ARTICLE IN PRESS Protist, Vol. 158, 295—324, July 2007 http://www.elsevier.de/protis Published online date 2 July 2007 ORIGINAL PAPER Phylogeny, Evolution, and Taxonomy of Vannellid Amoebae Alexey V. Smirnova,1, Elena S. Nassonovab, Ema Chaoc, and Thomas Cavalier-Smithc aFaculty of Biology and Soil Sciences, St. Petersburg State University, Universitetskaja nab. 7/9, 199034 St. Petersburg, Russia bLaboratory of Cytology of Unicellular Organisms, Institute of Cytology RAS, Tikhoretsky ave. 4, 194064 St. Petersburg, Russia cDepartment of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK Submitted October 24, 2006; Accepted April 21, 2007 Monitoring Editor: C. Graham Clark We sequenced 18S rRNA genes from 21 vannellid amoebae (Amoebozoa; Vannellidae), including nearly all available type cultures, and performed a comprehensive phylogenetic analysis for 57 Vannellidae sequences. The results show that species of Vannella and Platyamoeba are completely mixed and do not form distinct clades. Several very closely related species pairs exist, each with a Vannella and a Platyamoeba species differing in only a few nucleotides. Therefore, presence (Vannella) or absence (Platyamoeba) of glycostyles in the cell surface coat is an invalid generic distinction; the genera must be merged. As Vannella has priority, we formally transferred Platyamoeba species into Vannella, except for the non-vannellid P. stenopodia, here renamed Stenamoeba stenopodia gen. n. comb. n. and transferred to the family Thecamoebidae. Our trees show that Vannella glycostyles were probably easily and repeatedly evolutionarily lost. We have established a new genus Ripella, with distinct morphology and sequence signatures for Vannella platypodia and morphologically similar species that form a clearly separate clade, very distant from other Vannellidae. Vannellids form four well-separated single-genus clades: Vannella sensu stricto, Ripella, Clydonella, and Lingulamoeba. Species of the revised genus Vannella comprise four closely related, well-supported subclades: one marine and three freshwater. Here, we provide an illustrated checklist for all 40 known Vannellidae species. & 2007 Elsevier GmbH. All rights reserved. Key words: glycocalyx evolution; molecular identification; phylogeny; species problem; taxonomy. Introduction Flattened, fan-shaped amoebae of the family Discosea (Cavalier-Smith et al. 2004). Initially, they Vannellidae have only recently been accepted as were dispersed among various genera and an important major amoebozoan taxon. Once families. Probably the first reliably described classified by Page (1987) in the class Lobosea vannellid was ‘Amoeba platypodia’(Gla¨ ser 1912). subclass Gymnamoebia, they are now considered Schaeffer (1926) included vannellid amoebae then as members of the phylum Amoebozoa, class known in three genera, all in different families: Flabellula, where he placed ‘F. mira’ (now Vannella 1 Corresponding author; mira); Rugipes, where he placed R. vivax (now e-mail [email protected] (A.V. Smirnov). Clydonella vivax), and Unda with a not yet & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2007.04.004 ARTICLE IN PRESS 296 A.V. Smirnov et al. redescribed species U. maris. Wohlfarth-Botter- included only two genera: Vannella and Platya- mann (1960) added a fourth genus to this moeba (Page 1987). Later Rogerson and Patter- collection: ‘Hyalodiscus simplex’ (now Vannella son (2002) included Clydonella and Pessonella in simplex). Bovee (1965) established the genus the family Vannellidae together with the little- Vannella, for three of these species — ‘Flabellula known genera Discamoeba Jahn, Bovee and mira’, ‘Amoeba platypodia’, and ‘Hyalodiscus Griffith 1979 and Unda Schaeffer 1926, but simplex’; he redefined the genus Flabellula, omitted the genus Lingulamoeba, thus accepting restricting it to amoebae producing trailing uroidal Page’s suggestion that it is a synonym of filaments and short conical subpseudopodia, Platyamoeba (Page 1983). neither character being present in Vannella. How- These taxonomic perturbations well illustrate ever, he considered the genus Vannella as closely the difficulty of morphological systematics of related to flabellulid amoebae, so placed it in the vannellid amoebae. The primary reason for this family Flabellulidae; later he created a separate is that all vannellids are flattened, fan-shaped subfamily Vannellinae within Flabellulidae (Bovee organisms with a very limited number of light- 1970). microscopic (LM) morphological characters. Page F.C. Page initially did not accept the genus (1983) noted that a vannellid amoeba with slender, Vannella and provided a new diagnosis for tapering pseudopodia on the floating form can be Schaeffer’s species ‘Flabellula mira’ (Page 1968). safely classified as Vannella; however, further The same paper described a new species, studies show that some vannellas do not form Rugipes placidus, thus accepting the validity of such pseudopodia when afloat (Smirnov 2001), Schaeffer’s genus Rugipes. However, a year later while some platyamoebian floating forms may be Page accepted Bovee’s revision of Flabellula and very similar to those of vannellas (Smirnov 1999). noted that species redescribed by him as Flabel- Therefore, when electron-microscopic (EM) stu- lula must be identified as Vannella; he also dies revealed clear differences in cell surface established the genus Platyamoeba to accommo- structure between the then known species of date Rugipes placidus and a newly described Vannella (having glycostyles) and Platyamoeba species, P. stenopodia (Page 1969). He believed (lacking glycostyles) (Page and Blakey 1979), this these amoebae to be the closest relatives of cell surface structure immediately became the thecamoebids, not flabellulids, so placed Platya- key, and virtually only, character used to distin- moeba in the family Thecamoebidae. Pussard guish these genera and to classify all newly (1973) created a genus Pessonella for a single discovered vannellids into one of them. Page species of Vannella-like amoeba — Pessonella (1979) provided a new diagnosis of the genus marginata, possessing characteristic lobes on the Vannella that included cell surface structures. surface of the frontal hyaloplasm, placing it in the However, this character was not always congruent family Mayorellidae; later Page (1976) transferred with LM features of a species. For example, Page all these genera into Thecamoebidae. (1980b, p. 939) wrote that it was a ‘considerable Sawyer (1975a,b) established two more genera surprise’ to find that a Platyamoeba-like strain of a of vannellid amoebae — Clydonella and Lingula- vannellid amoeba nowadays known as Vannella moeba — to accommodate Vannella-like amoe- anglica possesses glycostyles. bae differing from both Vannella and Platyamoeba Molecular phylogenies showed vannellid amoe- in locomotive morphology and floating form. The bae as a distinct monophyletic lineage of Amoe- type species of Clydonella was Schaeffer’s bozoa, well separated from the other groups Rugipes vivax. Differentiation of these two genera (Bolivar et al. 2001; Fahrni et al. 2003; Sims et from Vannella and Platyamoeba was difficult, since al. 1999). Comprehensive analyses of amoe- no ultrastructure was provided at the time of bozoan SSU rRNA trees by Cavalier-Smith et al. description. Page (1983) considered Lingula- (2004), Kudryavtsev et al. (2005), and Smirnov et moeba a junior synonym of Platyamoeba (the al. (2005) strongly confirmed that all vannellids combined name Platyamoeba leei was provided), form a distinct clade on the SSU tree, consisting and listed Sawyer’s Clydonella among ‘other of four genera: Vannella, Platyamoeba, Clydonella, genera of Thecamoebidae’. Later Page (1987) and Lingulamoeba. Peglar et al. (2003) provided started to consider all these genera (and Pesso- SSU rRNA sequences and ultrastructure of Cly- nella) as possible synonyms of Vannella or donella and Lingulamoeba, which confirmed the Platyamoeba (Page 1988). His revised classifica- validity of both genera and the monophyly tion of naked amoebae raised the rank of Bovee’s and distinctiveness of vannellid amoebae as a subfamily Vannellinae to family Vannellidae, which whole. However, relationships between the genera ARTICLE IN PRESS Phylogeny, Evolution, and Taxonomy of Vannellid Amoebae 297 Vannella and Platyamoeba remain ill-resolved. It branch attraction artefact). The bipartition was shown that Platyamoeba stenopodia is not a between Glycostylida/Himatismenida (‘core Dis- vannellid, and is closely related with thecamoe- cosea’) and all the other taxa was reproducible but bids (Fahrni et al. 2003). Sims et al. (2002) using had only moderate to low support. Some trees partial SSU rDNA sequences found that Vannella suggest that Cochliopodium is sister to Dactylo- and Platyamoeba are mixed in the tree and podida plus Vannellidae, but others put it as sister suggested that their phylogeny is related more to of Vannellidae alone, but all trees grouped these LM features and to habitat (freshwater or marine) three taxa together, albeit with only weak to than to cell surface structure. Finally, Dykova´ et al. moderate support. Figure 1 strongly confirms that (2005a) obtained sequences of fish-associated Multicilia belongs to Varipodida (into which we vannellids identified only to genus, confirming that now transfer it from Glycostylida), making the strains with a surface structure formerly attributed class Flabellinea (Smirnov et al. 2005) and the to Vannella and Platyamoeba do not form distinct order