is part of clade 5, but indicate bootstrap support values (a–c) or posterior probabilities (d–f). Pseudostylochus is not included in the superfamily’sdefini- Acotylea and Cotylea sensu Faubel 1983 and 1984 are written in blue and tion given by Bahia et al. (2017). All of our 28Sshort6 trees red fonts, respectively. Species recovered as Acotylea or Cotylea in our trees are displayed with blue and red background, respectively. Branches show that the genus Leptoplana is monophyletic, and that and nodes are given the same colour as their respective taxon Notoplanidae as a whole and also its genera Notoplana and Notocomplana are not monophyletic. In 58% of the 28Sshort6 trees and also the corresponding 28Sshort6 trees. Prosthiostomum appears polyphyletic in 83% MRE tree, clade 6 is monophyletic, appears as sister group to of our 28Sshort trees and also the 28Sshort MRE tree, as clade 5 and includes Discocelis, Adenoplana, Ilyella, Amakusaplana and Lurymare cluster within. Phaenocelis and Amemiyaia. In our 18S28Slong trees and Leptoplanoidea sensu Faubel 1983 (in whose definition the corresponding MRE tree, clade 6 is represented only by Hoploplana and Theama are included) is not supported in Discocelis and always recovered as the sister group of clade 5. any of our trees. We have termed Leptoplanoidea sensu Clade 4 can be subdivided into two clades, clades 7 + 8 and Faubel 1983, but without Hoploplana and Theama,clade3 their sister group Callioplana.Thistopologyissupportedby (supported by all trees), which we further subdivided into two two of twelve 28Sshort6 trees, as well as the respective MRE clades (clades 5 and 6). tree, while in ten 28Sshort6 trees, either a polytomy exists

Clade 1 Chromoplana sp. 0.98 Anonymus ruber b 28Sshort3 a 1Anonymus virilis Euryleptodes galikias MUSCLE BI 1 Cycloporus gabriellae Clade 2 28Sshort1 1 Stylostomum ellipse Cycloporus variegatus without Gblocks 1 Maritigrella fuscopunctata MUSCLE BI 1 0.92 Maritigrella newmanae Cycloporus variegatus Prostheceraeus roseus Euryleptidae Chromoplana sp. 0.91 without Gblocks 1Maritigrella crozieri 2 1Anonymus ruber Clade 1 0.97 0.67Maritigrella crozieri 3 Anonymus virilis 1 1 Maritigrella crozieri 1 Callioplana marginata Prostheceraeus vittatus 2 1Leptoplana sp. 0.98 Pseudoceros velutinus 0.51 Leptoplana tremellaris 1 Pseudoceros astrorum 0.82 1Notoplana australis 1 1 Pseudoceros rawlinsonae Notoplana australis 2 Pseudoceros velutinus Leptoplana tremellaris 2 1 1Pseudoceros stimpsoni 0.98 Notocomplana sp. Pseudoceros nipponicus 0.69 Notocomplana japonica 1 1 1Notocomplana koreana 0.99 Pseudoceros bimarginatus 0.63 1 Notocomplana humilis Pseudoceros bicolor marcusorum 1Pseudostylochus obscurus 0.53 Pseudoceros cf bicolor Pseudostylochus sp. 0.85 0.55 Pseudoceros contrarius 0.92 Notoplana delicata Pseudoceros bicolor 0.93 Armatoplana leptalea Clade 5 Pseudoceros jebborum Melloplana ferruginea Notoplana sp. Pseudoceros periaurantius 1 Pseudobiceros flowersi 1 1 1Echinoplana celerrima 2 Yungia sp. Clade 3 Echinoplana celerrima 1 1Pseudoceros cf maximus 1 Comoplana agilis 1 Adenoplana evelinae 1 1 Pseudobiceros sp. Pseudocerotidae 0.9 1 Discocelis tigrina 0.59 Pseudobiceros caribbensis Ilyella gigas Clade 6 0.96Thysanozoon brocchii 1 0.95 Amemiyaia pacifica 0.99 Pseudobiceros splendidus 1 Phaenocelis medvedica 0.91 1 0.94 Pseudobiceros evelinae 1 Hoploplana villosa Pseudobiceros hancockanus 0.64 Hoploplana californica Thysanozoon alagoensis 0.89 Hoploplana divae Thysanozoon brocchii 2 Planocera pellucida 0.94 1 1 Thysanozoon raphaeli 0.97 1 Paraplanocera sp. Pseudocerotoidea 1 0.91 Planocera multitentaculata 1 1 Maiazoon orsaki Idioplana australiensis Pseudobiceros nigromarginatus Clade 4 Leptostylochus gracilis 1 Pseudobiceros bedfordi 0.58 Stylochus ellipticus 1 Pseudobiceros wirtzi Stylochus sp. Pseudobiceros pardalis 1 Clade 8 1Imogine stellae 1 Monobiceros langi Stylochus oculiferus Phrikoceros mopsus 0.71 0.78 0.56 Stylochus zebra Pseudoceros harrisi 0.74 1Imogine refertus Prosthiostomum grande 1Paraplanocera oligoglena Prosthiostomum siphunculus 2 Stylochus ijimai 1 1 1 Prosthiostomum siphunculus 3 1 1 Cestoplana rubrocincta 2 0.74 1 Cestoplana rubrocincta 1 Prosthiostomum siphunculus 1 Cestoplana salar Cestoplanoidea 1 Lurymare katoi Prosthiostomoidea 0.9 0.58 Cestoplana techa Acotylea 1 Amakusaplana acroporae 0.94 Pericelis cata 0.54 Prosthiostomum vulgaris 1 Pericelis orbicularis 1Enchiridium evelinae 1 Pericelis tectivorum Periceloidea 1 Enchiridium sp. 1 0.68 Chromyella sp. 0.84 Enchiridium sp. 2 1 Boninia divae Chromyella sp. 0.53 1 Theama mediterranea Chromoplanoidea 0.99 1 Boninia divae Theama sp. 1 Theama mediterranea Chromoplanoidea 1Enchiridium evelinae Theama sp. 1 Enchiridium sp. 2 Enchiridium sp. 1 1 Pericelis cata 0.97 Prosthiostomum grande 1 Pericelis orbicularis Pericelis tectivorum Periceloidea 1 1Prosthiostomum siphunculus 2 Cestoplana rubrocincta 2 0.86 Prosthiostomum siphunculus 3 Prosthiostomoidea 1 0.94 Prosthiostomum siphunculus 1 1 Cestoplana rubrocincta 1 Cestoplanoidea Cotylea Lurymare katoi 1 Cestoplana salar Amakusaplana acroporae Cestoplana techa 1 Prosthiostomum vulgaris Callioplana marginata Euryleptodes galikias 1 1 Phaenocelis medvedica 1 Cycloporus gabriellae Clade 2 0.93 Amemiyaia pacifica 1 Stylostomum ellipse Ilyella gigas Acotylea Pseudoceros velutinus 1 Clade 6 Pseudoceros astrorum 0.93 Adenoplana evelinae Pseudoceros rawlinsonae 0.85 Discocelis tigrina 1 Pseudoceros velutinus 2 1 Leptoplana sp. 0.61 1 1Pseudoceros stimpsoni 0.96 Leptoplana tremellaris 1 0.99 Pseudoceros bicolor 1Notoplana australis 1 1 1 Pseudoceros bicolor marcusorum Notoplana australis 2 0.7 Pseudoceros cf bicolor Clade 3 0.92 Notocomplana sp. Pseudoceros contrarius 0.94 Notocomplana japonica 0.99 Pseudoceros sp. Notocomplana koreana 0.99 Pseudoceros bimarginatus 1 Notocomplana humilis Pseudoceros jebborum 0.57 Leptoplana tremellaris 2 0.94 0.65 Pseudoceros periaurantius 0.97 Armatoplana leptalea 0.82 Pseudobiceros flowersi Notoplana sp. Clade 5 0.81 Maiazoon orsaki Melloplana ferruginea 0.99 1Pseudobiceros nigromarginatus Pseudocerotidae Pseudostylochus obscurus 1Pseudobiceros bedfordi 1Pseudostylochus sp. 0.98 Pseudobiceros pardalis Echinoplana celerrima 2 0.76 Pseudobiceros wirtzi 1 0.99 Echinoplana celerrima 1 Yungia sp. 0.54 0.53 1Pseudoceros cf maximus Notoplana delicata 0.85 Pseudobiceros caribbensis Comoplana agilis 0.89 Pseudobiceros sp. Hoploplana villosa 0.99 Pseudobiceros evelinae 1 Hoploplana californica 1Pseudobiceros splendidus 1 Hoploplana divae 0.99 0.97Thysanozoon brocchii 1 0.87 Idioplana australiensis Pseudobiceros hancockanus Leptostylochus gracilis 1 1Thysanozoon brocchii 2 1 Thysanozoon raphaeli 0.97 Stylochus ellipticus 0.85 Thysanozoon alagoensis 0.99 Imogine stellae Monobiceros langi Clade 4 1 Stylochus oculiferus 1Phrikoceros mopsus 1 Stylochus sp. Pseudoceros harrisi Cotylea 0.9 1 0.63 Imogine refertus Clade 8 Maritigrella fuscopunctata Paraplanocera oligoglena 1 0.52 Maritigrella newmanae 0.97 Stylochus ijimai 0.98 Prostheceraeus roseus 0.69 Stylochus zebra Prostheceraeus vittatus 0.71 1 Euryleptidae Planocera pellucida 1Maritigrella crozieri 2 Paraplanocera sp. 0.77 Maritigrella crozieri 3 1 1 Planocera multitentaculata 0.1 Maritigrella crozieri 1 0.09 Macrostomum lignano Macrostomum lignano Fig. 2 Result of using versions 1 and 2 of sequences provided by Bahia 2in(b). Node numbers indicate posterior probabilities. Acotylea and et al. (2017). Bayesian inference tree reconstructions based on the Cotylea sensu Faubel 1983 and 1984 are written in blue and red fonts, 28Sshort1 (a) and 28Sshort3 (b) datasets, using MUSCLE alignments respectively. Species recovered as Acotylea or Cotylea in our trees are without Gblocks curation. The same taxa are used in (a)and(b), but with displayed with blue and red background, respectively. Branches and version 1 of sequences provided by Bahia et al. (2017)in(a) and version nodes are given the same colour as their respective taxon 596 Dittmann I.L. et al.

Chromoplana sp. Xenoprorhynchus sp. Callioplana marginata Chromoplana sp. 0.58 1Leptoplana sp. 0.79 Anonymus ruber Leptoplana tremellaris 1 1Anonymus virilis 0.96 1Notoplana australis 1 Callioplana marginata Notoplana australis 2 Leptoplana sp. Leptoplana tremellaris 2 a 0.79 1 Leptoplana tremellaris 1 b 0.65 0.87 Melloplana ferruginea Notoplana australis 1 1 Notoplana sp. 1Notoplana australis 2 1Echinoplana celerrima 2 Melloplana ferruginea 0.56 Echinoplana celerrima 1 Notoplana sp. Notoplana delicata 0.53 0.74 0.99 Notocomplana sp. 28Sshort2XM 1 Echinoplana celerrima 2 28Sshort2X 1 Notocomplana japonica 1 Echinoplana celerrima 1 0.99 1 Notocomplana koreana 0.9 Notocomplana sp. 0.99 Notocomplana humilis MUSCLE BI 0.56 Notocomplana japonica MUSCLE BI Pseudostylochus obscurus 1Notocomplana koreana 1 1Pseudostylochus sp. 0.98 Notocomplana humilis Armatoplana leptalea with Gblocks 0.95 1 Pseudostylochus obscurus with Gblocks Comoplana agilis 0.73 Pseudostylochus sp. 1 Adenoplana evelinae 1 0.99 Notoplana delicata 1 1Discocelis tigrina outgroups: Armatoplana leptalea Ilyella gigas Leptoplana tremellaris 2 outgroup: 0.97 Comoplana agilis 1 Amemiyaia pacifica 1 Phaenocelis medvedica Xenoprorhynchus sp. 0.58 1 Adenoplana evelinae Xenoprorhynchus sp. 0.53 0.51 Hoploplana villosa 1 Discocelis tigrina 1 Hoploplana divae Ilyella gigas 0.78 0.86 Hoploplana californica Macrostomum lignano 0.86 1 Amemiyaia pacifica Idioplana australiensis Phaenocelis medvedica 1 Planocera pellucida Hoploplana villosa 0.99 0.991 Paraplanocera sp. 1 Hoploplana californica Planocera multitentaculata 0.91 Hoploplana divae 0.64 Leptostylochus gracilis 1 Planocera pellucida 0.99 Stylochus ellipticus 0.64 0.93 Paraplanocera sp. 1 Stylochus sp. Planocera multitentaculata 0.99 1Imogine stellae Idioplana australiensis 0.87 Stylochus oculiferus Leptostylochus gracilis 0.55 Stylochus zebra 0.56 0.61 Stylochus ellipticus 0.90 Imogine refertus 1 Stylochus sp. 0.68 1 Paraplanocera oligoglena Imogine stellae 1 Stylochus ijimai 1 Stylochus oculiferus Cestoplana rubrocincta 2 0.53 Stylochus zebra 1 0.65 0.64 Cestoplana rubrocincta 1 0.61 Imogine refertus 0.95 0.61 Cestoplana salar 1 1Paraplanocera oligoglena Cestoplana techa Stylochus ijimai 0.52 Chromyella sp. 1 Cestoplana rubrocincta 2 1 Boninia divae 0.59 Cestoplana rubrocincta 1 1Theama mediterranea Cestoplana salar Theama sp. 0.89 0.58 Cestoplana techa 0.99 Pericelis cata 1 0.56 Chromyella sp. Pericelis cata 2 Boninia divae 1 Pericelis orbicularis 1 Theama mediterranea 1 Pericelis byerleyana Acotylea 1 1 Theama sp. 1Pericelis tectivorum Pericelis cata1 Anonymus ruber 0.92 Pericelis cata 2 1Anonymus virilis 1 Pericelis orbicularis 1Enchiridium evelinae 1 Pericelis byerleyana 0.73 0.99 Enchiridium sp. 2 1Pericelis tectivorum Enchiridium sp. 1 Enchiridium evelinae 0.99 Prosthiostomum grande 0.99 0.99 1 Prosthiostomum siphunculus 2 Enchiridium sp. 2 1 Enchiridium sp. 1 0.91 Prosthiostomum siphunculus 3 0.99 Prosthiostomum grande 0.98 Prosthiostomum siphunculus 1 Prosthiostomum siphunculus 2 Lurymare katoi 1 1 1 0.91 Prosthiostomum siphunculus 3 Amakusaplana acroporae 0.79 Prosthiostomum siphunculus 1 Prosthiostomum vulgaris Lurymare katoi Acotylea 0.80 Cycloporus japonicus 1 Amakusaplana acroporae Cycloporus variegatus 1 Prosthiostomum vulgaris 0.75 Maritigrella fuscopunctata 0.66 Pseudoceros velutinus 2 0.91 Maritigrella newmanae Pseudoceros astrorum 0.89 Prostheceraeus roseus Prostheceraeus vittatus 1Pseudoceros rawlinsonae 0.89 0.59 Pseudoceros rawlinsonae 2 1Maritigrella crozieri 3 1 Pseudoceros velutinus 1 0.76 0.99 Maritigrella crozieri 2 1Pseudoceros stimpsoni 0.73 Maritigrella crozieri 1 Pseudoceros velutinus 2 1 Pseudoceros bicolor 1 0.53 Pseudoceros bicolor 2 Pseudoceros astrorum 0.99 Pseudoceros bicolor marcusorum Pseudoceros rawlinsonae 1 0.78 1 1Pseudoceros rawlinsonae 2 0.76 Pseudoceros cf bicolor 1 Pseudoceros velutinus 1 0.98 Pseudoceros contrarius 1Pseudoceros stimpsoni 0.99 Pseudoceros nipponicus. Pseudoceros bicolor 1 Pseudoceros bimarginatus 1 0.99Pseudoceros bicolor 2 Pseudoceros jebborum 1 Pseudoceros bicolor marcusorum 0.98 Pseudoceros periaurantius 0.66 Pseudoceros cf bicolor 0.76 Pseudobiceros flowersi Pseudoceros contrarius Maiazoon orsaki 0.96 0.99 1 Pseudobiceros nigromarginatus 0.99 Pseudoceros nipponicus Monobiceros langi 0.80 Pseudoceros bimarginatus 1Phrikoceros mopsus Pseudoceros jebborum 0.57 0.65 Pseudoceros periaurantius 1Thysanozoon brocchii Pseudobiceros flowersi 1 0.94 Thysanozoon raphaeli 0.86 Yungia sp. 0.97 Maiazoon orsaki Thysanozoon alagoensis 1 Pseudoceros nigromarginatus 0.98 0.5 Pseudoceros cf maximus Yungia sp. 0.83 Pseudobiceros caribbensis Thysanozoon alagoensis 1 0.88 59 Thysanozoon brocchii 2 0.8 Pseudobiceros sp. 1Thysanozoon raphaeli Pseudobiceros evelinae 0. 0.74 Pseudobiceros evelinae 0.59 1 0.76 Pseudobiceros splendidus 1Pseudobiceros splendidus 0.71 Thysanozoon brocchii Pseudobiceros hancockanus 0.56 Thysanozoon brocchii 1 Pseudobiceros bedfordi 0.97 0.97 Pseudoceros cf maximus 0.93 0.92 Pseudobiceros wirtzi 1 Pseudobiceros caribbensis 1Pseudobiceros pardalis 1 Pseudobiceros sp. 0.99 52 Pseudobiceros hancockanus Pseudobiceros pardalis 2 0.97 Pseudobiceros bedfordi Pseudoceros harrisi 0. 1 Pseudobiceros pardalis 1 Euryleptodes galikias 1 Pseudobiceros wirtzi 1 Cycloporus gabriellae Cotylea Pseudobiceros pardalis 2 0.99 Stylostomum ellipse 0.88 Monobiceros langi Cycloporus japonicus 1Phrikoceros mopsus Cycloporus variegatus Pseudoceros harrisi Maritigrella fuscopunctata Euryleptodes galikias 0.08 Maritigrella newmanae 1 1Cycloporus gabriellae 0.86 0.81 Prostheceraeus roseus Stylostomum ellipse Cotylea Prostheceraeus vittatus Xenoprorhynchus sp. 0.87 Maritigrella crozieri 2 0.94 0.98 Maritigrella crozieri 3 0.68 Maritigrella crozieri 1 0.1 0.97 1Enchiridium evelinae Macrostomum lignano Enchiridium sp. 2 Enchiridium sp. 1 0.95 Prosthiostomum grande Cycloporus variegatus 1Prosthiostomum siphunculus 2 Chromoplana sp. 1 Prosthiostomum siphunculus 3 0.81 Anonymus ruber 0.92 Prosthiostomum siphunculus 1 1Anonymus virilis 0.95 Lurymare katoi Callioplana marginata Amakusaplana acroporae 1Leptoplana sp. 1 Prosthiostomum vulgaris 0.81 0.6 Leptoplana tremellaris 1 Chromoplana sp. Leptoplana tremellaris 2 0.93 Notoplana australis 1 1Anonymus ruber 1Notoplana australis 2 0.52 Anonymus virilis Melloplana ferruginea Cycloporus japonicus 0.77 Notoplana sp. Cycloporus variegatus 1 Echinoplana celerrima 2 0.72 Maritigrella fuscopunctata 1Echinoplana celerrima 1 1 Maritigrella newmanae 0.98 Notocomplana sp. Prostheceraeus roseus Notocomplana japonica 0.74 Prostheceraeus vittatus 0.88 1Notocomplana koreana 0.55 1 Maritigrella crozieri 2 d Notocomplana humilis 1 0.99 0.99 Maritigrella crozieri 3 1 Pseudostylochus obscurus 0.9 Maritigrella crozieri 1 Pseudostylochus sp. Pseudoceros velutinus 2 1 Notoplana delicata 0.65 Pseudoceros astrorum Armatoplana leptalea Pseudoceros rawlinsonae 1 28Sshort2M 1 Comoplana agilis 1Pseudoceros rawlinsonae 2 0.99 Adenoplana evelinae Pseudoceros velutinus 1 1 Discocelis tigrina 1 1Pseudoceros stimpsoni MUSCLE BI 1 Ilyella gigas Amemiyaia pacifica 0.99 Pseudoceros bicolor 1 1 Phaenocelis medvedica 1 Pseudoceros bicolor 2 Hoploplana villosa 1 Pseudoceros bicolor marcusorum without Gblocks 1 Hoploplana californica 0.69 Pseudoceros cf bicolor 0.59 0.71 Hoploplana divae 0.98 Pseudoceros contrarius outgroup: Idioplana australiensis Pseudoceros nipponicus Planocera pellucida 0.99 0.52 1 0.75 Pseudoceros bimarginatus 1 0.99 Paraplanocera sp. Pseudoceros jebborum Macrostomum lignano 0.59 Planocera multitentaculata Pseudoceros periaurantius Leptostylochus gracilis Pseudobiceros flowersi Stylochus ellipticus 1 0.81 Maiazoon orsaki 1 Stylochus sp. 0.87 1Pseudobiceros nigromarginatus 0.99 Imogine stellae Pseudobiceros bedfordi 0.51 Stylochus oculiferus Stylochus zebra 0.99 1 0.61 Pseudobiceros pardalis 1 0.65 Imogine refertus Pseudobiceros pardalis 2 Paraplanocera oligoglena 0.8 0.95 Pseudobiceros wirtzi 1 1 Stylochus ijimai Yungia sp. Cestoplana rubrocincta 2 Pseudoceros cf maximus Cestoplana rubrocincta 1 1 Pseudobiceros caribbensis 0.61 1Cestoplana salar 0.84 Pseudobiceros sp. Cestoplana techa 0.99 0.81 0.89 Pseudobiceros evelinae 0.96 Chromyella sp. 1 Pseudobiceros splendidus 1 1 Boninia divae 0.98 Thysanozoon brocchii 1 1 Theama mediterranea 1 Pseudobiceros hancockanus Theama sp. Thysanozoon alagoensis 0.99 Pericelis cata 1 0.98 Thysanozoon brocchii 2 Pericelis cata 2 1 Thysanozoon raphaeli 1 1 Pericelis orbicularis 1 Pericelis tectivorum 0.66 Monobiceros langi Pericelis byerleyana 1 Phrikoceros mopsus 1Enchiridium evelinae Pseudoceros harrisi 1 Enchiridium sp. 2 Euryleptodes galikias Cotylea Enchiridium sp. 1 1 Cycloporus gabriellae 1 Prosthiostomum grande 0.98 Stylostomum ellipse 1 1 Prosthiostomum siphunculus 2 Callioplana marginata 0.92 Prosthiostomum siphunculus 3 Leptoplana sp. 0.99 Prosthiostomum siphunculus 1 1 Lurymare katoi 0.79 0.51 Leptoplana tremellaris 1 Amakusaplana acroporae Acotylea Leptoplana tremellaris 2 1 0.5 Prosthiostomum vulgaris 1Notoplana australis 1 Pseudoceros velutinus 2 Notoplana australis 2 Pseudoceros astrorum 0.98 Notocomplana sp. Pseudoceros rawlinsonae 1 0.67 Notocomplana japonica 1Pseudoceros rawlinsonae 2 1 Notocomplana koreana Pseudoceros velutinus 1 Notocomplana humilis 1 1 0.83 1 0.57 0.99 Pseudoceros stimpsoni 1 Pseudostylochus obscurus 1 Pseudoceros bicolor 1 c 0.8 Pseudostylochus sp. 1 0.98 Pseudoceros bicolor 2 Notoplana delicata Pseudoceros bicolor marcusorum 0.91 Armatoplana leptalea 0.71 Pseudoceros cf bicolor Melloplana ferruginea Pseudoceros contrarius 0.99 Notoplana sp. 0.98 Pseudoceros nipponicus 28Sshort2M 1 0.99 Pseudoceros bimarginatus 1 1Echinoplana celerrima 2 Pseudoceros jebborum Echinoplana celerrima 1 0.99 Pseudoceros periaurantius MUSCLE BI Comoplana agilis Pseudobiceros flowersi 1 Adenoplana evelinae 0.76 Maiazoon orsaki 1 1Discocelis tigrina 1Pseudobiceros nigromarginatus with Gblocks Ilyella gigas Monobiceros langi Amemiyaia pacifica 1Phrikoceros mopsus 0.99 1 Phaenocelis medvedica Yungia sp. outgroup: Hoploplana villosa 0.56 Thysanozoon alagoensis 1 Hoploplana californica 0.53 1 Pseudoceros cf maximus Macrostomum lignano 0.58 0.74 Hoploplana divae 0.88 Pseudobiceros caribbensis 1 Idioplana australiensis 0.99 0.56 Pseudobiceros sp. Planocera pellucida 1 Pseudobiceros evelinae 0.55 1 Paraplanocera sp. 1 Pseudobiceros splendidus Planocera multitentaculata 0.55 Thysanozoon brocchii 1 0.99 Pseudobiceros hancockanus Leptostylochus gracilis Thysanozoon brocchii 2 0.55 Stylochus ellipticus 1Thysanozoon raphaeli 1 Stylochus sp. Pseudobiceros bedfordi Imogine stellae 0.97 0.91 Pseudobiceros wirtzi 0.79 1 Stylochus oculiferus 0.59 1Pseudobiceros pardalis 1 Stylochus zebra Pseudobiceros pardalis 2 0.85 Imogine refertus Pseudoceros harrisi 0.51 1 Paraplanocera oligoglena Eurylepodes galikias Cotylea 1Stylochus ijimai 1 Cycloporus gabriellae Cestoplana_rubrocincta 1 0.77 Stylostomum ellipse Cestoplana rubrocincta 2 Cycloporus japonicus 0.53 1 Cestoplana salar Maritigrella fuscopunctata Cestoplana techa 1 0.6 Maritigrella newmanae 0.99 Chromyella sp. Prostheceraeus roseus Boninia divae 0.9 Prostheceraeus vittatus 1 1 Maritigrella crozieri 2 1 Theama mediterranea 1 Maritigrella crozieri 3 Theama sp. 0.89 Maritigrella crozieri 1 Pericelis cata 1 Acotylea Macrostomum lignano 1 Pericelis cata 2 1 1 Pericelis orbicularis 1 Pericelis tectivorum Pericelis byerleyana 0.09 Macrostomum lignano 0.1 Polyclad phylogeny persists to be problematic 597

ƒFig. 3 Effect of outgroup selection and alignment curation on tree MRE tree) as sister group to Planocera pellucida. In two cases, topology. a–d Bayesian inference tree reconstructions based on the Planocera pellucida is clustering within clade 8, while in one 28Sshort2 datasets, using MUSCLE alignments with (a–c) and without (d) Gblocks. a 28Sshort2XM dataset including both Xenoprorhynchus case, it is unresolved as a polytomy. sp. and Macrostomum lignano as outgroups. b 28Sshort2X dataset only Clade 8 resembles Stylochoidea sensu Faubel (1983)and including Xenoprorhynchus sp. as outgroup. c, d 28Sshort2M dataset in the 28Sshort6 dataset, includes Leptostylochus, Stylochus, only including Macrostomum lignano as outgroup. Node numbers Imogine, Paraplanocera and Planocera, but excludes indicate posterior probabilities. Acotylea and Cotylea sensu Faubel ’ 1983 and 1984 are written in blue and red fonts, respectively. Species Faubel s Callioplana, Idioplana and Pseudostylochus. This recovered as Acotylea or Cotylea in our trees are displayed with blue and clade is monophyletic in a minority (two of twelve) red background, respectively. Branches and nodes are given the same 28Sshort6 trees and in the 28Sshort6 MRE tree. Clade 8 is colour as their respective taxon polytomic in seven 28Sshort6 reconstructions and paraphyletic (including Idioplana and/or Hoploplana)inthe between Callioplana, clade 3 and clades 7 + 8, or clade 7 or 8 remaining three 28Sshort6 trees. In the 18S28Slong dataset, are not monophyletic, or several of these cases together. clade 8 is represented by Stylochus, Paraplanocera, Imogine In half of the 28Sshort6 trees, andalsointhecorresponding and Planocera and thus conforming to Faubel’s(1983)defi- MRE tree, clade 7 is formed by Hoploplana clustering as sister nition. Clade 8 is supported by only two of twelve of the group to Idioplana, but in one third of 28Sshort6 trees, 18S28Slong trees as well, but not in the 18S28Slong MRE Hoploplana is sister group to Planoceridae in clade 8. In our tree, where Hoploplana is sister group to Planocera. 18S28Slong trees, clade 7 is only represented by Hoploplana In 75% of our 28Sshort6 trees and also the corresponding californica, in nine of twelve trees (and also in the 18S28Sshort MRE tree, Planocera is not monophyletic as Paraplanocera

Callioplana marginata a b 1Leptoplana sp. 0.8 Leptoplana tremellaris 1 0.86 Leptoplana tremellaris 2 Cycloporus variegatus 28Sshort5 Notoplana australis 1 Callioplana marginata 1Notoplana australis 2 0.61 1Leptoplana sp. Melloplana ferruginea Leptoplana tremellaris 1 MUSCLE BI 0.7 28Sshort4 0.81 Leptoplana tremellaris 2 1 Notoplana sp. Notoplana australis 1 with Gblocks 1Echinoplana celerrima 2 1Notoplana australis 2 Echinoplana celerrima 1 MUSCLE BI Melloplana ferruginea 0.73 0.98 Notocomplana sp. Clade 5 0.67 0.84 Notoplana sp. without Cycloporus variegatus Notoplana japonica with Gblocks Echinoplana celerrima 2 Notoplana koreana 1 1Echinoplana celerrima 1 1 1 Notocomplana humilis Notoplana delicata 0.99 Pseudostylochus obscurus with Cycloporus variegatus 0.94 Armatoplana leptalea 1Pseudostylochus sp. Notocomplana sp. 1 Notoplana delicata 0.98 Notocomplana japonica Armatoplana leptalea 1 1Notoplana koreana 1 Comoplana agilis 1 0.98 Notocomplana humilis 1 Adenoplana evelinae 1 Pseudostylochus obscurus Clade 3 1 Discocelis tigrina Pseudostylochus sp. Ilyella gigas Clade 6 1 Comoplana agilis 0.97 Amemiyaia pacifica 1 Adenoplana evelinae 1 Phaenocelis medvedica 1 Discocelis tigrina Hoploplana villosa Ilyella gigas 1 0.97 Amemiyaia pacifica Hoploplana californica Clade 7 1 0.79 0.58 Hoploplana divae Phaenocelis medvedica Idioplana australiensis 1 Hoploplana villosa 0.94 Planoceram pellucida Hoploplana californica 1 0.97 0.99 Hoploplana divae 1Paraplanocera sp. 0.74 Planocera pellucida Clade 4 0.62 Planocera multitentaculata 0.64 Paraplanocera sp. Leptostylochus gracilis 0.99 Planocera multitentaculata 0.94 Stylochus ellipticus Idioplana australiensis 1 Stylochus sp. Clade 8 Leptostylochus gracilis Imogine stellae 0.59 1 0.73 Stylochus ellipticus Acotylea 0.6 Stylochus oculiferus 1 Stylochus sp. Stylochus zebra 0.99 Imogine stellae 0.62 Imogine refertus 0.52 Stylochus oculiferus 1 Paraplanocera oligoglena Stylochus zebra Stylochus ijimai 0.59 0.54 1Imogine refertus 0.94 Paraplanocera oligoglena 1Anonymus ruber 1 Stylochus ijimai Anonymus virilis Cestoplana rubrocincta 2 Pericelis cata 1 1 Pericelis orbicularis 0.95 Cestoplana rubrocincta 1 Cotylea Pericelis tectivorum 1 0.63 Cestoplana salar Cestoplanoidea 0.97 0.6 Cestoplana techa Maritigrella fuscopunctata Chromyella sp. 0.76 Maritigrella newmanae 0.61 Prostheceraeus roseus 1 Theama mediterranea 0.99 1 Theama sp. Chromoplanoidea 0.98 Prostheceraeus vittatus Boninia divae 1 Maritigrella crozieri 3 Anonymus ruber 1 Maritigrella crozieri 2 1Anonymus virilis Clade 1 0.69 Maritigrella crozieri 1 0.62 0.89 Pericelis cata Pseudoceros velutinus 2 1 Pericelis orbicularis Periceloidea Pseudoceros astrorum 1 Pericelis tectivorum 1 0.77 Pseudoceros rawlinsonae 1Enchiridium evelinae 1 Pseudoceros velutinus 1 1 Enchiridium sp. 2 1Pseudoceros stimpsoni Enchiridium sp. 1 0.95 Pseudoceros bicolor 1 Prosthiostomum grande 1 Pseudoceros bicolor marcusorum 1 Prosthiostomum siphunculus 2 0.96 1 Prosthiostomum siphunculus 3 Prosthiostomoidea 0.75 Pseudoceros cf bicolor 0.9 Prosthiostomum siphunculus 1 0.99 Pseudoceros contrarius 0.91 Lurymare katoi 0.99 Pseudoceros nipponicus 0.51 Prosthiostomum vulgaris Acotylea Pseudocerotoidea Pseudoceros bimarginatus 1 Amakusaplana acroporae 0.92 Pseudoceros jebborum 0.52 Pseudoceros velutinus 2 Pseudoceros periaurantius Pseudoceros astrorum 0.54 Pseudobiceros flowersi Pseudoceros rawlinsonae Maiazoon orsaki Pseudoceros velutinus 1 1 Pseudoceros nigromarginatus 1 1Pseudoceros stimpsoni 0.94 Yungia sp. Pseudoceros bicolor Thysanozoon brocchii 2 1 1 Pseudoceros bicolor marcusorum 1Thysanozoon raphaeli 0.99 0.67 Pseudoceros cf bicolor 0.73 Pseudobiceros evelinae 0.6 0.92 Pseudoceros contrarius 1 Pseudobiceros splendidus 0.99 Pseudoceros nipponicus 0.93 0.7 Thysanozoon brocchii 1 Pseudoceros bimarginatus Pseudoceros cf maximus Pseudoceros jebborum 0.94 1 Pseudobiceros caribbensis Pseudoceros periaurantius Pseudobiceros flowersi 0.82 0.89 Pseudobiceros sp. 0.85 Pseudocerotidae 0.8 Pseudobiceros hancockanus Maiazoon orsaki 1 Thysanozoon alagoensis 1 Pseudoceros nigromarginatus Pseudobiceros bedfordi 0.99 Yungia sp. 0.67 Thysanozoon brocchii 2 Pseudobiceros wirtzi 1 Thysanozoon raphaeli 1 Pseudobiceros pardalis 0.96 0.76 Pseudobiceros evelinae 0.76 Monobiceros langi 0.56 1 Pseudobiceros splendidus 1 Phrikoceros mopsus Thysanozoon brocchii 1 Pseudoceros harrisi 0.65 Pseudoceros cf maximus Euryleptodes galikias 0.81 1 1 0.58 0.99 Pseudobiceros caribbensis Cycloporus gabriellae 0.87 Pseudobiceros sp. 0.96 Stylostomum ellipse Pseudobiceros hancockanus Enchiridium evelinae 1 Thysanozoon alagoensis 0.99 1 Enchiridium sp. 2 Pseudobiceros bedfordi 0.77 Enchiridium sp. 1 0.87Pseudobiceros wirtzi 0.66 0.99 Prosthiostomum grande 1Pseudobiceros pardalis 1 Prosthiostomum siphunculus 2 0.77 Monobiceros langi 1 1Phrikoceros mopsus 0.88 Prosthiostomum siphunculus 3 Pseudoceros harrisi 0.59 Prosthiostomum siphunculus 1 Euryleptodes galikias 0.97 Lurymare katoi 1 Prosthiostomum vulgaris 1 Cycloporus gabriellae Clade 2 0.96 Amakusaplana acroporae Stylostomum ellipse 0.93 Maritigrella fuscopunctata Chromyella sp. Maritigrella newmanae 0.99 Boninia divae 0.99 0.94 Prostheceraeus roseus 1 Theama mediterranea Prostheceraeus vittatus Euryleptidae Theama sp. 0.93 1 Maritigrella crozieri 2 Cestoplana rubrocincta 2 1 Maritigrella crozieri 3 Cestoplana rubrocincta 1 0.75 Maritigrella crozieri 1 Cotylea 1Cestoplana salar Macrostomum lignano Cestoplana techa 0.08 Macrostomum lignano 0.08 Fig. 4 Effect of taxon sampling on tree topology. a, b Bayesian inference sensu Faubel 1983 and 1984 are written in blue and red fonts, tree reconstructions based on the 28Sshort4 dataset including Cycloporus respectively. Species recovered as Acotylea or Cotylea in our trees are variegatus (a) and the 28Sshort5 dataset without Cycloporus variegatus displayed with blue and red background, respectively. Branches and (b), using MUSCLE alignments and Gblocks for alignment curation. nodes are given the same colour as their respective taxon Node numbers indicate posterior probabilities. Acotylea and Cotylea 598 Dittmann I.L. et al.

Stylostomum ellipse Clade 2 18S28Slong 75 Pseudoceros bimarginatus MRE 100 Pseudocerotidae Pseudobiceros flowersi 100 with and without Gblocks 100 Pseudocerotoidea Pseudobiceros hancockanus Maritigrella crozieri 100 Euryleptidae 100 Prostheceraeus vittatus Prosthiostomum siphunculus 1 50 100 Prosthiostomoidea Prosthiostomum siphunculus 2 Anonymus ruber 100 Clade 1 Cotylea Chromoplana sp. Cestoplana rubrocincta Cestoplanoidea Stylochoidea Stylochus ellipticus sensu Faubel 1983 100 Paraplanocera oligoglena 50 50 42 Imogine stellae 100 Stylochoidea 67 sensu Bahia et al. 2017 Stylochus zebra Hoploplana californica 75 100 Planocera pellucida Discocelis tigrina

50 100 Comoplana agilis Leptoplanoidea 100 Echinoplana celerrima sensu Faubel 1983 Leptoplanoidea 100 Leptoplana tremellaris sensu Bahia et al. 2017 100 Notoplana australis Pericelis tectivorum Periceloidea Theama mediterranea 50 100 Theama sp. 100 Chromoplanoidea Boninia divae 100 Acotylea Chromyella sp. Macrostomum lignano Fig. 5 Extended majority-rule consensus tree based on all 12 trees of the or Cotylea in our trees are displayed with blue and red background, 18S28Slong dataset shown in Suppl. Figs. S1–12. Numbers indicate per- respectively. Branches and nodes are given the same colour as their re- centage of support. Acotylea and Cotylea sensu Faubel 1983 and 1984 are spective taxon written in blue and red fonts, respectively. Species recovered as Acotylea sp. clusters within. Similarly, Paraplanocera is not monophy- Alignment is important letic in any of our 28Sshort6 trees, and Planoceridae sensu Faubel 1983 is not recovered as monophyletic in any tree. In MUSCLE (Edgar 2004) was the alignment method of choice in the majority (75%) of all trees, as well as in both MRE trees, both recently published polyclad phylogenies based on partial the genus Stylochus is not monophyletic and in all 28Sshort6 28S sequences (Bahia et al. 2017;Tsunashimaetal.2017), and trees, Imogine is not monophyletic. was also used in one of the best-scoring trees in both datasets shown here (Table 2, Suppl. Figs. S10, 13). We have also used two different variants of MAFFT (Katoh and Standley 2013); Discussion previously, MAFFT E-INS-i was selected for the polyclad phy- logeny based on mitochondrial sequences (Aguado et al. 2017) Taxon sampling and outgroup selection, as well as the choice of and for an all-flatworm phylogeny working with the nearly com- marker genes, the alignment method and the analysing statistical plete nuclear ribosomal marker genes, 18S and 28S (Laumer and models affect the resulting phylogenetic reconstructions signifi- Giribet 2017). The other best-scoring 28Sshort6 tree according to cantly (see e.g. Lockyer et al. 2003;PuslednikandSerb2008; our scoring in Table 2 is MAFFT E-INS-i aligned (Suppl. Figs. Aguado and Bleidorn 2010; Laumer and Giribet 2017). For S15), and another MAFFT E-INS-i tree (Suppl. Fig. S18)isalso polyclad interrelationships using mainly a rather short stretch of closest to the topology shown in the MRE 28Sshort6 tree the 28S rDNA marker gene, but also a longer sequence com- (Fig. 6). MAFFT Q-INS-i is by far the most computationally prised of both partial 18S and 28S rDNA, we show that the demanding alignment method, and was also employed quite ex- change of any of these parameters can vastly change the resulting tensively for resolving flatworm interrelationships on the level of tree topology (Figs. 1, 2, 3 and 4). A strong hypothesis about orders based on partial 18S and 28S, e.g. macrostomorphs valid polyclad interrelationships is thus challenging, and we have (Janssen et al. 2015), rhabdocoels (van Steenkiste et al. 2013; therefore used majority-rule consensus trees to help us decide Tessens et al. 2014) and proseriates (Casu et al. 2014; Scarpa between different topologies (Figs. 5 and 6)andalsomanually et al. 2015, 2016, 2017). The two best-scoring 18S28Slong trees analysed the support of different hypotheses (Table 2). To our are both based on a MAFFT Q-INS-i alignment (Table 2, Suppl. knowledge, this is the first time that these difficulties and incon- Figs. S2, 8). sistencies are discussed or even mentioned in regard to polyclad However, the two worst-scoring 28Sshort6 trees are also interrelationships. based on MAFFT Q-INS-i alignments (Table 2, Suppl. Figs. Polyclad phylogeny persists to be problematic 599

Comoplana agilis Armatoplana leptalea Notoplana delicata Notocomplana humilis 28short6 33 100 Notocomplana sp. 100 42 100 Notocomplana japonica 67 50 Notocomplana koreana MRE Pseudostylochus obscurus 50 100 Pseudostylochus sp. Leptoplana tremellaris 2 Clade 5 58 100 Leptoplana sp. with and without Gblocks 100 83 100 Leptoplana tremellaris 1 Notoplana australis 1 100 Notoplana australis 2 Melloplana ferruginea 42 Notoplana sp. 83 100 Echinoplana celerrima 2

Clade 3 Clade Echinoplana celerrima 1 Ilyella gigas 100 Adenoplana evelinae 67 92 Discocelis tigrina 100 Amemiyaia pacifica 67 Phaenocelis medvedica Clade 6 Callioplana marginata Leptostylochus gracilis Stylochus zebra 42 Imogine refertus 58 25 83 Paraplanocera oligoglena 50 58 Stylochus ijimai Imogine stellae 100 83 Stylochus oculiferus Clade 8 17 Stylochus sp. 50 Stylochus ellipticus Clade 4 Clade Planocera pellucida 50 100 100 Paraplanocera sp. 67 Planocera multitentaculata Idioplana australiensis ACOTYLEA 50 Hoploplana villosa 100 Hoploplana californica Clade 7 92 Hoploplana divae Cestoplana rubrocincta 1 100 Cestoplana rubrocincta 2 33 Cestoplana salar 50 Cestoplana techa Cestoplanoidea Boninia divae 75 Chromyella sp. Chromoplanoidea 100 Theama mediterranea 100 Theama sp. 83 Pericelis orbicularis 100 Pericelis byerleyana 100 100 Pericelis tectivorum Periceloidea Pericelis cata 1 92 Pericelis cata 2 Chromoplana sp. 92 Anonymus ruber Clade 1 83 100 Anonymus virilis Pseudoceros harrisi Yungia sp. Pseudobiceros hancockanus 25 Thysanozoon brocchii 1 67 83 Pseudobiceros evelinae 50 Pseudobiceros splendidus 67 Pseudoceros cf maximus 58 100 Pseudobiceros caribbensis 50 83 Pseudobiceros sp. Pseudobiceros bedfordi 92 Pseudobiceros wirtzi 58 Pseudobiceros pardalis 1 83 67 67 67 Pseudobiceros pardalis 2 Pseudobiceros flowersi 67 Maiazoon orsaki 67 Pseudobiceros nigromarginatus 100 Thysanozoon alagoensis 75 Thysanozoon brocchii 2 100 Thysanozoon raphaeli Monobiceros langi Pseudocerotidae 75 Phrikoceros mopsus 75 Pseudoceros periaurantius Pseudoceros contrarius 83 83 Pseudoceros bicolor 2 58 100 Pseudoceros cf bicolor 33 50 Pseudoceros bicolor 1 92 100 Pseudoceros bic. marcusorum Pseudoceros bimarginatus 83 83 Pseudoceros nipponicus Pseudoceros velutinus 2 92 100 100 Pseudoceros stimpsoni Pseudoceros rawlinsonae 1 100 Pseudoceros rawlinsonae 2 83 Pseudoceros jebborum 33 Pseudoceros astrorum 92 Pseudoceros velutinus 1 Euryleptodes galikias 100 Stylostomum ellipse 92 Cycloporus gabriellae 1 2 100 Cycloporus gabriellae 2 Clade Maritigrella fuscopunctata Prostheceraeus vittatus 100 67 100 Maritigrella crozieri 2

Pseudocerotoidea Maritigrella crozieri 3 83 67 83 Maritigrella crozieri 1 75 Maritigrella newmanae 67 Prostheceraeus roseus Euryleptidae Cycloporus japonicus 100 Cycloporus variegatus 2 100 Cycloporus variegatus 1 100 Cycloporus variegatus 3 100 Cycloporus variegatus 4 Enchiridium sp. 1 100 Enchiridium evelinae 100 Enchiridium sp. 2 100 Prosthiostomum grande Prosthiostomum siphunculus 3 100 100 Prosthiostomum siphunculus 2 Prosthiostomoidae 83 Prosthiostomum siphunculus 1 83 Amakusaplana acroporae COTYLEA 75 Lurymare katoi 33 Prosthiostomum vulgaris Macrostomum lignano Fig. 6 Extended majority-rule consensus tree based on all 12 trees of the or Cotylea in our trees are displayed with blue and red background, 28Sshort6 dataset shown in Suppl. Figs. S13–24. Numbers indicate per- respectively. Branches and nodes are given the same colour as their re- centage of support. Acotylea and Cotylea sensu Faubel 1983 and 1984 are spective taxon written in blue and red fonts, respectively. Species recovered as Acotylea

S17, 23), while the two worst-scoring 18S28Slong trees are recommend to use at least two different methods to check MAFFT E-INS-i aligned (Table 2, Suppl. Figs. S6, 12). for consistency. The tree topologies resulting from these widely used alignment methods are not consistent (Fig. 1, Suppl. Figs. S1–24), corrobo- Model choice is important rating the findings of Laumer and Giribet (2017), in which they re- analysed their differently aligned dataset from their earlier publi- In the work presented here, we consistently recovered incon- cation (Laumer and Giribet 2014) and also recovered trees with sistent BI and ML topologies using the same datasets and several major differences. In their re-analysis, they used MAFFT alignments (Table 2, Fig. 1). In the most recently published E-INS-i instead of RNAsalsa (Stocsits et al. 2009), and then re- polyclad phylogeny, both BI and ML trees gave congruent covered a tree very similar to two independently made results (Litvaitis et al. 2019). In other recent polyclad phylog- transcriptomic analyses of flatworm interrelationships (Egger enies based on partial 28S, only either BI (Rawlinson and et al. 2015,Laumeretal.2015), suggesting that MAFFT E-INS- Stella 2012) or ML (Bahia et al. 2017, Tsunashima et al. i provided a more robust alignment than RNAsalsa. 2017) were used, so no comparisons between different From this work, we cannot give an unambigious recom- models can be made. In two polyclad phylogenies, both BI mendation for the most suitable alignment method, but and ML analyses were run, and the trees show the same 600 Dittmann I.L. et al. topology in Rawlinson et al. (2011) and are ‘highly congru- Taxon sampling is important ent’ in a mitochondrial gene analysis with several switches within families, but not of the overall topology (Aguado et al. Not only the long-branching Chromoplana (therefore 2017). Both models were used to resolve interrelationships excluded from the analysis in Bahia et al. 2017), but also within other flatworm orders, and reported with very similar Cycloporus variegatus was prone to upend the tree topology or identical results using combined 18S and 28S datasets in the 28S trees (Figs. 3b, c and 4). Interestingly, both the (Casu et al. 2014; Tessens et al. 2014; Janssen et al. 2015; complete removal of Chromoplana and all Cycloporus se- Scarpa et al. 2015, 2016, 2017), in one case also including quences, and the addition of more variants of Cycloporus mitochondrial markers (Janssen et al. 2015). While these species yielded similar tree topologies (Figs. 4b and 6). We studies usually use a matrix of more than 3000 nt, our own have not tested removing taxa from the 18S28Slong dataset, large matrix with more than 3000 nt positions gives less con- but at least in theory, it should be more robust to taxon sam- gruent results among different models and alignments than pling artefacts than the much shorter 28Sshort dataset. In gen- our short matrix (ca. 800 nt) (Table 2,Figs.5 and 6), indicat- eral, and as stated above, taxon sampling seems to be more ing that taxon sampling may be even more important than important for resolving a stable polyclad phylogeny than ma- matrix length. trix length at this point. Again, we recommend to use both models (BI and ML) to check for consistency between the models. In our case, results Correct determination is important were not consistent, indicating that taxon sampling and matrix length were not sufficient yet. The correct identification of species is far-reaching for the interpretation of phylogenetic trees. During our analysis, we realised several inconsistencies in species determination of so Outgroup selection is important far published sequences. In several of our 28Sshort6 trees, as well as in the corresponding MRE (Fig. 6), a sequence tagged We have tested the influence of outgroup choice on tree topol- as Paraplanocera sp. (KY263699.2) on GenBank clusters ogy with Macrostomum lignano, a basally branching within Planocera. Therefore, Planocera does not appear rhabditophoran, and Xenoprorhynchus sp., a basally monophyletic (Table 2,Fig.6). However, according to branching prorhynchid—Prorhynchida being sister group of Bahia et al. (2017), this sequence and the associated accession Polycladida (Egger et al. 2015, Laumer et al. 2015), using the number belongs to Planocera sp.; hence, Planocera would be same alignment (MUSCLE) and alignment curation monophyletic also in our trees. We found several similar prob- (Gblocks), as well as the same model (BI) and the same lems with sequences listed as ‘Leptoplana sp. or Notoplana dataset (28Sshort2). We found markedly different tree topol- sp.’ in Table 1 of Bahia et al. (2017). In their table, these ogies between using both Macrostomum and sequences have the accession numbers KY263695, Xenoprorhynchus,onlyXenoprorhynchus or only KY263650, KY262696, KY263698 and KY263651. Macrostomum as ougroups (Fig. 3a–c). Especially the sister KY262696 is apparently a typo and should read KY263696, group relationships of either Chromoplana sp. or Cycloporus which together with KY263695 and KY263698 is tagged as variegatus with all other polyclads (Fig. 3b, c) were the reason ‘Leptoplana tremellaris’ on GenBank, while KY263650 and to also test the influence of taxon sampling on the polyclad KY263651 are labelled as ‘Notoplana sp.’ on GenBank. In tree topology (Fig. 4). their tree, Bahia et al. (2017)alsoshowanunlisted An almost identical dataset, aligned with the same algo- Notocomplana sp., but it is not clear to which accession num- rithm and tree reconstruction done with the same model and ber this species refers to. As usual, we only took one sequence by the same leading author yielded two different topologies: in of the same species from the same authors, and we have used the first account, both Cestoplana and Pericelis are basally KY263695 (Leptoplana tremellaris) and KY263650 branching Acotylea (Rawlinson et al. 2011), while these two (Notoplana sp.) in our phylogenetic reconstructions taxa switch to basally branching Cotylea in the second ac- (Table 1). Interestingly, in our 28Sshort6 MRE tree (Fig. 6), count (Rawlinson and Stella 2012). The only two differences this Notoplana sp. by Bahia et al. (2017) does not cluster with in the reconstructions are one instead of two outgroups and a any other Notoplana, Notocomplana (Notoplanidae) or third sequence of Amakusaplana acroporae in the second pa- Leptoplana (Leptoplanidae) species, but with Melloplana per (Rawlinson and Stella 2012), indicating that a higher num- (Pleioplanidae) and Echinoplana (Gnesiocerotidae). ber of outgroups gives more reliable results in their case. In Also Pseudoceros is not monophyletic in our analyses, as our own datasets, we found no clear preference for outgroup two species, Pseudoceros harrisi and Pseudoceros cf. selection (Fig. 3), making us default on a single, basally maximus are clustering outside the other 13 included branching outgroup (Macrostomum lignano) for our main Pseudoceros species (Fig. 6). Pseudoceros harrisi is consis- datasets (28Sshort6 and 18S28Slong). tently recovered as sister group to all other Pseudocerotidae in Polyclad phylogeny persists to be problematic 601 our trees and also by Bahia et al. (2017) and Tsunashima et al. recent publication, phylogeny may be even better without (2017). In its species description, which is based on a single using Gblocks or similar alignment curation programs (Tan damaged specimen, it is stated that ‘This species does not et al. 2015). In our own study, however, we find that the best- resemble any other species of Pseudoceros. However, scoring trees were made with datasets using Gblocks for align- P. harrisi may be confused with members of Cycloporus ment curation (Table 2). [...]’ (Bolaños et al. 2007). Hence, the phylogenetic position Some of the sequences published by Tsunashima et al. of Pseudoceros harrisi might be the result of a mis- (2017)appeartobequitedifferenttoallotherpolycladse- determination of its genus in the original description. The quences published, especially in the 5′ region: among these Pseudoceros cf maximus sequence (KY263708) we used are the above-mentioned Cycloporus japonicus (LC100092), was published by Bahia et al. (2017) and it appears with high Thysanozoon brocchii (LC100093) and Thysanozoon support within Pseudobiceros in our reconstructions (Fig. 6). japonicum (LC100094). We initially removed all of these se- We noticed that the species name Pseudoceros cf maximus quences from further analyses, but later added Cycloporus does not appear in Bahia et al.’s tree. On the other hand, they japonicus (28Sshort2 and 28Sshort6) despite the divergent show two branches labelled ‘Pseudobiceros spp.’ in their tree, sequence. Also Chromoplana sp. from Laumer and Giribet but only list a single Pseudobiceros sp. sequence in their (2014) was an unusual sequence and was therefore removed Table 1. Taking into account our own results, we believe it is from the tree of Bahia et al. (2017), but is included in most of possible that the sequence published as Pseudoceros cf our reconstructions (except 28Sshort4-5). maximus on GenBank is one of the ‘Pseudobiceros sp.’ in Although termed as ‘clones’ on GenBank, there is a con- their tree. siderable difference between the four published Cycloporus Several sequences have undergone name changes after re- variegatus sequences by Bahia et al. (2017); we believe these determination efforts by the authors, or have dubious affilia- sequences are not derived from clones, but from different tions. For example, Cestoplana rubrocincta from Australia specimens of the same species. (C. rubrocincta 2 in our tree, HQ659009.1) is labelled as Polyclad phylogenies based on partial 28S rDNA pub- C. australis in the tree provided by Rawlinson et al. (2011), lished by different authors used different primers, making but called C. rubrocincta in their table, and also on GenBank. the integration of all sequences a challenge, as the overlapping Other sequence names were updated without changing their regions get smaller. Especially Tsunashima et al. (2017)useda accession number versions. We originally downloaded the region of the 28S gene more towards the 3′ end than all other following sequences published in Tsunashima et al. (2017) studies, but we have still included most of their sequences, from GenBank in June 2017, but they were subsequently because they provide important taxa not covered by our own renamed: Discoplana sp. to Ilyella gigas (LC100080), or other previously published sequences. For future studies, Notoplana koreana to Notocomplana koreana (LC100086), we recommend amplifying 28S starting with expansion seg- Melloplana japonica to Notocomplana japonica ment D1 and stretching as long as possible, to maximise com- (LC100087), Cycloporus sp. to Cycloporus japonicus patibility with published sequences. (LC100092), Thysanozoon sp. 1 to Thysanozoon brocchii (LC100093), Thysanozoon sp. 2 to Thysanozoon japonicum Classification on suborder and superfamily level (LC100094), Pseudoceros sp. 1 to Pseudoceros velutinus (LC100095), Pseudoceros sp. 2 to Pseudoceros nipponicus On suborder level, our 28Sshort6 trees are mostly compatible (LC100096), and Pseudoceros sp. 3 to Pseudobiceros with the molecular phylogenetic hypothesis of Bahia et al. nigromarginatus (LC100097). (2017), supporting their redefinition of Cotylea and Acotylea (see Table 2 and Fig. 6). There, two traditional actoylean fam- Sequence problems ilies, Cestoplanidae and Theamatidae, switched from Acotylea to Cotylea. When we started with this study in 2017, we noticed gaps in The majority of the 18S28Slong trees, on the other hand, all newly generated sequences uploaded to GenBank by Bahia support Cestoplanidae and Theamatidae as acotyleans. Also, et al. (2017). The first set of 28Sshort trees we made was based the traditionally cotylean genera Pericelis, Boninia and on a dataset including these sequences. We later realised that Chromyella are recovered as acotyleans (Table 2, Fig. 5). In the gaps in the sequences were caused by alignment curation this scenario, a sucker would be a character at the base of using Gblocks (J. Bahia, pers. comm.), and all other trees Polycladida and would have been lost at least five times: in (using the 28Sshort2-6 sequence collections) were based on the traditional Acotylea, in some Cestoplanidae, in the the updated sequences (version 2 on GenBank). We provided anonymid Simpliciplana marginata (Kaburaki 1923), in reconstructions based on both, Gblocks curated and original Theamatidae, in Amakusaplana (Rawlinson et al. 2011), and alignments, and often recovered different topologies if all oth- possibly in Chromyella (Fig. 5 and Faubel 1983, 1984, er parameters stayed the same (Table 2,Fig.3). According to a Prudhoe 1985). In the 28Sshort6 scenario, a sucker would 602 Dittmann I.L. et al. be present at the base of Cotylea and would have been lost one topology, but that some sequences are prone to change tree time less, i.e. not in Acotylea (Fig. 6). According to Bahia topology, among them Chromoplana, Cycloporus variegatus et al. (2017), a ‘true sucker’ may have gradually evolved and Cycloporus japonicus (Figs. 3 and 4). As long as single and may be an apomorphy of Prosthiostomoidea and taxa included or excluded can drastically change tree topology Pseudocerotoidea. A true sucker is muscular and characterised even in the overall more consistent 28S-only trees, polyclad by a modified epithelium with a thin basement membrane, phylogeny remains only preliminarily resolved, calling for while the adhesive disc or pad found in Boninia and larger datasets like in transcriptomic phylogenies. Cestoplana is just a shallow depression of the epithelium not However, apart from the position of Cestoplanidae, differentiated from the parenchyma (Prudhoe 1985; Theamatidae, Pericelis, Boninia, Chromyella, Anonymidae Rawlinson and Litvaitis 2008). Both true sucker and adhesive and Chromoplana in the tree, we find that most polyclad taxa disc/pad are always located posterior of the genital openings. are included in very well-supported clades. Several Pericelis species (excluded from having a true sucker Our data support the following new superfamilies sensu in Bahia et al. 2017, but listed as having a true sucker in Bahia et al. (2017): Rawlinson and Litvaitis 2008) are described with a ‘distinct Pseudocerotoidea sensu Bahia et al. (2017); this superfam- sucker’ (Dittmann et al. 2019), so we suggest that the true ily includes Pseudocerotidae and two clades of Euryleptidae sucker behind the genital openings already is an apomorphy in their reconstruction. In this work, we termed one of these for the unnamed group including Periceloidea, Anonymus, clades ‘clade 2’ as all relevant trees show this non-monophyly Chromoplana, Prosthiostomoidea and Pseudocerotoidea (Table 2). This division can also be observed in the study of (Fig. 6). The acotylean genus Leptoplana has a sucker (a so- Bahia et al. (2017), where Cycloporus gabriellae represents called genital pit) between the genital openings (Prudhoe our clade 2 of Euryleptidae, while Cycloporus variegatus and 1985); therefore, it is excluded from the definition of a Cycloporus japonicus are part of the remaining Euryleptidae. cotylean sucker. Also in a cladistic analysis, Euryleptidae was not recovered as Based on this scenario of sucker evolution in polyclads, it is monophyletic (Rawlinson and Litvaitis 2008). As already sug- more parsimonious to support the 28Sshort6 tree topology, al- gested before, the genus Cycloporus needs to be revised, but though the 18S28Slong alignment with ca. 3000 nt is almost four no obvious characters to distinguish between described times as long as the 28Sshort6 alignment with ca. 900 nt. Also, Cycloporus species could be determined so far (Bahia et al. the support values of the trees rejecting Cotylea and Acotylea 2017). Our data show that the separation of the Cycloporus sensu Bahia et al. (2017) are consistently lower than those species not only results from potential inconsistencies within supporting them (Suppl. Figs. S1–24). In five of the twelve the genus Cycloporus,asalsoStylostomum and Euryleptodes 18S28Slong trees, Cotylea and Acotylea sensu Bahia et al. appear within clade 2. Therefore, we propose the revision of (2017) are actually supported, and also in the 18S28Slong the whole family of Euryleptidae. As Eurylepta has been MRE tree without Gblocks (Suppl. Fig. S26). Only the shown to cluster as sister group of other Euryleptidae in a 18S28Slong dataset using Gblocks skews the picture towards a phylogeny based on mitochondrial genes (Aguado et al. weakly supported topology making Cestoplanidae, Theamatidae, 2017), the family name Euryleptidae should be retained for Pericelis, Boninia and Chromyella acotyleans (Suppl. Fig. S25), the group containing Maritigrella, Prostheceraeus, also in the combined 18S28Slong MRE tree (Fig. 5). Cycloporus variegatus and Cycloporus japonicus (Fig. 7). In all but two 28Sshort6 trees, Cotylea and Acotylea sensu Cycloporus japonicus has been shown to group with Bahia et al. (2017) are well supported (Fig. 6, Suppl. Figs. Maritigrella in Tsunashima et al. (2017) as well. We propose S13–16, 18–22, 24). On the other hand, we have shown that the new family name Stylostomidae fam. nov. for clade 2, this topology is very much dependant on taxon sampling, including at least Stylostomum, Euryleptodes and outgroup selection, alignment method and curation, and mod- Cycloporus gabriellae. In the recently published work by el choice (Figs. 1, 2, 3 and 4). Possibly, the most important Litvaitis et al. (2019), both Euryleptidae and Cycloporus parameter is taxon sampling, and this would explain why a appear as monophyletic, but neither Stylostomum, nor much larger alignment (18S28Slong) with 27 polyclad termi- Euryleptodes are included in their study. As in our study, nals and 26 different polyclad species gives less consistent Litvaitis et al. (2019) have recovered both, Prostheceraeus results than the shorter matrix (28Short6) with 118 polyclad and Maritigrella, as non-monophyletic and consequently they terminals and 100 different polyclad species. Bahia et al. have synonymised Maritigrella as junior synonym with (2017) show 136 polyclad terminals, but only 55 different Prostheceraeus. polyclad species, and Tsunashima et al. (2017)use53 Pseudoceros, Pseudobiceros and Thysanozoon are not re- polyclad terminals and 50 polyclad species in their phyloge- covered as monophyletic in our study, agreeing with Bahia netic trees. While we have not tested their original datasets et al. (2017) and Tsunashima et al. (2017), stressing the need with different parameters here, their results suggest that nei- of further revision of the family Pseudocerotidae (Litvaitis and ther the number of taxa, nor sequences are decisive for tree Newman 2001; Rawlinson and Litvaitis 2008). Polyclad phylogeny persists to be problematic 603

Comoplana agilis Armatoplana leptalea Notoplana delicata 28short6 Notocomplana humilis 33 100 Notocomplana sp. 100 42 100 Notocomplana japonica MRE 67 50 Notocomplana koreana Pseudostylochus obscurus 50 100 Pseudostylochus sp. Leptoplana tremellaris 2 with and without Gblocks 58 100 Leptoplana sp. Leptoplanoidea 100 83 100 Leptoplana tremellaris 1 Notoplana australis 1 100 Notoplana australis 2 Melloplana ferruginea 42 Notoplana sp. 83 100 Echinoplana celerrima 2

Clade 3 Clade Echinoplana celerrima 1 Ilyella gigas 100 Adenoplana evelinae 67 92 Discocelis tigrina 100 Amemiyaia pacifica 67 Phaenocelis medvedica Discoceloidea superfam. nov. Callioplana marginata Leptostylochus gracilis Stylochus zebra Stylochoidea 42 Imogine refertus 58 25 83 Paraplanocera oligoglena 50 58 Stylochus ijimai Imogine stellae 100 83 Stylochus oculiferus 17 Stylochus sp. Clade 8 50 Stylochus ellipticus Planocera pellucida 50 100 100 Paraplanocera sp. 67 Planocera multitentaculata Idioplana australiensis Idioplanidae fam. nov. 50 Hoploplana villosa ACOTYLEA 100 Hoploplana californica 92 Hoploplana divae Clade 7 Cestoplana rubrocincta 1 100 Cestoplana rubrocincta 2 33 Cestoplana salar 50 Cestoplana techa Cestoplanoidea Boninia divae 75 Chromyella sp. Boninioidea superfam. nov 100 Theama mediterranea 100 Theama sp. 83 Pericelis orbicularis 100 Pericelis byerleyana 100 100 Pericelis tectivorum Periceloidea Pericelis cata 1 92 Pericelis cata 2 Chromoplana sp. 92 Anonymus ruber Anonymoidea superfam. nov. 83 100 Anonymus virilis Pseudoceros harrisi Yungia sp. Pseudobiceros hancockanus 25 Thysanozoon brocchii 1 67 83 Pseudobiceros evelinae 50 Pseudobiceros splendidus 67 Pseudoceros cf maximus 58 100 Pseudobiceros caribbensis 50 83 Pseudobiceros sp. Pseudobiceros bedfordi 92 Pseudobiceros wirtzi 58 Pseudobiceros pardalis 1 83 67 67 67 Pseudobiceros pardalis 2 Pseudobiceros flowersi 67 Maiazoon orsaki 67 Pseudobiceros nigromarginatus 100 Thysanozoon alagoensis 75 Thysanozoon brocchii 2 100 Thysanozoon raphaeli Monobiceros langi Pseudocerotidae 75 Phrikoceros mopsus 75 Pseudoceros periaurantius Pseudoceros contrarius 83 83 Pseudoceros bicolor 2 58 100 Pseudoceros cf bicolor 33 92 50 Pseudoceros bicolor 1 100 Pseudoceros bic. marcusorum Pseudoceros bimarginatus 83 83 Pseudoceros nipponicus Pseudoceros velutinus 2 92 100 100 Pseudoceros stimpsoni Pseudoceros rawlinsonae 1 100 Pseudoceros rawlinsonae 2 83 Pseudoceros jebborum 33 Pseudoceros astrorum 92 Pseudoceros velutinus 1 Euryleptodes galikias 100 Stylostomum ellipse 92 Cycloporus gabriellae 1 100 Cycloporus gabriellae 2 Stylostomidae fam. nov. Maritigrella fuscopunctata Prostheceraeus vittatus 100 67 Maritigrella crozieri 2 100 Maritigrella crozieri 3 83 Pseudocerotoidea 67 83 Maritigrella crozieri 1 75 Maritigrella newmanae 67 Prostheceraeus roseus Euryleptidae Cycloporus japonicus 100 Cycloporus variegatus 2 100 Cycloporus variegatus 1 100 Cycloporus variegatus 3 100 Cycloporus variegatus 4 Enchiridium sp. 1 100 Enchiridium evelinae 100 Enchiridium sp. 2 100 Prosthiostomum grande Prosthiostomum siphunculus 3 100 100 Prosthiostomum siphunculus 2 Prosthiostomoidea 83 Prosthiostomum siphunculus 1 83 Amakusaplana acroporae COTYLEA 75 Lurymare katoi 33 Prosthiostomum vulgaris Macrostomum lignano Fig. 7 Same tree as shown in Fig. 6, but with newly defined and named or Cotylea in our trees are displayed with blue and red background, groups indicated. Acotylea and Cotylea sensu Faubel 1983 and 1984 are respectively. Branches and nodes are given the same colour as their written in blue and red fonts, respectively. Species recovered as Acotylea respective taxon

Prosthiostomoidea was erected by Bahia et al. (2017)and synonymise Amakusaplana with Prosthiostomum. Our data only contains a single family, Prosthiostomidae. All our data support this decision. The position of Lurymare within support the monophyly of this family/superfamily and most data Prosthiostomum was already assumed by Poulter (1975). He (Table 2) also their sister group relationship to Pseudocerotoidea proposed a subdivision of the genus Prosthiostomum into the as described by Bahia et al. (2017). Similar to our results, also in subgenera P. (Lurymare)andP. (Prosthiostomum), distinguish- the study of Tsunashima et al. (2017), Prosthiostomum is not able by the constitution of the prostatic vesicle (Poulter 1975). monophyletic, as Amakusaplana (and in our case, also Faubel (1984) remarks that this definition also includes Lurymare) clusters within. In Aguado et al. (2017), two different Enchiridium and elevates both subgenera back as genera. At species of Lurymare do not form an adelphotaxon. The fact that least Enchiridium may be monophyletic, as suggested by Amakusaplana clusters within Prosthiostomum is not very sur- Bahia et al. (2017), Litvaitis et al. (2019)andourowntrees. prising, as Faubel (1984) remarks that the genus Amakusaplana Together, the molecular phylogenies do not support any of the has to be eliminated, as it is too similar to Prosthiostomum.The previously proposed genera (Kato 1938;Poulter1975; Faubel genus Amakusaplana is distinguished from Prosthiostomum 1984) except Enchiridium, i.e. the revision of the genera mainly by body shape and the arrangement of eyes (Kato Prosthiostomum and Lurymare is required. 1938;Faubel1984), and also by the absence of the ventral Our clade 1, consisting of Anonymus and Chromoplana,is sucker (Kato 1938; Rawlinson et al. 2011). Only in two of extremely well supported and always monophyletic, except in twelve 28Sshort6 reconstructions is Prosthiostomum monophy- one case, where it appears polytomic (Suppl. Fig. S21). We letic (Suppl. Figs. S17, 23), and Litvaitis et al. (2019) propose a new superfamily Anonymoidea superfam. nov. 604 Dittmann I.L. et al.

(Fig. 7), including the families Anonymidae (Lang 1884)and Moreover, the name of the superfamily has been erected Chromoplanidae (Bock 1922). based on the oldest family of the three included genera, Cestoplanoidea was defined by Poche (1926), emended by Theama, Chromyella and Boninia (Bahia et al. 2017). Prudhoe (1985) and supported by Bahia et al. (2017)and According to Bahia et al. (2017), the corresponding families Litvaitis et al. (2019); a majority of the 28Sshort6, but only of these genera are Theamatidae, Amyellidae and a minority of the 18S28Slong analyses support its sister group Chromoplanidae. Theama is a member of Theamatidae relationship with all other Cotylea, as suggested by Rawlinson Marcus 1949, Chromyella isamemberofeitherAmyellidae and Stella (2012) and Bahia et al. (2017), even if the family Faubel 1983 or Chromoplanidae Bock 1922,butBoninia is a was originally assigned to Acotylea (Lang 1884;Faubel1983; member of Boniniidae Bock 1923. Also, the epynomous ge- Prudhoe 1985) and appears as an acotylean in a majority of the nus of Chromoplanidae, Chromoplana, is not clustering with 18S28Slong analyses, as well as in Rawlinson et al. (2011). In Chromyella in any tree containing both of the genera (see also his monograph, Faubel remarks that organisation features of Laumer and Giribet 2014; Tsunashima et al. 2017). Therefore, Cestoplana, like the forward direction of the male complex, the family name Chromoplanidae should stay with the multiplication of the female apparatus in Cestoplana Chromoplana,andChromyella should be retained in the fam- polypora, or the presence of an adhesive organ in some (but ily Amyellidae, making Boniniidae the oldest family of the not all) Cestoplana species, could imply that Cestoplanidae three clustering genera. Here, we propose a new superfamily, have possibly arisen from a cotylean ancestor (Laidlaw 1903; Boninioidea superfam. nov., with the morphological defini- Bock 1922;Faubel1983; Bahia et al. 2017). tion of Chromoplanoidea sensu Bahia et al. 2017, but includ- In all of our reconstructions, Cestoplanoidea are monophyletic ing the families Theamatidae, Amyellidae and Boniniidae. (Table 2), although the only representing genus is Cestoplana. Cryptoceloidea sensu Bahia et al. (2017) include the families Periceloidea was also erected by Bahia et al. (2017)and Discocelidae (represented by Adenoplana in Bahia et al. 2017 and also contains a single, monotypic family, Pericelidae. Our data by Discocelis and Adenoplana in our 28Sshort6 trees), and support the monophyly of this group. Additionally, our Cryptocelidae (represented by Phaenocelis in Bahia et al. 2017, 28Sshort6 MRE tree (Fig. 6) supports its sister group relation- and by Cryptocelis, Phaenocelis and Amemiyaia in our 28Sshort6 ship with all remaining Cotylea except Cestoplanidae, as al- trees). While Faubel (1983) puts the genus Amemiyaia into the ready assumed by Bahia et al. (2017) and Rawlinson and family Stylochoplanidae, Prudhoe (1985) considers it to be a Stella (2012). In Tsunashima et al. (2017), Pericelis is also Cryptocelididae, the latter being consistent with our results recovered as a cotylean, but as sister group to Boninia + (Figs. 6 and 7). Thus, we reject the family Cryptocelidae sensu Chromyella (Theamatidae), although Cestoplana is absent in Faubel (1983). Our clade 6 contains members of Discocelidae and Tsunashima et al.’s reconstruction. In Rawlinson et al. (2011) Cryptocelididae sensu Prudhoe (1985), and with Ilyella gigas an and our 18S28Slong MRE tree (Fig. 5), Periceloidea are Ilyplanidae (Faubel 1983). We therefore reject Cryptoceloidea grouping with Acotylea, however. Litvaitis et al. (2019)in- sensu Bahia et al. (2017) as it contains Cryptocelidae sensu clude Diposthus in their phylogenetic reconstruction, which Faubel (1983) and redefine the superfamily with the inclusion of emerges as sister group of Pericelis, and they argue for the family Cryptocelididae sensu Prudhoe (1985), and the families abolishing both Pericelidae and Periceloidea in favour of the Ilyplanidae and Discocelidae. This in turn means that Discocelidae family Diposthidae. Laidlaw (1903) is the oldest family constituting the superfamily, Our data do not support the following superfamilies sensu and accordingly, the superfamily is named Discoceloidea, includ- Bahia et al. (2017): ing the families Cryptocelididae, Discocelidae and Ilyplanidae. The position of Chromoplanoidea within Cotylea is supported Stylochoidea sensu Bahia et al. (2017) has nuchal tentacles in by most of our analyses (Table 2), although in the 18S28Slong common and includes the families Hoploplanidae, Stylochidae, MRE tree, Chromoplanoidea is recovered as acotylean (Fig. 5). Pseudostylochidae and Planoceridae. Faubel (1984)placedthe The superfamily always is monophyletic, but the interrelationships genus Hoploplana within Leptoplanoidea, mainly due to the pres- between the three included chromoplanoid genera are differently ence of an interpolated prostatic vesicle. This is in contrast to resolved. In Bahia et al. (2017), Theama + Chromyella form a Prudhoe (1985), who considered the genus to be part of sister group to Boninia, while in almost all of our trees, including Planoceridae and thus in the superfamily Stylochoidea. the MRE trees, Chromyella + Boninia are sister group to Theama. Hoploplana was sister to Planocera within Stylochoidea in Curiously, in the only trees of our dataset supporting Theama + Bahia et al. (2017) and Litvaitis et al. (2019). Also Aguado et al. Chromyella (Suppl. Figs. S13, 15, 21), we used the same align- (2017) proposed the inclusion of Hoploplana in Stylochoidea ment method (MUSCLE), the same reconstruction method based on the morphological differences of the prostatic vesicle (RAxML), a partial 28S matrix and Gblocks, just like Bahia (also see Noreña et al. 2015) between leptoplanoids and that of et al. (2017). In Laumer and Giribet (2014, 2017), the remaining Hoploplana, as well as on their molecular phylogeny. Our possibility is realised, i.e. Theama + Boninia are sister group to 28Sshort6 MRE tree supports the sister group relationship of Chromyella. Hoploplana with the pseudostylochid Idioplana (Fig. 6), while Polyclad phylogeny persists to be problematic 605 there is strong support of Hoploplana + Planocera in our Paraplanocera oligoglena always clusters within Stylochidae, 18S28Slong trees, where Idioplana is lacking (Fig. 5), but also even in our 18S28Slong trees (Table 2,Fig.5). This phylogenetic in some of the 28Sshort6 trees (Suppl. Figs. S14, 17, 20, 23). position of Paraplanocera oligoglena corresponds to the finding We reject the superfamily Stylochoidea sensu Bahia et al. of Tsunashima et al. (2017)andBahiaetal.(2017). As stated (2017) in the current form, as all our 28Sshort6 trees show that under the section ‘Correct determination is important’, the pseudostylochids Pseudostylochus sp. as well as Paraplanocera sp. is confusingly labelled as Planocera sp. in their Pseudostylochus obscurus appear within Leptoplanoidea sensu paper (Bahia et al. 2017), but published as Paraplanocera sp. in Bahia et al. (2017), thus forming our clade 5, whereas the re- GenBank. This Paraplanocera sp. sequence renders the genus maining pseudostylochid, Idioplana australiensis, recovers with- Planocera paraphyletic in most of our 28Sshort6 trees (Table 2). in Stylochoidea (sensu Bahia et al. 2017), see above. Leptoplanoidea sensu Bahia et al. (2017) includes Pseudostylochus is the type genus of Pseudostylochidae, so the Pleioplanidae, Leptoplanidae, Notoplanidae and family name is retained with the genus; consequently, we erect a Stylochoplanidae. As discussed above (in Stylochoidea new family for Idioplana, Idioplanidae fam. nov., currently with sensu Bahiaetal.2017), we also have to reject this superfam- the diagnosis of the genus. ily in its current form, as Pseudostylochidae (represented by A further indication that Pseudostylochidae belongs within Pseudostylochus) clusters in all of our 28Sshort6 trees within Leptoplanoidea sensu Bahia et al. (2017), rather than within Leptoplanoidea. Hence, the group including Pleioplanidae, Stylochoidea sensu Bahia et al. (2017), can be found in the study Leptoplanidae sensu Prudhoe 1985 (excluding Hoploplana), of Aguado et al. (2017). There, Pseudostylochus intermedius clus- Notoplanidae, Stylochoplanidae and Pseudostylochidae is to ters within Leptoplanoidea (Aguado et al. 2017). The authors trace be called Leptoplanoidea. this position back to a misidentified species by Sato et al. (2001). Within Leptoplanoidea, Stylochoplanidae sensu Faubel However, we think a misidentification is unlikely, as all of our (1983) (including Amemiyaia, Comoplana and Armatoplana) phylogenetic trees including Pseudostylochus sp. as well as appears polyphyletic in all of our 28Sshort6 trees (see Pseudostylochus obscurus confirm the position of Discussion about Cryptoceloidea), strongly suggesting the need Pseudostylochus within Leptoplanoidea—with different sampling of revision of the family. The only other molecular study includ- material, and different genes than provided by Sato et al. (2001). ing more than one member of Stylochoplanidae is Aguado et al. Also, Pseudostylochus is always recovered as monophyletic. (2017), in which mitochondrial sequences of Stylochoplana Already in the original description of the genus maculata and Comoplana agilis were used, which did not appear Pseudostylochus, it was placed within the same superfamily as as sister groups in their phylogenetic reconstruction. However, Leptoplanidae, Schematommata (Yeri and Kaburaki 1918). In the published sequence of S. maculata was found to be almost the study of Tsunashima et al. (2017), Pseudostylochus is shown identical to the sequence of Leptoplana tremellaris, leading the within Notoplanidae, and hence within Leptoplanoidea as well. authors to suggest that S. maculata was possibly misidentified As Pseudostylochus has nuchal tentacles, albeit ‘small and indis- and is actually L. tremellaris (Aguado et al. 2017). tinct’ (Yeri and Kaburaki 1918), the placement of the genus within All our 28Sshort6 trees show that Leptoplanidae (sensu theLeptoplanoidea(agroupwithout nuchal tentacles) contradicts Faubel 1983 or Prudhoe 1985), Notoplanidae (sensu Faubel the hypothesis that nuchal tentacles have only evolved once in 1985) and Notoplana are not monophyletic, while Polycladida, at the base of Stylochoidea (Bahia et al. 2017). Notocomplana and Leptoplana are always monophyletic. In As a result, we redefine the superfamily Stylochoidea Tsunashima et al. (2017), as well as in Bahia et al. (2017), (sensu Bahia et al. 2017) consisting of Hoploplanidae, Notoplanidae are not monophyletic as well. In their recently Idioplanidae nov. fam., Stylochidae and Planoceridae, but published phylogenetic reconstruction, Litvaitis et al. (2019) without Pseudostylochidae. revised several families and genera within this superfamily. Within the family Stylochidae (represented by the genera Stylochus, Imogine, Leptostylochus), only the minority of our 28Sshort6 trees recovers the genus Stylochus as monophyletic Conclusions (two of twelve), and none of our trees supports a monophy- letic Imogine, corroborating the results of Aguado et al. (2017) Success in resolving polyclad interrelationships was ham- and Bahia et al. (2017). This is not surprising, as both genera pered so far by different approaches using different genes or were formerly included as subgenera of Stylochus (Jennings different parts of the same gene, making a combination of and Newman 1996;Aguadoetal.2017). We therefore recom- published data difficult. Polyclad interrelationships are still mend to combine them in one genus—Stylochus—once more, only tentatively resolved using single or two gene phyloge- as the name Stylochus (Ehrenberg 1831) predates the name nies. We have identified some stable parts of the phylogeny, Imogine (Girard 1853). and also groups which need to be revisited with better taxon Additionally, Planoceridae sensu Faubel (1983) are never sampling and with longer alignments, ideally using a monophyletic in any of our 28Sshort6 trees, because transcriptomic-phylogenomic approach. 606 Dittmann I.L. et al.

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