FOLIA PARASITOLOGICA 55: 256–264, 2008 Phylogeny of Flabellulidae (Amoebozoa: Leptomyxida) inferred from SSU rDNA sequences of the type strain of Flabellula citata Schaeffer, 1926 and newly isolated strains of marine amoebae Iva Dyková1,2, Ivan Fiala1,2, Hana Pecková1 and Helena Dvořáková1 1Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic; 2Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic Key words: Amoebozoa, Leptomyxida, Flabellulidae, Flabellula citata, SSU rDNA phylogeny Abstract. New strains of non-vannellid flattened amoebae isolated from fish, an invertebrate and the marine environment were studied together with Flabellula citata Schaeffer, 1926 selected by morphology as a reference strain. The study revealed a pau- city of features distinguishing individual strains at the generic level, but clearly evidenced mutual phylogenetic relationships within the assemblage of strains as well as their affiliation to the Leptomyxida. In this study, the SSU rDNA dataset of lepto- myxids was expanded and a new branching pattern was presented within this lineage of Amoebozoa. Sequences of three newly introduced strains clustered in close relationship with the type strain of F. citata, the type species of the genus. Three strains, including one resembling Flamella sp., were positioned within a sister-group containing Paraflabellula spp. Results of phyloge- netic analysis confirmed doubts of previous authors regarding generic assignment of several Rhizamoeba and Ripidomyxa strains. Naked amoebae with fan-shaped trophozoites flat- In phylogenetic analyses based on SSU rDNA se- tened to a substrate were described in the early period of quences, two representatives of Flabellulidae, P. reni- amoeba research. At that time, Schaeffer (1926) estab- formis and P. hoguae (Sawyer, 1975) were presented as lished the genus Flabellula for marine amoebae with members of Leptomyxa-Hartmannella (LH) clade in triangular or flabelliform trophozoites. Amaral Zettler et al. (2000). The same phylogenetic Amendments to the generic diagnosis of Flabellula position of these two sequences was recognized by Bo- Schaeffer, 1926 by Bovee (1965) made it consistent livar et al. (2001) when they investigated the ancestry of with the diagnosis of the type species (F. citata Gymnamoebae. The LH lineage persisted in later analy- Schaeffer, 1926) and lead to the erection of the genus sis of the Gymnamoebia (Peglar et al. 2003) but con- Vannella Bovee, 1965. Bovee (1970) also removed Fla- tained only the sequence of P. hoguae. A clade denomi- bellula from the Mayorellidae and established the fam- nated “Leptomyxoidea” with sequences of two Parafla- ily Flabellulidae. The re-diagnosis of the genus Flabel- bellula spp., Rhizamoeba sp. and Leptomyxa reticulata lula that included also ultrastructural features was pub- Goodey, 1914 was presented by Cavalier-Smith et al. lished by Page in 1980. Page (1983) later unified three (2004) in their revision of the higher-level classification genera, Flabellula, Paraflabellula Page et Willumsen, of Amoebozoa. These authors identified this clade 1983, and Flamella Schaeffer, 1926, within the family within the superfamily Leptomyxoidea Pussard et Pons, Flabellulidae Bovee, 1970. In addition, Flabellula citata 1976 and divided it into Flabellulidae Bovee, 1970 and Schaeffer, 1926, Paraflabellula reniformis (Schmoller, Leptomyxidae Pussard et Pons, 1976. In the high-rank 1964) and Flamella magnifica Schaeffer, 1926 were phylogenetic classification of Amoebozoa, Smirnov et designated as the type species of the respective genera. al. (2005) mentioned the genus Flabellula as “probably The inclusion of Flamella Schaeffer, 1926 in the Flabel- belonging” to the order Leptomyxida. This was due to lulidae was confirmed by Michel and Smirnov (1999), the lack of representation of Flabellula gene sequences who amended the generic diagnosis and described two but evident morphological similarities of Flabellula new species (F. aegyptia and F. lacustris). The five spe- with other genera assigned to the families Leptomyxi- cies of Flabellula were used by Smirnov and Goodkov dae Pussard et Pons, 1976 and Flabellulidae Bovee, (1999) to exemplify “flabellate” morphotype of the 1970. In the sequence analysis of 27 strains of Lobosea, Gymnamoebia, whereas for species of the genera Para- Smirnov et al. (2008) introduced CCAP1570/42 strain flabellula and Flamella they defined “paraflabellulian” as a new species of Leptomyxida, most accurately rep- morphotype. resenting Rhizamoeba saxonica Page, 1974. In the same Address for correspondence: I. Dyková, Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic. Phone: +420 387 775 423; Fax: +420 385 310 388; E-mail: [email protected] 256 Dyková et al.: Phylogeny of Flabellulidae paper, Smirnov et al. (2008) questioned the generic as- UKNCC) were heavily contaminated with bacteria and they signment of the ATCC 50742 strain to Rhizamoeba, the failed to propagate, therefore only their morphology could be validity of the Ripidomyxa sp., and excluded Flamella observed. from Leptomyxida. Light microscopical observations of living trophozoites Although the search for correlation between phy- were made in hanging drop preparations, using the Olympus logenetic and morphological groupings of amoeboid Nomarski differential interference contrast (DIC). Hoechst protists has motivated the research on these organisms 33258 (Sigma) fluorescent dye was used to visualise nuclei in trophozoites after their fixation in 95% ethanol. The images of for more than a decade, gene sequences of the strain trophozoites characteristic of individual strains were selected representatives of many genera have not been included from sets counting 40 to 60 trophozoites for each strain. Sam- in phylogenetic tree reconstructions. Some clades are ples for transmission electron microscopy were prepared as poorly represented, and the gene sequences of some previously described (Dyková et al. 2003). Ultrathin sections species that are morphologically assigned to the same were examined with a JEOL JEM 1010 electron microscope genus are in distant phylogenetic positions and await operating at 80 kV. Images were collected with MEGAview II revision of their generic identification. soft imaging system using analySIS® software. Typical ultra- This study was undertaken with the aim to incorpo- structural features were studied in ultrathin sections from tro- rate into phylogenetic analyses the SSU rDNA sequence phozoites fixed in two to four different periods of subcultur- of the type strain of Flabellula citata Schaefer, 1926 ing. DNA was extracted from pelleted trophozoites using the and clarify phylogenetic positions of other strains with DNeasyTM Tissue Kit (Qiagen GmbH, Germany) according to similar light microscopical and ultrastructural features. the manufacturer’s protocol. Universal eukaryotic primers (5’ ACCTGGTTGATCCTGCCAG 3’ and 5’ CTTCCGCAG- MATERIALS AND METHODS GTTCACCTACGG 3’) (Barta et al. 1997) were used for am- The study is based on six strains of marine amoebae we plification of the SSU rRNA gene. PCR was carried out in isolated from the tissues of three species of fishes, turbot, 25 µl reaction volume using 10 pmol of each primer, 250 µM Psetta maxima (L.), (Pleuronectiformes: Scophthalmidae); of each dNTP, 2.5 µl 10 × PCR Buffer (Top-Bio, Czech Re- triggerfishes, Sufflamen verres (Gilbert et Starks) and Balistes public) and 1 Unit of TaqDNA polymerase (Top-Bio, Czech polylepis Steindachner (both Tetraodontiformes: Balistidae); Republic). The reactions were run on a Tpersonal Thermocy- starfish, Porania pulvillus (O. F. Müller) (Asteroida: Porani- cler (Biometra). The thermal cycling conditions consisted of dae); and from sediments and net material of sea floating initial denaturation at 95°C (5 min), 30 cycles of denaturation cages used in Atlantic salmon aquaculture (Table 1). These at 94°C (1 min), annealing at 48°C (1.5 min) and extension at strains were selected by their light microscopical features, 72°C (2 min) followed by a final extension at 72°C (10 min). which were similar to those of flabellulids. In addition, the Following visualisation of PCR products via gel electrophore- type strain of Flabellula citata (CCAP 1529/2), the type spe- sis, amplification products were extracted from the agarose cies of the genus, obtained from UK National Culture Collec- using JETQUICK Gel Extraction Spin Kit (Genomed, Ger- tion (UKNCC), was included in the study. New strains were many), then cloned into pCR® 2.1 TOPO Cloning vector using isolated using MY75S (Malt & Yeast Extract-75% seawater) the TOPO-TA Cloning Kit (Invitrogen) and sequenced on an agar (Catalogue of the UKNCC, 2001) with the content of automatic sequencer CEQTM 2000 using CEQ DTCS Dye Kit extracts reduced to half. Primary isolates of amoebae were (Beckman Coulter) according to the manufacturer’s protocol. either directly transferred onto MY75S agar or after the The complete SSU rDNA sequence was obtained stepwise amount of contaminating bacteria was reduced using MY75S using a combination of flanking and internal primers as men- agar with reduced content of extracts. Subculturing of strains tioned elsewhere (Dyková et al., in press). and clonal cultures derived from them were accomplished on SSU rDNA sequences were aligned by Clustal_X