Org Divers Evol (2017) 17:653–678 DOI 10.1007/s13127-017-0327-5 ORIGINAL ARTICLE Polycladida phylogeny and evolution: integrating evidence from 28S rDNA and morphology Juliana Bahia1,2 & Vinicius Padula3 & Michael Schrödl1,2 Received: 29 August 2016 /Accepted: 12 December 2016 /Published online: 11 May 2017 # Gesellschaft für Biologische Systematik 2017 Abstract Polyclad flatworms have a troubled classification Combining morphological and molecular evidence, we history, with two contradicting systems in use. They both rely redefined polyclad superfamilies. Acotylea contain on a ventral adhesive structure to define the suborders tentaculated and atentaculated groups and is now divided in Acotylea and Cotylea, but superfamilies were defined accord- three superfamilies. The suborder Cotylea can be divided in ing to eyespot arrangement (Prudhoe’s system) or prostatic five superfamilies. In general, there is a trait of anteriorization vesicle characters (Faubel’s system). Molecular data available of sensory structures, from the plesiomorphic acotylean body cover a very limited part of the known polyclad family diver- plan to the cotylean gross morphology. Traditionally used sity and have not allowed testing morphology-based classifi- characters, such as prostatic vesicle, eyespot distribution, cation systems on Polycladida yet. We thus sampled a suitable and type of pharynx, are all homoplastic and likely have mis- marker, partial 28S ribosomal DNA (rDNA), from led polyclad systematics so far. Polycladida (19 families and 32 genera), generating 136 new sequences and the first comprehensive genetic dataset on Keywords Platyhelminthes . Marine flatworms . Cotylea . polyclads. Our maximum likelihood (ML) analyses recovered Acotylea . Molecular phylogenetics . Morphology Polycladida, but the traditional suborders were not monophy- letic, as the supposedly acotyleans Cestoplana and Theama were nested within Cotylea; we suggest that these genera Introduction should be included in Cotylea. The partial 28S rDNA trees were generally well supported and robust but in conflict with Polycladida are free-living Platyhelminthes that inhabit ma- both Faubel’s and Prudhoe’s superfamilies. Therefore, we rine environments, as different as coral reefs, rocky shores, compiled morphological and anatomical characters for all taxa soft bottoms, and deepwater (Newman and Cannon 2003; used and examined their distribution on our molecular tree. Quiroga et al. 2006) as also artificial aquaculture structures (Bahia 2015). Around 1000 species of Polycladida are known Electronic supplementary material The online version of this article in the world (Rawlinson 2008; Tyler et al. 2016). The main (doi:10.1007/s13127-017-0327-5) contains supplementary material, characteristic of this group of Platyhelminthes is the simple which is available to authorized users. and dorsoventrally flattened body, with a much ramified in- testine (Hyman 1951). Characters used in taxonomy of the * Juliana Bahia order Polycladida are the hermaphrodite reproductive anato- [email protected] my and external morphology (eyespot arrangements, tenta- cles, and pharynx) (Hyman 1951;Faubel1984; Prudhoe 1 SNSB-Zoologische Staatssammlung München, Münchhausenstrasse 1985). Coloration pattern is also used to distinguish closely 21, 81247 Munich, Germany related species (Newman and Cannon 1995; Litvaitis et al. 2 GeoBioCenter LMU and Biozentrum of the Ludwig-Maximilians 2010). In general, polyclads have cryptic behavior, living un- Universität München, Munich, Germany der rocks, often associated with invertebrates on which they 3 Department of Marine Biotecnology, Instituto de Estudos do Mar feed (Marcus and Marcus 1951; Newman and Cannon 2003). Almirante Paulo Moreira (IEAPM), Arraial do Cabo, Brazil They are important predators in hard bottom environments 654 Bahia J. et al. (Rawlinson et al. 2011) and are models in studies involving systematic key, which was based on the scattered taxonomic regeneration (Egger et al. 2007), toxicology and predation polyclad bibliography (Schmmarda 1859;Lang1884; (Ritson-Williams et al. 2006), pharmacologically active com- Laidlaw 1903a, 1903b, 1903c; Bock 1923; Marcus 1950; pounds (Schupp et al. 2001), mimetism (Newman and Hyman 1955). This system was further improved in indepen- Cannon 1995), and aposematism (Ang and Newman 1998). dent approaches by Faubel (1983, 1984) and Prudhoe (1985). These animals can also damage mollusk aquaculture (Pearse Faubel (1984) also proposed a phylogenetic arrangement and Wharton 1938; Sluys et al. 2005). (Fig. 1c) for the order. Both Faubel and Prudhoe and previous Polyclads are a conspicuous group of marine invertebrates, authors divided polyclads in the suborders Acotylea and possibly the most charismatic members of the phylum Cotylea, differentiated by a ventral adhesive structure. Platyhelminthes, and even so are still poorly studied However, Faubel’sandPrudhoe’s concepts resulted in mas- (Littlewood et al. 1999). Their position within sive conflict on superfamily level. Platyhelminthes is controversial, as historically polyclads Faubel (1983) divided Acotylea in superfamilies based on were considered basal in the phylum, in Trepaxonemata as the absence of true prostatic vesicle (Ilyplanoidea = sister group to the Neoophora (Ehlers 1985, 1986) or together Emprosthommatidae), presence of true free prostatic vesicle with Macrostomorpha (Litvaitis and Rhode 1999; Janssen (Stylochoidea = Craspedommatidea), or true interpolated et al. 2015). However, recently, they were proposed as sister prostatic vesicle (Leptoplanoidea). Prudhoe (1985)usedin- group to Lecithoepitheliata as a whole (Egger et al. 2015;Sòla stead the distribution of eyespots dividing Acotylea in super- et al. 2015) or to Prorhynchida (Laumer et al. 2015). families with frontal eyespots (Cestoplanoidea), with frontal, Polycladida systematics has had a troubled history as well. tentacular, and cerebral eyespots (Stylochoidea = One of the first phylogenetic hypotheses suggested that the Craspedommatidea) and with tentacular and cerebral eyespots genera Cestoplana and Prosthiostomum (Fig. 1b) were the (Planoceroidea = Schemmathomatidea). The suborder most derived ones in Acotylea and Cotylea, respectively Cotylea was divided by Faubel (1984) based on different char- (Lang 1884). Laidlaw (1903c) illustrated another hypothesis acters, two monospecific superfamilies were created, one with of relationship between cotyleans (Fig. 1a). Later, Marcus and male apparatus behind female structures (Opisthogenoidea) Marcus (1966) developed the first comprehensive Polycladida and the other with triclad-like digestive system Fig. 1 Traditional morphology-based hypotheses on the phylogeny of Polycladida. a Laidlaw (1903c). b Lang (1884). c Faubel (1984) Polycladida phylogeny and evolution 655 (Ditremagenidea). The other cotyleans were divided in a Table 1 Species list, localities, and number of samples included in the group with ruffled pharynx (Pseudocerotoidea) and other with phylogeny and respective GenBank accession numbers tubular pharynx (Euryleptoidea). Prudhoe did not divide the Species Localities GenBank cotyleans in superfamily groupings, since there was not an accession important difference between eyespot arrangements within number Cotylea. Adenoplana evelinae Marcus, Brazil KY263647 A recent morphology-based cladistic study by Rawlinson and 1950 Litvaitis (2008) focused on Cotylea, with interesting results about Phaenocelis medvedica Marcus, Brazil KY263701 genus relationships, many of them not being monophyletic. But 1952 KY263702 KY263703 so far, no molecular study has ever tested the homology of the KY263704 characters used in traditional systematic taxonomy. There is no KY263705 molecular phylogenetic hypothesis on Polycladida or its major KY263706 subclades either. Some molecular data, mainly 28S ribosomal Idioplana australiensis Australia HQ659008 DNA (rDNA) and some 18S rDNA sequences, are available Woodworth, 1898 from a couple (not more than seven) of polyclad species that Imogine refertus Du Brazil KY263694 Bois-Reymond Marcus, 1965 were included into Platyhelminthes phylogenies (Campos et al. Stylochus sp. Australia AF131707 1998;Littlewoodetal.1999; Litvaitis and Rhode 1999; Litvaitis Stylochus sp. Peru KY263743 and Newman 2001). Within Polycladida, a phylogeny of the Imogine zebra (Verrill, 1882) US Atlantic coast AF342800 family Pseudocerotidae included 18 species (Litvaitis and Imogine oculifera Girard, 1853 Florida HQ659007 Newman 2001) and focused on relations between genera, but Hoploplana californica Hyman, California KC869850 the information is not on an online database. There are also 1953 molecular data available from a study focused on a species com- Hoploplana divae Marcus, 1950 Brazil KY263692 plex (Litvaitis et al. 2010). Egger et al. (2015)changedthepre- KY263693 vailing hypothesis on the origin of polyclads and, recently, some Armatoplana leptalea (Marcus, Brazil KY263648 1947) KY263649 polyclad COI and 16S sequences were included in other Leptoplana sp. or Notoplana sp. Spain, Brazil KY263695 Platyhelminthes phylogeny papers (Laumer and Giribet 2014; KY263650 Laumer et al. 2015). The first mitogenomic information support- KY262696 ed the monophyly of Polycladida and its suborders (Aguado KY263698 et al. 2015). However, this latter study included just three species KY263651 and one of them unidentified. Molecular phylogeny of polyclads Notoplana australis (Schmarda, Australia AY157153 1859) HQ659015 is thus in an initial stage mainly