ORIGINAL PAPERPhylogeny of Novel Naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea Revised
Introduction
The protozoan phylum Cercozoa (Cavalier-Smith and Chao 2003) is a major component of marine, freshwater, and especially soil ecosystems (Bass and Cavalier-Smith 2004; Richards and Bass 2005). One subphylum (Filosa) comprises many filose amoebae (e.g. euglyphids, tectofilosids, chlorarachnean algae) and numerous zooflagellates, mostly of gliding life style and frequently strong pseudopodial tendencies – often filopodial (Bass et al. 2007b; Howe et al. 2008). Increasing numbers of previously unassigned zooflagellates (e.g. Cavalier-Smith et al. 2008; Hoppenrath and Leander, 2006a, Hoppenrath and Leander, 2006b; Vickerman et al. 2005) are now in the subphylum Filosa, as are two groups that catch prey by axopodia: desmothoracid pseudoheliozoa and phaeodarian radiolaria (Nikolaev et al. 2004; Polet et al. 2004). By contrast, zooflagellates are currently unknown in the other cercozoan subphylum, Endomyxa (Cavalier-Smith, 2000, Cavalier-Smith, 2002; Cavalier-Smith and Chao 2003), which comprises two parasitic groups (Phytomyxea and Ascetosporea) and several uncharacterised free-living lineages, at least one amoeboid: the testate reticulose Gromiidea (Burki et al. 2002; Cavalier-Smith and Chao 2003). The mostly heterotrophic euglyphids (e.g. Euglypha, Paulinella) and the heterotrophic tectofilosids (e.g. Pseudodifflugia and Lecythium) are testate filose amoebae, though not directly related, grouping instead with certain zooflagellates in the separate classes Imbricatea and Thecofilosea respectively (Cavalier-Smith and Chao 2003). In contrast to the three testate groups, naked cercozoan amoebae are poorly understood.
Indeed, naked heterotrophic filose and reticulose protozoa in general are little known, despite being common in freshwater, soil, and marine sediments. For over a century they were variously placed in taxa like Proteomyxidea (Lankester 1890) or Reticulosa (Cash 1905; Rhumbler 1904) that have long been thought to be polyphyletic. Though some filose amoebae were originally included, the classical core of the proteomyxids comprised reticulose taxa such as Biomyxa, Arachnula, Gymnophrys, Vampyrella, Pontomyxa and Protomyxa (Lankester 1890; Rhumbler 1904; Valkanov 1940) for which there is no molecular evidence, making the status of Proteomxyidea unclear. Many general treatments of Protozoa simply ignored them (Levine et al. 1980), grouped them piecemeal and arbitrarily with larger better established groups, e.g. filose testate amoebae, Mycetozoa, or Foraminifera, or placed them as protists incertae sedis (Lee et al. 2002). This paper characterises eight novel rhizopodial strains by microscopy and ribosomal rDNA sequencing, showing that all are Cercozoa. We also expand knowledge of the diversity of rhizopodial Cercozoa by sequencing many partial 18S rRNA genes amplified from environmental DNA samples using cercozoan-specific primers (Bass and Cavalier-Smith 2004). Phylogenetic analysis indicates a sharper phylogenetic distinction than was previously recognised between naked filose and naked reticulose cercozoan rhizopods.
The only published molecular data for naked heterotrophic reticulose protists are for Reticulomyxa, which turned out to be a foraminiferan (Pawlowski et al. 1999), and ‘Corallomyxa’ tenera (Tekle et al. 2007), which is an endomyxan cercozoan, but in our view was assigned to the wrong genus. To test the suggestion that other reticulose heterotrophs are Cercozoa (Cavalier-Smith and Chao 2003), we sequenced 18S rDNA of five non-flagellate reticulose amoebae (four newly discovered). All prove to be endomyxan Cercozoa, but belong to two not directly related clades: two are Arachnula-like and terrestrial; three are new species of a new marine genus Filoreta, morphologically similar to and grouping robustly with the recently described ‘Corallomyxa’ tenera (Tekle et al. 2007), which we now place in Filoreta. A culture previously named Gymnophrys cometa (Cavalier-Smith and Chao 2003; Mikrjukov and Mylnikov 1998; Nikolaev et al. 2003) was misidentified and misleadingly implied to be reticulose; it is actually filose, so we name it Limnofila borokensis.
Naked heterotrophic filose amoebae belong to at least two phyla; molecular evidence recently established that Nuclearia, which has non-granular filopodia and no cilia, belongs to the opisthokonts (Amaral Zettler et al. 2001), whereas four protozoa with granular filopodia (three of them also biciliate) belong in Cercozoa (Cavalier-Smith and Chao 2003). These known cercozoan naked filose amoebae are Massisteria marina, two previously unnamed species (formally named here as Nanofila marina and Mesofila limnetica), and L. borokensis. The clade comprising these four filose taxa and numerous unidentified environmental sequences was earlier referred to as Proteomyxidea (because of the confusion over ‘Gymnophrys’/Limnofila), but is here treated as a new naked filosan class Granofilosea, which is a deeply divergent branch of the cercozoan subphylum Filosa (Bass and Cavalier-Smith 2004; Bass et al. 2005; Cavalier-Smith and Chao 2003). As recent environmental DNA studies showed that the granofilosean clade is immensely diverse in all habitats (Bass and Cavalier-Smith 2004), yet is grossly under-represented by available cultures, we characterised three new non-marine granofilosean strains with exceedingly thin granular pseudopodia (all new Limnofila species); our study demonstrates the phylogenetic and phenotypic unity of Granofilosea.
Our microscopic studies and critical review of the literature revealed many misidentifications and past usage of invalid names. We therefore substantially revise the nomenclature and classification of many rhizopodial Cercozoa and discuss the evolutionary implications of our results. Their diversity in size, form, and feeding habits emphasizes that we are only beginning to appreciate the true heterogeneity of form and function in Cercozoa and the consequent diversity of their ecological roles and environmental impact.
Section snippets
Results
We first describe the positions of the rhizopodial strains in 18S rDNA phylogenies, using their new names throughout to avoid confusion with obsolete names. Then we describe the morphology of the new isolates and of previously sequenced but undescribed Mesofila limnetica (ATCC 50522), together with the taxonomic and classificatory revisions. Revised classifications of Granofilosea and Endomyxa are in Table 1, Table 2. Because a crucial element of this work was an extensive assimilation of
Class Ascetosporea
Order Paradinida Cavalier-Smith ord. n. Diagnosis: marine endomyxan parasites of Crustacea with multinucleate plasmodial trophic phase; unlike other ascestosporans have a biciliate dispersal phase with two unequal cilia. Not a typified name. Sole family Paradiniidae Schiller, 1935. Paradinium (Skovgaard and Daugbjerg 2008) and the spot prawn parasite (Bower and Meyer 2002).
Discussion
We draw three primary conclusions. First, all cultured members of the previously identified major filosan clade now named Granulofilosea are naked protists with extremely slender, often branching, filopodia or axopodia that invariably bear numerous obvious granules that are only barely mobile – nothing like the vigorous cytoplasmic movement in some Endomyxa; only some have apparently anastomosing filopodia (which contain microtubules in all cases studied by electron microscopy) and the degree
Methods
Isolation and maintenance of cultures: The new cultures were isolated as follows: Limnofila oxoniensis (DB isolate) – garden soil, Oxfordshire; L. anglica and L. longa – benthic sediment at edge of Priest Pot, Cumbria, UK (DB); Filoreta turcica – coastal marine sediment from rabbit island, Gümüslük, Turkey (EC); Filoreta marina – coastal marine sediment from near Kartesh, White Sea, Russia (A. P. Mylnikov); Filoreta japonica – coastal marine sediment from Fujie fishery port, Fukuyama,
Acknowledgements
We thank A.P. Mylnikov for the Filoreta marina culture, light micrographs of Clathrulina elegans and Hedriocystis reticulata, and discussion and advice. TC-S thanks NERC and the Canadian Institute for Advanced Research Evolutionary Biology Program for Fellowship support and NERC for a research grant, which supports DB and EC in part, and the Leverhulme Foundation for a research grant supporting CB. SN thanks Jan Pawlowski for hospitality in his laboratory where he sequenced Filoreta marina,
References (97)
- et al.
Fine structure of a marine proteomyxid and cytochemical changes during encystment
J Ultrastruct Res
(1979) - et al.
Polyubiquitin insertions and the phylogeny of Cercozoa and Rhizaria
Protist
(2005) - et al.
Phylogenetic position of Gromia oviformis Dujardin inferred from nuclear-encoded small subunit ribosomal DNA
Protist
(2002) Amoeboflagellates and mitochondrial cristae in eukaryotic evolution: megasystematics of the new protozoan subkingdoms Eozoa and Neozoa
Arch Protistenkd
(1997)- et al.
Phylogeny and classification of phylum Cercozoa (Protozoa)
Protist
(2003) - et al.
Molecular phylogeny of Amoebozoa and the evolutionary significance of the unikont Phalansterium
Europ J Protistol
(2004) Corallomyxa nipponica n. sp. and the phylogeny of plasmodial protists
Arch Protistenkd
(1991)Supplementary observations on Thalassomyxa jamaicensis Grell
Arch Protistenkd
(1992)Reticulamoeba gemmmipara n. gen., n. sp., an “amoebo-flagellate” with reticulopodia and zoosporogenesis
Arch Protistenkd
(1994)- et al.
Ebriid phylogeny and the expansion of the Cercozoa
Protist
(2006)
Molecular phylogeny of Cercomonadidae and kinetid patterns of Cercomonas and Eocercomonas gen. nov. (Cercomonadida, Cercozoa)
Protist
Fine structure of an unusual rhizopod, Penardia cometa, containing extrusomes and kinetosomes
Europ J Protistol
Cell division in the heliozoan Dimorpha mutans and evolution of centrosomal organization during the cell cycle
Biol Cell
Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata
Mol Phylogenet Evol
Small-subunit ribosomal RNA gene sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria
Protist
Molecular screening of free-living microbial eukaryotes: diversity and distribution using a meta-analysis
Curr Opin Microbiol
Identity and systematic position of Paradinium poucheti and other Paradinium-like parasites of marine copepods based on morphology and nuclear-encoded SSU rDNA
Protist
Correct identification of species makes the amoebozoan rRNA tree congruent with morphology for the order Leptomyxida Page 1987; with description of Acramoeba dendroida n. g., n. sp., originally misidentified as ‘Gephyramoeba sp.’
Europ J Protistol
A multigene analysis of Corallomyxa tenera sp. nov. suggests its membership in a clade that includes Gromia, Haplosporidia and Foraminifera
Protist
Phylogenetic placement of diverse amoebae inferred from multigene analyses and assessment of clade stability within ‘Amoebozoa’ upon removal of varying rate classes of SSU-rDNA
Mol Phylogenet Evol
Aurigamonas solis n. gen., n. sp., a soil-dwelling predator with unusual helioflagellate organisation and belonging to a novel clade within the Cercozoa
Protist
Parallel Metropolis-coupled Markov chain Monte Carlo for Bayesian phylogenetic inference
Bioinformatics
The nucleariid amoebae: more protists at the animal–fungal boundary
J Eukaryot Microbiol
Phylum-specific environmental DNA analysis reveals remarkably high global biodiversity of Cercozoa (Protozoa)
Int J Syst Evol Microbiol
Yeast forms dominate fungal diversity in the deep oceans
Proc R Soc B
DNA evidence for global dispersal and probable endemicity of protozoa
BMC Evol Biol
The SSU rDNA coding region of a filose amoeba contains a group I intron lacking the universally conserved G at the 3′-terminus
J Eukaryot Microbiol
Studies on helioflagellates. I. Morphology and fission of Dimorpha floridanis n. sp
Arch Protistenkd
Morphology and ultrastructure of a protistan pathogen in the haemolymph of shrimp (Pandalus spp.) in the northeastern Pacific Ocean
Can J Zool
Caractéristiques ultrastructurales de l’Hélioflagellé Tetradimorpha (Hsiung) et leur intérêt pour l’étude phylétique des héliozoaires
J Protozool
Les caractéristiques ultrastructurales de l’hélioflagellé Dimorpha mutans Gruber (Sarcodina – Actinopoda) et leur intérêt phylétique
Protistologica
The cell characters of two helioflagellates related to the centrohelidian lineage: Dimorpha and Tetradimorpha
Origins Life
Phylogenomics reshuffles the eukaryotic supergroups
PLoS ONE
Introduction to the Study of the Foraminifera
British freshwater rhizopoda
J Linn Soc Zool
The British Freshwater Rhizopoda and Heliozoa
Kingdom Protozoa and its 18 phyla
Microbiol Rev
Flagellate Megaevolution: The Basis for Eukaryote Diversification
The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa
Int J Syst Evol Microbiol
Phylogeny and megasystematics of phagotrophic heterokonts (Kingdom Chromista)
J Mol Evol
Un protéomyxée, Cinetidomyxa nucleoflagellata nov. gen. nov sp., à cinétide intracytoplasmique, et sa multiplication
C R Sci Soc Biol
Ueber einige Rhizopoden und verwandte Organismen
Arch Mikr Anat
Sur une protéomyxée (protiste plasmodial) développée dans des cultures d’algues marine et sur son rattachement au genre Gymnophrydium de P. A. Dangeard
C R Acad Sci Paris
Beitrag zur Kenntnis der Rhizopoden: morphologische und systematische Untersuchungen und ein Klassifikationsversuch
Mem Mus Roy d’Hist Nat Belgique
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