Molecular Phylogenetics and Evolution 89 (2015) 130–150 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev A new phylogeny of the Cephalaspidea (Gastropoda: Heterobranchia) based on expanded taxon sampling and gene markers q ⇑ Trond R. Oskars a, , Philippe Bouchet b, Manuel António E. Malaquias a a Phylogenetic Systematics and Evolution Research Group, Section of Taxonomy and Evolution, Department of Natural History, University Museum of Bergen, University of Bergen, PB 7800, 5020 Bergen, Norway b Muséum National d’Histoire Naturelle, UMR 7205, ISyEB, 55 rue de Buffon, F-75231 Paris cedex 05, France article info abstract Article history: The Cephalaspidea is a diverse marine clade of euthyneuran gastropods with many groups still known Received 28 November 2014 largely from shells or scant anatomical data. The definition of the group and the relationships between Revised 14 March 2015 members has been hampered by the difficulty of establishing sound synapomorphies, but the advent Accepted 8 April 2015 of molecular phylogenetics is helping to change significantly this situation. Yet, because of limited taxon Available online 24 April 2015 sampling and few genetic markers employed in previous studies, many questions about the sister rela- tionships and monophyletic status of several families remained open. Keywords: In this study 109 species of Cephalaspidea were included covering 100% of traditional family-level Gastropoda diversity (12 families) and 50% of all genera (33 genera). Bayesian and maximum likelihood phylogenet- Euthyneura Bubble snails ics analyses based on two mitochondrial (COI, 16S rRNA) and two nuclear gene markers (28S rRNA and Cephalaspids Histone-3) were used to infer the relationships of Cephalaspidea. The monophyly of the Cephalaspidea Molecular systematics was confirmed. The families Cylichnidae, Diaphanidae, Haminoeidae, Philinidae, and Retusidae were Taxonomy found non-monophyletic. This result suggests that the family level taxonomy of the Cephalaspidea war- rants a profound revision and several new family and genus names are required to reflect the new phy- logenetic hypothesis presented here. We propose a new classification of the Cephalaspidea including five new families (Alacuppidae, Colinatydidae, Colpodaspididae, Mnestiidae, Philinorbidae) and one new genus (Alacuppa). Two family names (Acteocinidae, Laonidae) and two genera (Laona, Philinorbis) are reinstated as valid. An additional lineage with family rank (Philinidae ‘‘Clade 4’’) was unravelled, but no genus and species names are available to reflect the phylogeny and formal description will take place elsewhere. Ó 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction 2008). Most species inhabit soft bottoms of sand or mud, but sev- eral occur in close association with seagrass and algae and others The Cephalaspidea (bubble snails) is a major lineage of marine in coral rubble and sponges (Burn and Thompson, 1998; Gosliner heterobranch gastropods with about 634 species (Jörger et al., et al., 2008). A single genus is known to inhabit exposed rocky 2010; WoRMS, 2015) distributed worldwide across all latitudes, shores (Smaragdinella A. Adams, 1848: Gosliner et al., 2008). reaching highest diversity in tropical and subtropical waters of Malaquias et al. (2009) suggested that diet specialization played the Indo-West Pacific (Lin and Qi, 1985; Bouchet et al., 2002; a chief role in the evolution and diversification of cephalaspids. Gosliner et al., 2008; Too et al., 2014). Cephalaspids occur from Within the group there are herbivorous and carnivorous lineages, the intertidal zone (e.g. Haminoea Turton & Kingston, 1830) to which feed preferentially upon diatoms, filamentous algae, the deep-sea (e.g. Inopinodon Bouchet, 1975, Meloscaphander foraminiferans, small bivalves, gastropods and polychaetes (see Schepman, 1913, Scaphander Montfort, 1810: Bouchet, 1975; Malaquias et al., 2009; Göbbeler and Klussmann-Kolb, 2011; Valdés, 2008; Eilertsen and Malaquias, 2013a), but seem to be Eilertsen and Malaquias, 2013b, for reviews). more abundant in shallow areas to 40 m deep (Gosliner et al., Mikkelsen (1993) highlighted the difficulties on establishing synapomorphies and the sharp discrepancies between proposed classifications. Consequently, the first attempts to study the rela- q This paper has been recommended for acceptance by Jan Strugnell. tionships of the group within a cladistic framework were ham- ⇑ Corresponding author. E-mail addresses: [email protected] (T.R. Oskars), [email protected] pered not only by incomplete taxon sampling, but mostly by (P. Bouchet), [email protected] (M.A.E. Malaquias). difficulties on the interpretation of morphological characters and http://dx.doi.org/10.1016/j.ympev.2015.04.011 1055-7903/Ó 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). T.R. Oskars et al. / Molecular Phylogenetics and Evolution 89 (2015) 130–150 131 resulted in poorly resolved hypotheses (Mikkelsen, 1996, 2002; (4) to test and revise the classification proposed by Malaquias Dayrat and Tillier, 2001; Wägele and Klussmann-Kolb, 2005). et al. (2009) for the Cephalaspidea. Nevertheless, these studies backed the view that some traditional cephalaspidean groups such as Acteonidae, Aplustridae, 2. Methods Bullinidae Gray, 1850, and Ringiculidae Philippi, 1853 were likely not closely related to the remaining lineages, a hypothesis origi- 2.1. Sampling nally suggested by Haszprunar (1985). Since the late 1990s the advent of molecular phylogenetics Novel material used in the present study was self-collected in a yielded significant new insights about the relationships of series of shore based expeditions and deep water cruises con- euthyneuran gastropods and the Cephalaspidea was no exception ducted by the Museum National d’Histoire Naturelle (MNHN) in (reviewed in Malaquias et al., 2009). Molecular studies contributed the tropical Indo-Pacific; and from the University Museum of to an objective redefinition of the concept of Cephalaspidea and Bergen (Natural History), Norway (ZMBN). Whenever possible at become well established that traditional taxa such as the least four species per genus were included in the analyses. In some Acteonoidea (Acteon Montfort, 1810, Hydatina Schumacher, 1817, situations we have added additional species, namely for gen- Micromelo Pilsbry, 1895, Pupa Röding, 1798, Rictaxis Dall, 1871) era/families of uncertain systematic status (e.g. Retusidae; and Runcinacea (Runcina Forbes, 1851, Ilbia Burn, 1963) are not Malaquias et al., 2009) and when previous unpublished prelimi- members of the clade Cephalaspidea. Also, the Diaphanoidea nary work undertaken by us have hinted possible cases of unex- Odhner, 1914 (Diaphana Brown, 1827, Toledonia Dall, 1902) repre- pected non-monophyly (e.g. Chelidonura A. Adams, 1850, sents the basal lineages of the Cephalaspidea sensu stricto (sensu Haminoea, Philinidae). Furthermore, when specimens did not have Malaquias et al., 2009; Jörger et al., 2010; Göbbeler and full gene coverage (the four genes used in this study), we have used Klussmann-Kolb, 2011). The ‘‘Cephalaspidea’’ went from being additional representatives to complement the gene diversity (see considered as basal ‘‘primitive’’ opisthobranchs (e.g. Gosliner, Table 1). 1981) to derived and specialized members of the Euthyneura DNA extracts of Philinoglossa praelongata and Pluscula cuica Spengel, 1881 within the monophyletic Euopisthobranchia (sensu were provided by colleagues from The Bavarian State Collection Jörger et al., 2010; Schrödl et al., 2011; Brenzinger et al., 2013; of Zoology, Germany (ZSM). Additionally 153 sequences from 60 Wägele et al., 2014). specimens were mined from GenBank (Table 1). Yet, the systematics and evolutionary relationships of the Outgroup taxa consisting of 30 species from 24 genera repre- Cephalaspidea remain far from completely understood. The study senting nine higher euthyneuran clades of ranking similar to that by Malaquias et al. (2009) is still the only one to specifically focus of Cephalaspidea (Jörger et al., 2010) were included in the analyses, on the internal relationships of the Cephalaspidea sensu stricto namely Acochlidia, Acteonoidea, Anaspidea, Nudipleura, Pteropoda within a molecular phylogenetic framework. The authors used (Gymnosomata and Thecosomata), Pyramidelloidea, Runcinacea, three gene markers (mitochondrial COI and nuclear 18S rRNA Sacoglossa, and Umbraculida. The trees were rooted with the and 28S rRNA) and included representatives of nine families, 22 caenogastropod species Littorina littorea a sister lineage to the genera, and 41 species. They found the families Cylichnidae, Heterobranchia molluscs (Zapata et al., 2014). In total this study Diaphanidae, Haminoeidae, and Retusidae to be polyphyletic and includes 177 specimens (145 Cephalaspidea, 31 Euthyneura out- in need of revision and reinstated the family names Rhizoridae groups, and 1 Caenogastropoda) and a total of 596 sequences (for Volvulella Newton, 1891) and Scaphandridae (for (Table 1). Scaphander). The family relationships were generally poorly resolved, but sister relationships were found between Philinidae and Aglajidae and between Philinoglossidae and Gastropteridae. 2.2. DNA extraction, amplification, and sequencing A possible