* Canadian Institute for Advanced Research, Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada
Département de Zoologie et Biologie Animale, University of Geneva, Geneva, Switzerland
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Abstract |
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Key Words: Cercozoa eukaryotes Foraminifera polyubiquitin genes
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Introduction |
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We have examined the structure and evolution of polyubiquitin genes from two important, yet evolutionarily enigmatic, protist lineages, Cercozoa and Foraminifera. The Foraminifera are a diverse group of extraordinarily abundant marine and freshwater protists, which are characterized by granulose, reticulating pseudopodia and organic or mineralized tests (shells) (Lee 1990). The evolution of Foraminifera has been extensively studied, and they have perhaps the best-characterized fossil record of any protist lineage. However, their evolutionary origin and relationships to other eukaryotes remains controversial because foraminiferan ribosomal RNA gene sequences are generally divergent, show dramatic fluctuations in evolutionary rates, and conflict with fossil evidence (Pawlowski et al. 1996, 1997). The Cercozoa are another very large and diverse group of eukaryotes that includes euglyphid amoebae, cercomonad amoeboflagellates, thaumatomonads, and chlorarachniophyte algae, among others. These organisms are morphologically so diverse that they were only recently recognized as being related through phylogenetic analysis (e.g., Bhattacharya, Helmchen, and Melkonian 1995; Cavalier-Smith and Chao 1997; Cavalier-Smith 1998; Keeling, Deane, and McFadden 1998; Keeling 2001). As is the case with Foraminifera, the relationships of Cercozoa to other eukaryotes has largely been a matter of speculation, as different gene trees conflict in their placement of Cercozoa relative to other eukaryotic groups (e.g., Bhattacharya, Helmchen, and Melkonian 1995; Cavalier-Smith and Chao 1997; Keeling, Deane, and McFadden 1998; Keeling et al. 1999; Keeling 2001). Here we show that cercozoan and foraminiferan polyubiquitin genes contain a unique insertion with important functional implications for polyubiquitin processing. All evidence indicates that the insertion is a shared derived character and that Foraminifera and Cercozoa share a common origin. These two groups represent a significant fraction of eukaryotic biodiversity, and their union marks the emergence of a new eukaryotic supergroup.
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Materials and Methods |
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Cloning and Sequencing of Polyubiquitin Genes
Multiple cDNAs encoding polyubiquitin gene fragments from Chlorarachnion sp. CCMP 621 were sequenced in the course of an ongoing EST sequencing project. The fragments were assembled to form three complete and distinct polyubiquitin genes, the sizes of which were confirmed by PCR using the universal primer sites flanking the multiple cloning site of the vector. Polyubiquitin gene fragments were amplified from Lotharella amoeboformis, L. globosa, Cercomonas sp., Cercomonas sp. 18, Cercomonas sp. 22, Euglypha rotunda, Reticulomyxa filosa, and Haynesina germanica using the following primers: UBIQ1: 5'-GGCCATGCARATHTTYGTNAARAC-3'; IUB2: 5'-GATGCCYTCYTTRTCYTGDATYTT-3'. The UBIQ1/IUB2 primer pair generates a ladder of ubiquitin gene products ranging from a half-monomer fragment to increasing numbers of tandem repeats of the polyubiquitin tract. Polyubiquitin fragments between 1.5 and 3.5 repeat units were isolated and cloned into pCR2.1 using the Topo TA cloning kit (Invitrogen). Multiple independent clones were sequenced from each species. Spliceosomal introns were present in several of the cercomonad polyubiquitins. For a given organism, amino acid sequences inferred from independent clones were generally identical, although synonymous substitutions were often observed between clones. For the foraminiferan Haynesina germanica, nonforaminiferan polyubiquitins were also sequenced, likely corresponding to genes amplified from food organisms. The foraminiferan sequences determined here were found to share several unique amino acid substitutions with ubiquitin monomers sequenced from other foraminiferans in a previous study (Wray and DeSalle 1994). New ubiquitin sequences were deposited in GenBank under the accession numbers AY099115AY099148 and AY101385.
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Results and Discussion |
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Polyubiquitin Genes in Cercozoa and Foraminifera
Chlorarachnion possesses a previously uncharacterized insertion in an otherwise invariant region of the ubiquitin molecule, which has important functional implications. Characteristics such as this are rare and can be powerful indicators of evolutionary relationships. We therefore isolated polyubiquitin genes from other members of the Cercozoa in an attempt to determine the phylogenetic distribution of this novel feature. We amplified and sequenced polyubiquitin gene fragments from two additional chlorarachniophytes that are distantly related to Chlorarachnion (Lotharella amoeboformis and L. globosa), as well as from three cercomonads (Cercomonas sp., Cercomonas sp. 18, and Cercomonas sp. 22) and from a euglyphid (Euglypha rotunda). All six were found to contain insertions at the same position as Chlorarachnion. The L. amoeboformis and L. globosa polyubiquitins have a single alanine (A) or S insertion at their monomer-monomer junctions, while the cercomonads and E. rotunda contain an SG or SA doublet (fig. 2). This character provides strong evidenceindependent of phylogenetic analysisfor the sisterhood of cercomonads and euglyphids, consistent with small subunit ribosomal RNA (SSU rRNA) phylogenies (Cavalier-Smith and Chao 1997; Wylezich et al. 2002). More generally, the insertion supports a specific relationship between chlorarachniophytes and cercomonads, as has been observed in analyses of SSU rRNA, tubulins, and actin (Bhattacharya, Helmchen, and Melkonian 1995; Cavalier-Smith and Chao 1997; Keeling, Deane, and McFadden 1998; Keeling et al. 1999; Keeling 2001).
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The absolute conservation of length and high degree of amino acid sequence conservation characteristic of the ubiquitin molecule across the full breadth of eukaryotic diversity (fig. 2) makes the possibility of independent insertions at such a functionally critical position of polyubiquitins from cercomonads/euglyphids, chlorarachniophytes, and Foraminifera extremely improbable. The cercozoan/foraminiferan ubiquitin insertion is thus very likely a synapomorphy uniting the two groups. Nevertheless, the fact that there are both single and double amino acid insertions in the polyubiquitins from these organisms indicates that multiple insertion/deletion events have occurred. Several scenarios could explain the variation in insertion size within the Cercozoa/Foraminifera lineage, but the significance of this insertion is in its presence. An insertion of any length at this location indicates a unique tolerance of size heterogeneity or, perhaps more likely, the presence of a novel processing pathway that removes the extra residues. Either way, the result is an increased tolerance for variation at this site of the polygene.
Indeed, this tolerance is also suggested by our observation of two separate instances of variability in amino acid sequence within the polyubiquitin insertions of a given organism (fig. 2). In both Lotharella amoeboformis and Cercomonas sp. 18, sequence heterogeneity was found between different insertions within the same polyubiquitin gene. This is somewhat unexpected, given the high degree of conservation of the amino acid sequence flanking the junctions. Combined with the heterogeneity in insertion length between cercomonads and Euglypha versus Foraminifera and chlorarachniophytes, this is consistent with the possibility (discussed above) that the extra amino acids are in fact removed during polyubiquitin processing and are thus under somewhat reduced evolutionary constraints.
Concluding Remarks
The origins of both Cercozoa and Foraminifera have been evolutionary puzzles, but for very different reasons. On one hand, the evolution and systematics of Foraminifera have been extensively studied using morphological, paleontological, and molecular approaches, and various suggestions for their evolutionary position have been made. On morphological grounds, they have been suggested to be related to various amoebae or heterokonts (Lee 1990). Molecular data from Foraminifera have also generated conflicting conclusions; rRNA gene trees have suggested that Foraminifera are closely related to slime moulds and amoebae (Pawlowski et al. 1994) or, alternatively, that they are an extremely ancient eukaryotic lineage (Pawlowski et al. 1996). Detailed analyses of SSU rRNA have led to the conclusion that the rate of substitution in foraminiferan sequences is very high, confounding any conclusions as to the position of Foraminifera in rRNA trees (Pawlowski et al. 1997). Cercozoa, on the other hand, have presented a very different puzzle since the group has only recently been recognized. Prior to the recognition of the Cercozoa, the evolutionary origin of each of its members was naturally considered independently. The cercomonads have been hypothesized to be related to bodonids (e.g., Hollande 1952), whereas thaumatomonads were thought to be related to heterokonts (e.g., Hollande 1952; Beech and Moestrup 1986). Chlorarachniophytes have been allied with heterokonts (Geitler 1930) and even tentatively with forams (Grell 1990), and various cercozoan amoebae have been considered most closely related to other amoeboid groups (e.g., Lee et al. 2000). The recognition that these morphologically diverse lineages were in fact related did little to suggest how they fit into the larger picture of eukaryotic evolution. Molecular phylogenies have been largely inconclusive, suggesting that Cercozoa (or some of its members) might be related to heterokonts (Van de Peer et al. 1996; Cavalier-Smith and Chao 1997) or revealing no stable position whatsoever (e.g., Keeling, Deane, and McFadden 1998; Dacks et al. 2002). Most recently, analyses of actin genes showed Cercozoa branching with Foraminifera (Keeling 2001), and now the shared presence of a unique insertion in a functionally critical position of their polyubiquitins significantly reinforces this conclusion.
It is now generally recognized that single-gene phylogenetic analyses often fail to correctly infer the relationships among the major groups of eukaryotes. This has led to the analysis of large concatenated sequence data sets (e.g., Martin et al. 1998; Baldauf et al. 2000; Bapteste et al. 2002). These analyses are helping to reshape eukaryotic diversity into a relatively small number of very diverse lineages, dubbed "supergroups." Unfortunately, many key eukaryotic lineages are still extremely poorly sampled from a molecular perspective, resulting in their exclusion from combined data analyses. In such cases, molecular markers that are independent of phylogenetic reconstruction, such as the polyubiquitin insertion characterized in this report, can be useful predictors of large-scale evolutionary relationships. The cercozoan/foraminiferan supergroup proposed here unites two large and diverse eukaryotic groups and represents a major advance towards a comprehensive and realistic picture of eukaryotic phylogeny.
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Acknowledgements |
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Footnotes |
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