Molecular Phylogenetics and Evolution 40 (2006) 585–608 www.elsevier.com/locate/ympev A modern approach to rotiferan phylogeny: Combining morphological and molecular data Martin V. Sørensen ¤, Gonzalo Giribet Department of Organismic and Evolutionary Biology, Museum of Comparative Zoology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA Received 30 November 2005; revised 6 March 2006; accepted 3 April 2006 Available online 6 April 2006 Abstract The phylogeny of selected members of the phylum Rotifera is examined based on analyses under parsimony direct optimization and Bayesian inference of phylogeny. Species of the higher metazoan lineages Acanthocephala, Micrognathozoa, Cycliophora, and potential outgroups are included to test rotiferan monophyly. The data include 74 morphological characters combined with DNA sequence data from four molecular loci, including the nuclear 18S rRNA, 28S rRNA, histone H3, and the mitochondrial cytochrome c oxidase subunit I. The combined molecular and total evidence analyses support the inclusion of Acanthocephala as a rotiferan ingroup, but do not sup- port the inclusion of Micrognathozoa and Cycliophora. Within Rotifera, the monophyletic Monogononta is sister group to a clade con- sisting of Acanthocephala, Seisonidea, and Bdelloidea—for which we propose the name Hemirotifera. We also formally propose the inclusion of Acanthocephala within Rotifera, but maintaining the name Rotifera for the new expanded phylum. Within Monogononta, Gnesiotrocha and Ploima are also supported by the data. The relationships within Ploima remain unstable to parameter variation or to the method of phylogeny reconstruction and poorly supported, and the analyses showed that monophyly was questionable for the fami- lies Dicranophoridae, Notommatidae, and Brachionidae, and for the genus Proales. Otherwise, monophyly was generally supported for the represented ploimid families and genera. © 2006 Elsevier Inc. All rights reserved. Keywords: Acanthocephala; Bayesian inference; Cladistics; Rotifera; Hemirotifera; Seison; Syndermata; Micrognathozoa; Cycliophora 1. Introduction teristics. A total of nine diVerent types of trophi (Fig. 1), plus some intermediate forms, are recognized. Rotifera is a group of mostly microscopic, aquatic inver- Three major clades are usually recognized within Rotif- tebrates with about 1900 described species (Segers, 2002a). era: Seisonidea, Bdelloidea, and Monogononta (Wallace They are generally characterized by the presence of a and Colburn, 1989; Wallace and Snell, 1991; Melone et al., corona (or wheel organ) formed of ciliary bands in the 1998a; Sørensen, 2002). Seisonidea comprises only three cephalic region. They also have a complex pharyngeal species in the genera Seison and Paraseison (Sørensen et al., apparatus, the mastax, composed of hard parts (trophi), 2005). They are epizoic symbionts on the members of the connective musculature, and ligaments (Nogrady et al., leptostracan crustacean genus Nebalia, and are character- 1993). In a phylogenetic and taxonomic context, the mor- ized by possessing a special fulcrate trophus type (Fig. 1E), phology of the trophi is among the most signiWcant charac- and gamogenetic reproduction (Ricci et al., 1993; Ahlrichs, 1995a; Segers and Melone, 1998). Bdelloidea comprises ca. 400 species with a rather conserved body plan. The body consists of a trunk and telescopic retractable head and foot * Corresponding author. Present address: Ancient DNA and Evolution Group, Niels Bohr Institute and Biological Institute, University of Copen- regions. The head carries a retractable rostrum with adhe- hagen, Juliane Maries Vej 30, 2100 Copenhagen, Denmark. sive glands used for crawling, and the trophi are of the E-mail address: [email protected] (M.V. Sørensen). ramate type (Fig. 1A). Furthermore, all bdelloids reproduced 1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.04.001 586 M.V. Sørensen, G. Giribet / Molecular Phylogenetics and Evolution 40 (2006) 585–608 Fig. 1. Scanning electron micrographs showing the nine rotiferan trophus types. (A) Ramate trophi of Rotaria neptunia (Bdelloidea), caudal view. (B) Uncinate trophi of Stephanoceros Wmbriatus (Collothecacea), ventral view. (C) Malleoramate trophi of Filinia longiseta (Flosculariacea), dorsal view. (D) Malleate trophi of Brachionus plicatilis (Ploima: Brachionidae), ventral view. (E) Fulcrate trophi of Seison nebaliae (Seisonidea), ventral view. (F) Incudate trophi of Asplanchnopus dahlgreni (Ploima: Asplanchnidae), ventral view. (G) Virgate trophi of Eothinia elongata (Ploima: Notommatidae), right lateral view. (H) Forcipate trophi of Encentrum astridae (Ploima: Dicranophoridae), ventral view. (I) Cardate trophi of Lindia torulosa (Ploima: Lindiidae). Abbreviations: fu, fulcrum; ma, manubrium; im, intramalleus; pc, posterior manubrial chamber; ra, ramus; sm, supramanubrium; un, uncus. solely by parthenogenesis and most species have cryptobi- Early microscope pioneers such as van Leeuwenhoek, otic capabilities (Donner, 1965; Ricci, 1987, 1998a; Mark Linnaeus, and Müller originally classiWed rotifers Welch and Meselson, 2000; Ricci and Melone, 2000). The among the diVuse group “Vermes” or the protozoan Infu- third clade, Monogononta, with ca. 1500 described species, soria. Later, Rotifera were assigned to the polyphyletic is by far the most diverse rotifer group and it also displays Aschelminthes, but more recently an increasing amount the greatest morphological disparity. Monogononts are of evidence supports a close relationship between Rotif- mostly free-swimming, even though sessile taxa are repre- era and Gnathostomulida, and together with Acantho- sented as well. The remaining seven trophus types are cephala and Micrognathozoa they are united in the clade found in this clade. Contrary to the exclusively parthenoge- Gnathifera, supported by the presence of jaws with netic bdelloids, many monogonont rotifers follow a com- identical ultrastructure (Ahlrichs, 1995a,b; Rieger and plex cycle composed of a parthenogenetic amictic phase Tyler, 1995; Haszprunar, 1996; Giribet et al., 2000; Søren- and a mictic phase that include sexual reproduction and the sen et al., 2000; Kristensen and Funch, 2000; Giribet, presence of haploid dwarf males (Nogrady et al., 1993; 2002a; Sørensen, 2003; Funch et al., 2005). The jaws Melone and Ferraguti, 1999). have been lost secondarily in the highly specialized, endo- Also related to rotifers are the members of a parasitic parasitic acanthocephalans, but the presence of a syncy- clade of metazoans, Acanthocephala. While still considered tial epidermis with an intraskeletal lamina supports a a phylum in their own, recent molecular data derived from close relationship with Rotifera (HaVner, 1950; Koehler, nuclear ribosomal genes suggest that acanthocephalans are 1966; Storch and Welsch, 1969, 1970). The two related to rotifers (Herlyn et al., 2003), perhaps nesting groups have traditionally been considered sister taxa, but within them as the sister group to bdelloids (García-Varela recently the rotifer monophyly with respect to Acantho- et al., 2000; Giribet et al., 2004) or sister to Seisonidea cephala has been questioned. Morphological data (Zrzavý, 2001a; Herlyn et al., 2003). As such, we cannot have suggested various combinations of sister-group rela- attempt to evaluate rotiferan relationships without consid- tionships between Acanthocephala and the three main ering acanthocephalans. rotifer clades, Seisonidea, Bdelloidea, and Monogononta M.V. Sørensen, G. Giribet / Molecular Phylogenetics and Evolution 40 (2006) 585–608 587 (Lorenzen, 1985; Ahlrichs, 1995a, 1997, 1998; Ferraguti 2. Materials and methods and Melone, 1999; Sørensen et al., 2000), but all these have been based on dubious homology statements or con- 2.1. Taxon sampling tradictory details in sperm anatomy (Melone et al., 1998a; Ricci, 1998b; Funch et al., 2005). Hence, morphological The ingroup comprises 48 monogononts, four bdelloids, data cannot at present provide critical support for Acan- four acanthocephalans, Seison nebaliae (Seisonidea), and L. thocephala as a rotifer ingroup, although a steadily grow- maerski (Micrognathozoa), spanning most of the diversity ing amount of molecular data strongly support its within the phylum Rotifera and potential ingroups. Out- inclusion (Garey et al., 1996, 1998; Near et al., 1998; Giri- group taxa include representatives of other gnathiferan and bet et al., 2000, 2004; Mark Welch, 2000; Miquelis et al., platyzoan phyla, including Wve Gnathostomulida, four 2000; Near, 2002). Platyhelminthes, and the two described species of Cyclio- The discovery of the intriguing Micrognathozoa has phora. Most of the ingroup taxa were collected and placed a question mark to rotiferan monophyly. Kristensen sequenced speciWcally for the present study, while most out- and Funch (2000) originally described it as the sister group group sequences were obtained by the authors for two to Rotifera and Acanthocephala, whereas De Smet (2002) related studies (Giribet et al., 2004; Sørensen et al., 2006). interpreted the single micrognathozoan species Limnogna- Details on collecting localities and GenBank accession thia maerski as an aberrant monogonont rotifer; 18S rRNA numbers are reported in Table 1. and 28S rRNA sequence data suggested a position closer to Cycliophora and Gnathostomulida (Giribet et al., 2004). 2.2. DNA sequencing DiVerent morphological studies have dealt with the internal phylogeny of Rotifera and most