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We are currently focusing on four aspects of the evolution of eukaryotic microbes:
Eukaryotes, cells with nuclei, existed exclusively as microorganisms for at least 500 million years before the evolution of plants and animals. These microbial eukaryotes, or protists, represent a tremendously diverse assemblage of organisms. Protists are characterized by numerous innovations in cell biology (e.g. multiple acquisitions of chloroplasts), play an essential role in ecosystems (e.g. carbon fixation in marine systems), and some are causative agents of prevalent infectious diseases (e.g. malaria) that impact the social and economic fortunes of entire countries.
Dr. Katz is the lead PI on a five-year NSF grant entitled "Reconstructing Eukaryotic Phylogeny through Multigene Analyses of Microbial Eukaryotes." We are elucidating relationships among lineages of predominantly free-living microbial eukaryotes through analyses of DNA sequences of nine genes characterized from ~200 protist species. Our sampling plan includes well-circumscribed taxonomic diversity of eukaryotes, but also extends to taxa of as yet unknown affinities. Phylogenetic analyses of these data combine existing approaches with newly developed methods for partitioning multigene data. The resulting comprehensive eukaryotic phylogeny is essential for: i) unifying the universal tree of life that includes both prokaryotes and eukaryotes, ii) understanding the multiple origins of multicellular eukaryotes, and iii) interpreting the origins of disease-causing protists.
Central to our project is a conservative approach to taxonomy. This emerges from a recognition of the paucity of taxon sampling to date, and the difficulties in reconstructing deep nodes in gene genealogies. Using this approach, we have found strong support in both the literature (Parfrey et al. 2006) and in multigene analyses (Yoon et al., submitted) for clades that also have ultrastructural identies (e.g. stramenopiles, alveolates, heterolobosea) while weak to moderate support for many putative supergroups
The project is based in six laboratories at five institutions and the other PIs are: Marian McKee (American Type Culture Collection), Debashish Bhattacharya and John Logsdon (University of Iowa), David J. Patterson (Marine Biological Laboratory), and John Huelsenbeck (University of California – San Diego). More information can be found at http://www.eutree.org.
Phylogeography of Coastal Choreotrich and Oligotrich Ciliates
In collaboration with an ecologist at Univ. Conn. (George McManus), we are collecting molecular data to assess the phylogeography of Oligotrich and Choreotrich ciliates (Snoeyenbos-West et al. 2002). Current ideas on protist biogeography fall into one of two camps: (1) all protists are cosmopolitan (high gene flow) and we have discovered the bulk of protist species, and (2) there are many endemic species (low gene flow) of protists yet to be discovered. Our data suggest the need to redefine this debate as we find repeated evidence of ciliate lineages with both high gene flow and high genetic diversity (Katz et al. 2005). Sequences of the ITS region of the rDNA locus reveal that both the freshwater morphospecies Halteria grandinella and the marine tide pool species Strombidium oculatum include numerous genetically-isolated clades and yet show high levels of gene flow (Katz et al. 2005).
We are now turning to culture-independent analyses of ciliates in near shore environments. To date, we have analyzed clone libraries generated from three sites in the Northwest Atlantic Ocean, each sampled across two time points (Doherty et al., submitted). In summary, our analyses reveal (1) similarly high levels of diversity at each site; and (2) distinct membership within these assemblages. Our next steps are to increase sampling for both clone library and DGGE approaches.
Molecular Systematics of Ciliates
To interpret data on ciliate genome evolution, and to map morphological features in this clade, we are generating gene genealogies at a variety of phylogenetic levels (among all ciliates, within classes, within orders, etc). As an example, we collected the first molecular data from the multicellular ciliate Sorogena stoianovitchae (Lasek-Nesselquist and Katz 2001). Like Dictyostelium, this ciliate lives as individual cells when food is plentiful and aggregates to form a multicellular fruiting body during starvation (Olive 1978). Our analyses indicate that Sorogena is a relatively recently derived member of the class Colpodea, a group of predominantly soil-dwelling ciliates (Lasek-Nesselquist and Katz 2001). We are now in the process of analyzing a broader sampling of ciliates in the class Colpodea (Dunthorn et al., in preparation).
We also analyzed ssu-rDNA sequences to evaluate both the monophyly of the ciliate class Phyllopharyngea de Puytorac et al. (1974), and relationships among subclasses. Our analyses of diverse members representing three of the four subclasses of Phyllopharyngea provide strong support for the monophyly of the Phyllopharyngea, and indicate that the Chonotrichia emerge from within the Phyllopharyngia (Snoeyenbos-West et al. 2004). Further, we report the discovery of group I introns in the Suctoria Acineta sp. and Tokophrya lemnarum. These introns represent only the second examples of group I introns in a ciliate ribosomal gene, since the discovery of ribozymes in the LSU rRNA gene of Tetrahymenathermophila. Phylogenetic analyses of Group I introns suggest a complex evolutionary history involving either multiple loses or gains of introns within endogenously budding Suctoria (Snoeyenbos-West et al. 2004).
Ciliates are defined by the presence of two distinct genomes, each contained within its own nucleus. One of these nuclei, the ‘germline’ micronucleus (MIC), undergoes meiosis and mitosis but is transcriptionally inactive. In contrast, the macronucleus (MAC) is the site of virtually all transcription within ciliates. The macronuclear genome develops from a zygotic genome through a series of sometimes-extensive chromosomal rearrangements, including fragmentation of chromosomes, elimination of specific sequences and amplification of the remaining chromosomes.
My lab has focused on exploring patterns of chromosomal rearrangements among diverse ciliate lineages. We demonstrated that extensively fragmented genomes occur in three classes of ciliates: in addition to the well-studied class Spirotrichea, the Armophorea and Phyllopharyngea extensively fragment their MAC genome to generate ‘gene-sized’ chromosomes (Riley and Katz 2001). For example, in these lineages the ~1.6kb a-tubulin gene resides on a chromosome 1.8-2.2 kb in length. The appearance of several lineages containing extensively fragmented genomes suggests multiple origins of gene-sized MAC chromosomes (Katz 2001; Riley and Katz 2001).
Having established that extensively processed genomes evolved multiple times in ciliates, we are now characterizing the mechanisms of genome rearrangements from diverse ciliates. We identified potential cis-acting regulatory sequences involved in the elimination of internally excised sequences by comparing homologous MIC (unprocessed) and MAC (processed) chromosomes (Katz et al. 2003). Analogous to introns, IESs are sequences located within coding regions and are spliced out as DNA during the development of MAC genomes. Our analyses suggest that the dimorphic nature of ciliate genomes enables ciliates to cross the 'valleys' in the adaptive landscape of protein evolution -- ciliates may be able to maintain deleterious copies of paralogs in their MICs while ‘hiding’ them from selection in their MACs (Katz et al. 2004; McGrath et al. 2006; Zufall et al. 2006). Processed MAC genomes: (1) break up linkage groups (fate of paralogs less affected by fate of other polymorphisms on linked chromosomes); (2) allow assortment of paralogs in the MAC but not in the MIC (deleterious mutations may be hidden in the MAC even though they are present in the MIC); and (3) redefine ‘genetic load’ through differential amplification of MAC chromosomes (may make it relatively inexpensive for ciliates to carry duplicated genes).
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REFERENCES
Katz, L. A. 2001. Evolution of nuclear dualism in ciliates: a reanalysis in light of recent molecular data. Int. J. Syst. Evol. Microbiol. 51:1587-1592.
Katz, L. A., J. Bornstein, E. Lasek-Nesselquist, and S. V. Muse. 2004. Dramatic diversity of ciliate histone H4 genes revealed by comparisons of patterns of substitutions and paralog divergences among eukaryotes. Mol Biol Evol. 21:555-562.
Katz, L. A., E. Lasek-Nesselquist, and O. L. O. Snoeyenbos-West. 2003. Structure of the micronuclear a-tubulin gene in the phyllopharyngean ciliate Chilodonella uncinata: implications for the evolution of chromosomal processing. Gene 315:15-19.
Katz, L. a., G. B. McManus, O. L. O. Snoeyenbos-West, A. Griffin, K. Pirog, B. Costas, and W. Foissner. 2005. Reframing the 'Everything is everywhere' debate: evidence for high gene flow and diversity in ciliate morphospecies. Aquatic Microbial Ecology 41:55-65.
Lasek-Nesselquist, E., and L. A. Katz. 2001. Phylogenetic position of Sorogena stoianovitchae and relationships within the class Colpodea (Ciliophora) based on SSU rDNA sequences. Journal of Eukaryotic Microbiology 48:604-607.
McGrath, C., R. A. Zufall, and L. A. Katz. 2006. Genome evolution in ciliates in L. A. Katz and D. Bhattacharya, eds. Genomics and Evolution of Eukaryotic Microbes. Oxford University Press.
Olive, L. S. 1978. Sorocarp development by a newly discovered ciliate. Science 202:530-532.
Parfrey, L. W., E. Barbero, E. Lasser, M. Dunthorn, D. Bhattacharya, D. J. Patterson, and L. A. Katz. 2006. Evaluating support for the current classification of eukaryotic diversity. Plos Genetics 2:2062-2073.
Riley, J. L., and L. A. Katz. 2001. Widespread distribution of extensive genome fragmentation in ciliates. Mol. Biol. Evol. 18:1372-1377.
Snoeyenbos-West, O. L. O., J. Cole, A. Campbell, D. W. Coats, and L. A. Katz. 2004. Molecular phylogeny of phyllopharyngean ciliates and their group I introns. Journal of Eukaryotic Microbiology 51:441-450.
Snoeyenbos-West, O. L. O., T. Salcedo, G. B. McManus, and L. A. Katz. 2002. Insights into the diversity of choreotrich and oligotrich ciliates (Class: Spirotrichea) based on genealogical analyses of multiple loci. International Journal of Systematic and Evolutionary Microbiology 52:1901-1913.
Zufall, R. A., C. McGrath, S. V. Muse, and L. A. Katz. 2006. Genome architecture drives protein evolution in ciliates. Mol. Biol. Evol. 23:1681-1687.
For more information, contact Laura A. Katz: LKatz@smith.edu
Click here for alignment of tyrosine recombinases, including newly-isolated sequences from E. crassus.
