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

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Abstract

Placing amoeboid lineages on the eukaryotic tree of life is difficult due to the paucity of comparable morphological characters and the limited molecular data available for many groups. This situation has led to the lumping of distantly related lineages into large inclusive groups, such as Sarcodina, that do not reflect evolutionary relationships. Previous analyses of molecular markers with limited taxon sampling reveal members of Sarcodina are scattered in five of the six proposed supergroups. We have used multigene analyses to place seven diverse amoeboid lineages—two Nolandella spp., Rhizamoeba sp., Pessonella sp., Arcella hemisphaerica, Arachnula sp. and Trichosphaerium sp.—on the eukaryotic tree of life. Bayesian analysis of the concatenated data of the four genes sequenced (SSU-rDNA, actin, alpha-tubulin and beta-tubulin), including diverse representatives of eukaryotes, indicates that all seven taxa group within the ‘Amoebozoa’ supergroup. We further performed separate analyses of the well-sampled SSU-rDNA and actin genes using Bayesian and Maximum Likelihood analyses to assess the positions of our newly characterized taxa. In the case of SSU-rDNA, we performed extensive analyses with removal of the fastest rates classes to evaluate the stability and resolution of various taxonomic hypotheses within ‘Amoebozoa’. Five of our seven amoeboid lineages fall within well-supported clades that are corroborated by morphology. In contrast, the positions of Arachnula sp. and Trichosphaerium sp. in the SSU-rDNA gene trees are unstable and vary by analyses. Placement of these taxa will require additional data from slowly evolving genes combined with taxon-rich phylogenetic analyses. Finally, the analyses without the fastest rate classes demonstrate that SSU-rDNA has a limited signal for deep relationships within the ‘Amoebozoa’.

Introduction

The term amoeba is used to describe the many unicellular eukaryotes that move by temporary cytoplasmic projections known as pseudopods. The taxonomy of amoeboid lineages has had a turbulent history creating substantial confusion in the literature (Schaeffer, 1926, Singh, 1955, Loeblich and Tappan, 1961, Jahn and Bovee, 1965, Jahn et al., 1974, Bovee, 1985, Page and Blanton, 1985, Page, 1986, Page, 1987, Rogerson and Patterson, 2002, Cavalier-Smith, 2000, Cavalier-Smith, 2003, Cavalier-Smith et al., 2004, Smirnov et al., 2005, Adl et al., 2005). Amoeboid lineages traditionally placed in broad taxonomic groups such as the Sarcodina (Schmarda, 1871) are now found scattered in five of the six proposed eukaryotic supergroups (Baldauf et al., 2000, Simpson and Roger, 2002, Nikolaev et al., 2004, Adl et al., 2005), though many lineages remain homeless (Patterson, 1999, Smirnov et al., 2005, Nikolaev et al., 2006, Cavalier-Smith et al., 2004). Comparative systematics has failed for over a century to provide a robust comprehensive higher-level taxonomic framework for amoeboid protists, despite considerable effort (Bovee, 1953, Flickinger, 1974, Page, 1978, Page, 1980, Page, 1985, Page, 1986, Page, 1987, Page, 1988, Page, 1991, Page and Blakey, 1979, Page and Blanton, 1985, Rogerson and Patterson, 2002). This is due to few comparable morphological characters, though ultrastructural identities have been reported for lower-level taxonomic groups (Page, 1983, Page, 1988, Patterson, 1999, Rogerson and Patterson, 2002). Molecular systematics is introducing new comparable characters that will ultimately yield insights into a more natural higher-level classification of amoeboid protists.

The supergroup ‘Amoebozoa’, whose members are the focus of this study, emerged from analyses of molecular data (e.g. Cavalier-Smith, 1998, Fahrni et al., 2003, Kudryavtsev et al., 2005, Nikolaev et al., 2006). The ‘Amoebozoa’ lack a clear morphological synapomorphy though they share a few broadly defined morphological characters such as dynamic pseudopodia and branched tubular mitochondrial cristae (Cavalier-Smith, 1998). This putative supergroup includes many of the naked amoebae (e.g. Amoeba proteus), testate lobose amoebae (e.g. Arcella) and pelobionts (e.g. Pelomyxa, Entamoeba histolytica) as well as cellular (dictyostelid), acellular (e.g. myxogastrid) and protostelid slime molds.

We performed multigene analyses to investigate the phylogenetic placement of seven newly characterized taxa within the eukaryotic tree of life. Multigene analyses are proving useful as a means of placing eukaryote lineages within phylogenies where morphological evidence and single gene analyses have not been successful (e.g. Nikolaev et al., 2004, Tekle et al., 2007). We obtained sequences of SSU-rDNA as well as three protein-coding genes (actin, alpha-tubulin and beta-tubulin) of several amoebae. Specifically, we characterized genes from two amoebae of unknown phylogenetic position: Arachnula sp. and Trichosphaerium sp. and other taxa including two Nolandella spp., Pessonella sp., Rhizamoeba sp. and Arcella hemisphaerica for which no molecular data were previously available.

We further performed single gene analyses of the relatively well-sampled genes (actin and SSU-rDNA) to determine the position of our newly characterized sequences within the supergroup ‘Amoebozoa’. Particularly, the SSU-rDNA has been used as the basis for identifying major clades within the ‘Amoebozoa’ including Tubulinea, Flabellinea and Conosea (see Smirnov et al., 2005), though the relationship among these lineages is unknown and many of these clades are poorly supported. It is not known whether the lack of resolution in ‘Amoebozoa’ SSU-rDNA gene trees is due to the considerable rate heterogeneity and length variation among members of this supergroup. To better understand the evolution of ‘Amoebozoa’ and to test the validity of the different hypotheses, we performed extensive analyses of the SSU-rDNA data set with systematic removal of the fastest evolving rate classes (Philip et al., 2005). Ultimately, the validity of the clades based on SSU-rDNA analyses that conflict with morphology will require corroboration from other sources, including additional genes.

Section snippets

Taxa studied and morphology

Arachnula sp. ATCC® 50593, Nolandella sp. ATCC® 50913, Nolandella sp. ATCC® PRA-27, Pessonella sp. ATCC® PRA-29, 50933, Trichosphaerium sp. ATCC® 40318 and Rhizamoeba sp. ATCC® 50933 were obtained from the American Type Culture Collection (Manassas, VA). Arcella hemisphaerica was isolated from a culture of Arcella vulgaris (Carolina Biological Supply Company Catalogue No. 13-1310).

All species, except Arcella hemisphaerica, were identified only to the genus level based on preliminary gross light

Concatenated four-gene analysis

Bayesian analysis of the concatenated data (SSU-rDNA and amino acid sequences of actin, alpha-tubulin and beta-tubulin) including a total of 100 representatives from diverse eukaryotic groups (Fig. 3) is generally concordant with previously published trees (e.g. Baldauf et al., 2000). Given our taxon selection, which includes only a limited sample of non-amoeboid lineages, most of the traditionally well-defined lower taxonomic groups (i.e. dinoflagellates, apicomplexa, ciliates, stramenopiles,

Placement of the newly characterized amoeboid lineages

A concatenated multigene analysis places the seven diverse amoeboid lineages including Arachnula sp. and Trichosphaerium sp. within the supergroup ‘Amoebozoa’ (Fig. 3). All of our taxa share the broad morphological features characteristic of most of this supergroup including the presence of dynamic pseudopodia and branched tubular mitochondrial cristae. However, these characteristics are also found elsewhere on the eukaryotic tree of life, including in some members of the supergroup ‘Rhizaria’

Acknowledgments

The authors thank Erika Barbero for providing actin sequences of Arcella hemisphaerica. This work is supported by the National Science Foundation Assembling the Tree of Life Grant (043115) to Debashish Bhattacharya, D.J.P., and L.A.K. This is Lamont-Doherty Earth Observatory Contribution Number 7117.

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