*Marine Biological Laboratory, Woods Hole, Massachusetts;
School of Biological Sciences, University of Sydney;
Protistology Collection, American Type Culture Collection, Manassas, Virginia
Pelobionts (Archamoebae sensu Cavalier-Smith 1991
) lack mitochondria and are mostly free-living, heterotrophic, amoeboid, flagellated protists that inhabit organically enriched, microoxic, or anoxic freshwater and marine environments (Schulze 1877
; Penard 1921
; Brugerolle 1993
). Because of their ultrastructural simplicity and the basal position of many amitochondriate parasitic protists in ribosomal RNA phylogenies (Sogin 1997
), pelobionts were hypothesized to represent one of the earliest diverging eukaryotic lineages (Margulis 1970
; Whatley 1976
; Griffin 1979
; Cavalier-Smith 1983
; Griffin 1988
; Whatley and Chapman-Andresen 1990
; Cavalier-Smith 1991
; Brugerolle 1993
; Patterson 1994
). However, molecular data support conflicting hypotheses about the monophyly and phylogenetic placement of pelobionts. Early analyses of small subunit rRNAs (SSU rRNA) (Hinkle et al. 1994
) and partial sequences of large subunit rRNAs (LSU rRNA) (Morin and Mignot 1995
) show that the pelobiont Mastigamoeba balamuthi (=Phreatamoeba balamuthi [Simpson et al. 1997
]) diverged relatively late among eukaryotes. In contrast, a phylogenetic analysis of DNA-dependent RNA polymerase II (RPB1) genes (Stiller, Duffield, and Hall 1998
) placed a sequence from an organism identified as M. invertens as one of the most basal eukaryotes.
Discrepancies between inferences from cytology and RPB1 (Stiller, Duffield, and Hall 1998
) versus LSU rRNA (Morin and Mignot 1995
) and SSU rRNA data (Hinkle et al. 1994
) prompted Stiller and Hall (1999)
to examine the position of M. invertens in SSU rRNA trees. Their initial maximum likelihood (ML)based analysis did not cluster M. invertens with the other pelobiont sequences, nor were they basal to other eukaryotes. Stiller and Hall hypothesized that long-branch attractions (LBA) or skewed base compositions in rRNAs account for conflicting phylogenies of SSU rRNAs and RNA polymerases. When the rapidly evolving lineages were removed from that analysis, the two pelobionts formed a monophyletic group basal to other eukaryotes, but the GC content of half of the basal branches was not aberrant and was unlikely to impact the analysis.
To reexamine the evolutionary position and monophyly of pelobionts, we obtained new pelobiont sequences that were PCR amplified (Sogin 1990
) from Mastigella commutans (isolated from sediments of a domestic wastewater tank at the Cape Tribulation Tropical Research Station, Queensland, Australia16°05'S, 145°27'EGenBank AF421219), Mastigamoeba simplex (as studied by Bernard, Simpson, and Patterson 2000; Walker et al. 2001
GenBank AF421218), and Mastigamoeba sp. (strain wac-6, ATCC 50617GenBank AF421220).
We performed the likelihood analysis shown in figure 1
using PAUP* version 4.0.0b4a (Swofford 1999
) with TBR branch swapping and 117 random-addition replicates. Modeltest version 3.0 (Posada and Crandall 1998
) revealed that all models more simple than the general time-reversible model of nucleotide substitution, with invariable sites and a gamma distribution of among-site rate variation (GTR + I +
) had a significantly poorer fit to the data. This model was used for both ML and minimum evolution (ME) distance bootstrap analyses with 100 and 1,000 replicates, respectively. Empirical base frequencies were used, the proportion of invariable sites was estimated at 0.173, and the value of the shape parameter for the gamma distribution was 0.615. Under ML we discovered 10 locally optimal tree topologies. The best tree overall (24 out of 117 replicates) had a likelihood value of -lnL = 18,188.18. According to the Shimodaira-Hasegawa test (Shimodaira and Hasegawa 1999
), the alternative trees were not significantly different from the tree with the best likelihood value (P values ranging from 0.59 to 0.89). In the likelihood topology (-lnL = 18,191.53 found in 22 replicates) most congruent with the bootstrap consensus tree, M. invertens diverged nearly simultaneously, with many lineages from the crown group (fig. 1 ). Despite poor resolution, it does not appear to be specifically related to other pelobionts in any of the 10 locally optimal trees. The major pelobiont clade (M. balamuthi, M. commutans, Mastigamoeba sp., and M. simplex) also includes Endolimax nana with low to moderate bootstrap support (52, 77, and 70 in ML, ME, and parsimony, respectively). Bootstrap support for the E. nana-M. simplex relationship is 62, 86, and 70 and that for the grouping of these two taxa with M. balamuthi, M. commutans, and Mastigamoeba sp. is 52, 77, and 70. Robust groups are recovered in this data set (e.g., red algae, green plants, stramenopiles, alveolates, and fungi), but their relative branching order is difficult to resolve (e.g., Hasegawa and Hashimoto 1993
; Hinkle and Sogin 1993
; Kumar and Rzhetsky 1996
; Stiller and Hall 1999
; Philippe and Germot 2000
). Moderate bootstrap support separates pelobionts from diplomonads and trichomonads; however, there is no significant support separating the main pelobiont group from the crown taxa in any analysis of this data set. Intervening nodes consistently separate M. invertens from other pelobionts, although bootstrap values for nodes between M. invertens and the other pelobionts are not always robust. This result was obtained with the taxon sampling or alignment of Stiller and Hall (1999)
, or both. The failure of the two pelobionts to cluster together in Stiller and Hall's analysis (1999) may reflect evolutionary history (see also Milyutina et al. 2001
). A significant difference was found (S-H Test, P < 0.002) between the topology found in our analysis above and the best tree found under ML criterion with pelobionts constrained as a monophyletic group, further supporting the above result.
|
We were intrigued by the poorly supported relationship between pelobionts and the Entamoebidae. A wide range of analyses utilizing different data sets and phylogenetic methods consistently indicated that pelobionts have a recent common evolutionary history with the Entamoebidae (see http://www.mbl.edu/Astrobiology/PelobiontProject/PelobiontProject.htm for details). Similar suggestions have been made based on analyses that employed fewer taxa (Silberman et al. 1999
; Milyutina et al. 2001
). The late branching position of pelobionts in our analyses is consistent with secondary loss of mitochondria or conversion into degenerate organellar forms. Entamoebae have cryptons, which may be degenerate mitochondria (Clark and Roger 1995
; Mai et al. 1999
; Tovar, Fischer, and Clark 1999
). The detection of small, electron-dense organelles in some pelobionts (Walker et al. 2001) is consistent with a loss of mitochondria deep within the super clade containing E. nana plus M. simplex and all other entamoebae. Because no electron microscopical data is available for E. nana, we do not know if this organism displays ultrastructural similarities with the pelobionts.
The relationship of pelobionts with the Entamoebidae was tested in the context of a smaller data set that included other proposed relatives of pelobionts (Karpov 1997
; Walker et al. 2001). This ML analysis included the myxogastrid Physarum polycephalum (Cavalier-Smith 1997
; Walker et al. 2001) and its putative relative Hyperamoeba, the pelobionts, Hartmannella vermiformis, the entamoebae in our previous analyses, and Oxytricha nova as an outgroup. With this reduced data set, we included a larger number of aligned sites (1,348). The single best tree (-lnL = 9,367.14) shows pelobionts and entamoebae clustering together with bootstrap support of 97% under ML (fig. 2
). LogDet analysis confirmed these results (data not shown). We suggest that entamoebae and pelobionts are sister taxa to the exclusion of other eukaryotes form a clade to the exclusion of other eukaryotes. Further research will be required to determine whether Endolinax (fig. 1
) or possibly all Entamoebae (fig. 2
) are descended from within pelobionts rather than the pelobionts and the entamoebae forming sister taxa, as is usually assumed.
|
The notion that ancestral eukaryotic morphologies are represented by cytologically simple, extant groups (Cavalier-Smith 1983
; Patterson 1994
), conflicts with interpretations of molecular data. Phylogenetic analyses of SSU rDNA (e.g., this study), tubulins from deep-branching eukaryotes (Edlind et al. 1996
; Keeling and Doolittle 1996
; Philippe et al. 2000
; Edgcomb et al., unpublished data), and RNA polymerases (e.g., Hirt et al. 1999
), all contradict the archaezoa notion, which posits that ultrastructural simplicity is diagnostic of early diverging eukaryotes. There are other examples of protist groups originally thought to be ancient eukaryotes on the basis of morphological characters but which turn out to be derived based on molecular phylogenetic analysis (e.g., Clark and Roger 1995
; Edlind et al. 1996
). Discrepancies have also been found between phylogenies based on different genes (e.g., Baldauf and Palmer 1993
; Keeling and Doolittle 1996
; Germot, Philippe, and Le Guyader 1997
; Philippe and Laurent 1998
; Hirt et al. 1999
; Keeling, Luker, and Palmer 2000
; Van de Peer, Ben Ali, and Meyer 2000
). All the comparisons of phylogenetic methods, alignments, taxon selection, and sites used in our analysis illustrate subtle differences between methodologies but uniformly suggest that M. invertens does not group with other pelobionts.
The apparent sister group relationship between the pelobionts and the entamoebae in our analyses is consistent with the emerging view of the secondarily amitochondriate status of several groups previously thought to be primitively amitochondriate. Together, these results suggest that the ultrastructural simplicity of the pelobionts is most likely the result of degeneracy instead of an ancestral trait.
Acknowledgements
The authors thank John Stiller for providing his alignment, Charles O'Kelly for preliminary data on the ultrastructure of M. invertens, AUSTROP for hospitality, Andrew G. McArthur and David T. Kysela for helpful discussions. We acknowledge financial assistance from NASA Exobiology grant NRA97-OSS-11, NASA Astrobiology Cooperative Agreement NCC2-1054, and continuing support from the Unger G. Vetlesen Foundation to M.L.S and NSF BIO-DBI 9806681 to T.A.N. D.J.P. thanks ABRS and ARC for financial support.
Footnotes
1 Research performed at the Marine Biological Laboratory, Woods Hole, Massachusetts.
2 Present address: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
Keywords: pelobionts
SSU rRNA
amitochondriate amoebae
evolution
Mastigamoeba
phylogeny
Address for correspondence and reprints: Mitchell L. Sogin, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. sogin{at}mbl.edu
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