Characterization of Selenaion koniopes n. gen., n. sp., an Amoeba that Represents a New Major Lineage within Heterolobosea, Isolated from the Wieliczka Salt Mine

A new heterolobosean amoeba, Selenaion koniopes n. gen., Characterization of Selenaion koniopes n. gen, n. sp, an Amoeba that Represents a New Major Lineage within Heterolobosea, Isolated from the Wieliczka Salt Mine JONG SOO PARK, a JOHAN F. DE JONCKHEERE b, c and ALASTAIR G. B. SIMPSON d a Department of Oceanography and Institute for Phylogenomics and Evolution, Kyungpook National University, Sangju, 742- 711, Korea, and b Research Unit for Tropical Diseases, de Duve Institute, B- 1200, Brussels, Belgium, and c Scientific Institute of Public Health, B- 1050, Brussels, Belgium, and d Canadian Institute for Advanced Research, Program in Integrated Microbial Diversity, and Department of Biology, Dalhousie University, Halifax, B3H 4R2, Canada ABSTRACT. A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73& saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater- like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm- these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_ 1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep- branching position being moderate ( i. e. maximum likelihood bootstrap support- 67%; posterior probability- 0.98). Cells grew at 15- 150 & salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group. Key Words. Cyst, Discicristata, excavate, halophile, ITS, pores, protist, protozoa, 18S rDNA. T HE taxon Heterolobosea Page and Blanton 1985 is a diverse group that includes amoebae ( e. g. Vahlkampfia, Neovahlkampfia), amoeboflagellates ( e. g. Naegleria, Tetramitus, Heteramoeba), typical flagellates ( e. g. Percolomonas), the pseudociliate Stephanopogon, and the aggregative slime moulds Acrasis and Pocheina ( Brown et al. 2012; Cavalier- Smith and Nikolaev 2008; Patterson et al. 2000). Most are free living, although one species, Naegleria fowleri, is an opportunistic pathogen that very rarely causes Primary Amoebic Meningoencephalitis ( PAM), a lethal brain infection ( De Jonckheere 2002). Heterolobosea are most commonly thought of as inhabitants of soils and freshwater, and they are also well documented from marine waters and sediments ( Page 1983, 1988; Patterson et al. 2000). Nonetheless, the group is also a showcase of the abilities of microbial eukaryotes to live under more extraordinary environmental conditions. Numerous species, including the pathogen N. fowleri, are moderate thermophiles that are capable of growing above 40 C ( De Jonckheere 2002). The recently described Euplaesiobystra hypersalinica and Marinamoeba thermophila can grow at 50 C ( De Jonckheere et al. 2009; Park et al. 2009), whereas Tetramitus thermacidophilus and Oramoeba fumarolia can grow even at 54- 55 C ( Baumgartner et al. 2009; De Jonckheere et al. 2010). Most impressively, M. thermophila and O. fumarolia actually grow optimally at 48 C and 50 C, respectively ( De Jonckheere et al. 2009, 2010). Tetramitus thermacidophilus is also an acidophile that can grow below pH 1.5 ( Baumgartner et al. 2009), whereas an environmental 18S rRNA gene sequence from a heterolobosean has been amplified from the Rio Tinto at pH 2 ( Amaral Zettler et al. 2002). Another assemblage of Heterolobosea are anaerobes/ microaerophiles. This group includes Psalteriomonas, Sawyeria, Monopylocystis, and Harpagon, and phylogenetically represents a single clade ( O'Kelly et al. 2003; Pa ' nek et al. 2012). Finally, organisms that are likely to be heteroloboseans have been observed repeatedly in very hypersaline samples, such as solar salterns, Dunaliella salina production facilities, natural salt lakes, and salt mines ( e. g. Hamburger 1905; Namyslowski 1913; Post et al. 1983; Rogerson and Hauer 2002; Ruinen 1938; Volcani 1943; - for summaries see Hauer and Rogerson 2005b; Park et al. 2009). Several halophilic heteroloboseans have now been cultured and examined using modern techniques, including Euplaesiobystra, Pharyngomonas, Pleurostomum, and Tulamoeba ( Park et al. 2007, 2009; Park and Simpson 2011). Isolates of all of these taxa can grow in media of least 250& salinity, with Pleurostomum flabellatum apparently showing optimal growth at 300& salinity ( Park et al. 2007). The halophilic heteroloboseans cultured so far represent several distinct clades in 18S rRNA gene trees. Pleurostomum and Tulamoeba are relatively closely related to Naegleria, whereas Euplaesiobystra is more closely related to Heteramoeba ( Park et al. 2009), and Pharyngomonas represents the deepest branch within Heterolobosea, and is without known specific relatives ( Nikolaev et al. 2004; Park and Simpson 2011). This indicates that there is a substantial sample of the high- level diversity of Heterolobosea present in hypersaline habitats, and suggests the possibility that additional phylogenetically important heterolobosean groups might await discovery there. The Wieliczka mine in Poland is one of the oldest salt mines in Europe, and is of historical importance for the study of halophilic eukaryotes. Almost a century ago, Namyslowski ( 1913) reported several protozoa from the waters in this salt mine. We here characterize a new heterolobosean amoeba that was isolated from a sample of intermediate hypersalinity ( i. e. 73& salinity) from the Wieliczka salt mine. This halotolerant amoeba is not closely related to previously characterized halophiles in 18S rRNA gene phylogenies, and represents a previously undetected higher level lineage within Heterolobosea. MATERIALS AND METHODS Isolation and cultivation. Amoeba isolate A5 was isolated from 73& salinity water collected in June 2008 from the Wieliczka salt mine, Poland ( 49 58' 59. N, 20 3' 21. W). Park and Simpson ( 2010) previously reported the isolation of the Corresponding Author: A. Simpson, Department of Biology, Dalhousie University, 1355 Oxford St, Halifax, Nova Scotia B3H 4R2, Canada- Telephone number: + 902 494 1247; FAX number: + 902 494 3736; e- mail: Alastair. Simpson @ dal. ca J. Eukaryot. Microbiol., 59( 6), 2012 pp. 601- 613 c 2012 The Author( s) Journal of Eukaryotic Microbiology c 2012 International Society of Protistologists DOI: 10.1111/ j. 1550- 7408.2012.00641. x 601 Published by the International Society of Protistologists Eukaryotic Microbiology The Journal of. sp., was isolated from 73 parts per thousand saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater-like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasmthese may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep-branching position being moderate (i.e. maximum likelihood bootstrap support 67%; posterior probability 0.98). Cells grew at 15-150 parts per thousand salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.