The Journal of Published by the International Society of Eukaryotic Microbiology Protistologists J. Eukaryot. Microbiol., 57(2), 2010 pp. 189–196 r 2010 The Author(s) Journal compilation r 2010 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2009.00466.x Invalidation of Hyperamoeba by Transferring its Species to Other Genera of Myxogastria ANNA MARIA FIORE-DONNO,a AKIKO KAMONO,b EMA E. CHAO,a MANABU FUKUIb and THOMAS CAVALIER-SMITHa aZoology Department, University of Oxford, South Parks Road, OX1 3PS Oxford, United Kingdom, and bThe Institute of Low Temperature Science, Hokkaido University, Kita 19, Nishi 8, Kita-ku, Sapporo, Hokkaido 010-0819, Japan ABSTRACT. The genus Hyperamoeba Alexeieff, 1923 was established to accommodate an aerobic amoeba exhibiting three life stages— amoeba, flagellate, and cyst. As more species/strains were isolated, it became increasingly evident from small subunit (SSU) gene phylo- genies and ultrastructure that Hyperamoeba is polyphyletic and its species occupy different positions within the class Myxogastria. To pinpoint Hyperamoeba strains within other myxogastrid genera we aligned numerous myxogastrid sequences: whole small subunit ribo- somal (SSU or 18S rRNA) gene for 50 dark-spored (i.e. Stemonitida and Physarida) Myxogastria (including a new ‘‘Hyperamoeba’’/ Didymium sequence) and a 400-bp SSU fragment for 147 isolates assigned to 10 genera of the order Physarida. Phylogenetic analyses show unambiguously that the type species Hyperamoeba flagellata is a Physarum (Physarum flagellatum comb. nov.) as it nests among other Physarum species as robust sister to Physarum didermoides. Our trees also allow the following allocations: five Hyperamoeba strains to the genus Stemonitis; Hyperamoeba dachnaya, Pseudodidymium cryptomastigophorum, and three other Hyperamoeba strains to the genus Didymium; and two further Hyperamoeba strains to the family Physaridae. We therefore abandon the polyphyletic and redundant genus Hyperamoeba. We discuss the implications for the ecology and evolution of Myxogastria, whose amoeboflagellates are more widespread than previous inventories supposed, being now found in freshwater and even marine environments. Key Words. Amoebozoa, molecular phylogeny, Mycetozoa, Myxomycetes, slime molds, SSU rRNA gene. genus Hyperamoeba being polyphyletic, its ‘‘raison d’eˆtre’’ is INCE its description in 1923, 12 amoeboflagellates have been questionable. To avoid taxonomic confusion, a more precise iden- S allocated to the genus Hyperamoeba Alexeieff. They have tification of Hyperamoeba taxa and isolates is highly desirable. been found in very diverse terrestrial and freshwater habitats Three strains are still available in culture (Table 1, and Hyper- (Karpov and Mylnikov 1997; Michel, Walochnik, and Aspo¨ck amoeba sp. ATCC 50247 deposited by T.K. Sawyer, not included 2003; Walker et al. 2003; Walochnik, Michel, and Aspo¨ck 2004; in this study) thus deserving a better determination for future use. Zaman and Adoutte 1999). An organism phylogenetically close to Also, Hyperamoeba SSU rRNA gene sequences have been de- Hyperamoeba dachnaya has been found in sea urchins of the Ad- posited in GenBank, and confuse the BLAST results when iden- riatic Sea (Dykova´ et al. 2007). Hyperamoeba is characterized by tifying environmental sequences (Kamono and Fukui 2006; the alternation of three life stages, amoeboid, flagellate, and Kamono et al. 2009a, b; Win Ko Ko et al. 2009). To avoid tax- cyst—a feature that is found in many unrelated protistan groups, onomic and phylogenetic confusion, clarifying the nomenclature such as Cercozoa, Heterolobosea, and Amoebozoa. of the two taxa for which EST projects are in progress—H. dach- The classification of Hyperamoeba has been controversial, es- naya, which has been often mis-spelled as ‘‘dachnya,’’ and pecially because a significant character, the second flagellum, Hyperamoeba sp. ATCC 50570—is especially important, since sporadic and adhering to the cell, has sometimes been overlooked the massive data that will be generated are and will be used to (see the excellent introduction by Walochnik et al. 2004 and ref- infer evolutionary hypotheses (Watkins and Gray 2008). We used erences therein; Dykova´ et al. 2007). Ultrastructural studies have our extensive database of dark-spored myxogastrian SSU rRNA revealed that a second basal body is always present, whether the gene sequences to find the closest relative to each isolate of second flagellum is developed or not, and striking ultrastructural Hyperamoeba, including a new one sequenced here, and renamed similarities with Myxogastria have been underlined (Dykova´ et al. all as precisely as possible. We abandon this redundant genus, 2007; Karpov and Mylnikov 1997; Walker et al. 2003; Walochnik which is a life form not a taxon, and establish new combinations et al. 2004). The flagellar apparatus in myxogastrids (including within the genera Stemonitis, Didymium, and Physarum for all Ceratiomyxa fruticulosa) is highly conservative (Karpov and My- named Hyperamoeba species. lnikov 1997; Nelson and Scheetz 1975) and is characterized by two centrioles mutually oriented at a roughly orthogonal angle varying from 601 to 1201, connected by a ‘‘short striated fibre’’ whose variations among taxa may have phylogenetic meaning MATERIALS AND METHODS (see Karpov, Novozhilov, and Chistiakova 2003, Walker et al. 2003) for detailed illustrations and discussion). This particular Culture, DNA extraction, and sequencing. We found an flagellar apparatus is probably an apomorphy of the clade My- Hyperamoeba-like flagellate as a contaminant in plates of Mi- xogastria1C. fruticulosa—Myxogastrea sensu (Cavalier-Smith, cronuclearia, whose origin could not be established. The culture, Chao, and Oates 2004), as confirmed by molecular phylogeny named Didymium sp. OX13PS, is maintained in Prof. Cavalier- (Fiore-Donno et al. 2010). Smith’s laboratory in Volvic natural mineral water (available in Phylogenies based on the small subunit (SSU) rRNA gene have most shops) together with bacteria carried over during subcultur- confirmed that all Hyperamoeba sequenced to date belong to ing. DNA was extracted with the MoBio UltraClean Soil DNA kit different groups of Myxogastria: some cluster in the order Physar- (MO BIO Laboratories Inc., Carlsbad, CA). The SSU rRNA gene ida in the families Physaridae and Didymiidae, and some in the ( 1,900 bp long) was amplified with primers G and F (G: 50-AA order Stemonitida (Dykova´ et al. 2007; Michel et al. 2003; Walker CCTGGTTGATCCTGCCAGTAGTCATATGC-30;F:50-GATC et al. 2003; Walochnik et al. 2004; Zaman and Adoutte 1999). The CTTCTGCAGGTTCACTAC-30). The sequence was obtained in three overlapping fragments, using the primer pairs G-9R (9R: 50-TT 0 0 Corresponding Author: A. M. Fiore-Donno, Zoology Department, AGAGCTGGAATTACCG-3 ), 4F-12R (4F: 5 -CCGCGGTAATT 0 0 0 University of Oxford, South Parks Road, OX1 3PS Oxford, United CCAGCTCC-3 ; 12R: 5 -ACCGGCCATGCACCACC-3 ) and 6F- Kingdom—Telephone number: 1441865281322; FAX number: F (6F: 50-GGTGGTGCATGGCCG-30). Although these primers 144165281310; e-mail: afi[email protected] have some mismatches with the sequence, this has not impaired 189 190 J. EUKARYOT. MICROBIOL., 57, NO. 2, MARCH–APRIL 2010 Table 1. New names given to Hyperamoeba species and strains. Asterisks indicate EST ongoing projects. New name Previous name Reference Origin Substrate GenBank # Stemonitis sp. Hyperamoeba sp. strain A1K Walochnick et al. DE, Melsbach Maple tree bark AY321107 (2004) Stemonitis sp. Hyperamoeba sp. strain AH1 Walochnick et al. DE, Melsbach Maple tree bark AY321108 (2004) Stemonitis aff. flavogenita Hyperamoeba sp. strain B1/2 Walochnick et al. DE, Plauen Drinking water AY321109 (2004) treatment plant Stemonitis sp. Hyperamoeba sp. strain BuP Walochnick et al. DE, Melsbach Beech tree bark AY321110 (2004) Stemonitis aff. flavogenita ÃHyperamoeba sp. ATCC50570 Zaman & Adoutte PK, Karachi Human fecal sample AF093247 (1999) Physarum flagellatum (Alexeieff) Hyperamoeba flagellata Karpov & Mylnikov RU, Yaroslavl Surface ice of a pond AF411289 Fiore-Donno, Kamono & Alexeieff ATCC 50637 (1997) Cavalier-Smith 2009, comb. nov. Undetermined Physaridae Hyperamoeba sp. G1a Walochnick et al. DE, Giessen Physiotherapy bath AY321113 (2004) Undetermined Physaridae Hyperamoeba sp. W2i Walochnick et al. DE, Wielbad Physiotherapy bath AY321115 (2004) Didymium sp. Hyperamoeba sp. E2/8 Walochnick et al. DE, Dresden Drinking water AY321111 (2004) treatment plant Didymium sp. Hyperamoeba sp. E3P Walochnick et al. DE, Dresden Drinking water AY321112 (2004) treatment plant Didymium sp. Hyperamoeba sp. Hpl Walochnick et al. DE, Andernach Sycamore tree bark AY321114 (2004) Didymium dachnayum (Walker, ÃHyperamoeba dachnaya Walker et al. RU, St-Petersburg Sediments under the AY062881 Karpov, Frolov & Patterson) Walker, Karpov, Frolov & (2003) ice, lake Osinovoye Fiore-Donno, Kamono & PattersonCCAP1535/1 Cavalier-Smith Didymium cryptomastigophorum Pseudodymium Michel et al. (2003) DE, Wildbad Physiotherapy bath AY207466 (Michel, Walochnik & Aspock) cryptomastigophorum Fiore-Donno, Kamono & Michel, Walochnik & Aspock Cavalier-Smith CCAP1573/1 their effectiveness. The sequence is deposited in GenBank under added where longer sequences had insertions, in order to keep as accession number GQ249857. many homologous positions as possible in the final alignment Sequence alignments. (Fig. 1). The length of Helix 44 was larger in Didymiidae (mean Large dark-spored clade alignment. A first dataset of length: