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4.2 Origins of eukaryotes (Fitz-Gibbon, House, Johnson, Lake, Porter, Rivera)
Szathmáry and Maynard Smith (1995) identified eight major milestones in the history of life on Earth: cells (compartments), chromosomes (genomes for cells), nucleic acids and proteins (separation of function at the molecular level), endosymbiosis (combination of function), sexuality (genetic recombination), multicellularity (separation of function at the cellular level), social organization (separation of function at the organismic level), and civilization (communication via language). The rise of eukaryotes began before the fourth of these thresholds was crossed, but eukaryotes progressively passed all of the other milestones and are the only branch of terrestrial life to have done so. Understanding the early part of this progressive history may therefore reveal general principles that are applicable to the growth of complexity in any living system. In this section, biologists and geologists will study early evolution of eukaryotes using information from molecular biology and the fossil record. The goals are to better understand the order in which important universal properties of eukaryotes (nucleus, sterols, cytoskeleton, endoplasmic reticulum, organelles, multicellularity, etc.) were acquired, and to try to time these events using both the fossil record and molecular clocks. Unless ongoing horizontal (lateral) gene transfer has completely obliterated deep historical information from modern genomes (§3.6.1), one clear possibility is that the last common ancestor of the primary line of descent leading to eukaryotes - the so-called eukaryote "host" - was also the last common ancestor of the Archaea (Cavalier-Smith 2002; House et al., submitted) or, perhaps, some members of the Archaea, if the Archaea is not a monophyletic group (Rivera and Lake 1992). In this scenario, this event (the origin of the eukaryote total group) would be unrecognizable in the microfossil and biomolecular fossil records because both descendant species would have been essentially identical at the time of divergence. Only subsequently, did they independently acquire those metabolic and other attributes that allow them to be differentiated into distinct biological categories. A second class of hypotheses for the origin of eukaryotes invokes biological convergence - an amalgamation of two or more fundamentally different kinds of organisms instead of an ordinary evolutionary divergence (Gupta et al. 1994; Lake and Rivera 1994; Martin and Müller 1998; Horiike et al. 2001; Hartman and Federov 2002). If this were true, the originating event would represent a genomic singularity in the tree of life and, perhaps, also be visible in the fossil record. In terms of molecular sequence comparisons, the amalgamation event can, in principal, be seen as a time when a signifcant number of foreign genes entered the genome of the "host" organism. The nature of the "host" organism and the source of the prokaryotic "donations" to the genome(s) of the last common ancestor of all living eukaryotes are outstanding problems in evolutionary biology that will be addressed by the research proposed here. There are obvious contributions from the endosymbiotic events that gave rise to mitochondria and plastids (chloroplasts etc.), but it is still not clear whether mitochondria and other similar energy-producing organelles known as hydrogenosomes were incorporated before the origin of the crown group (Roger and Silberman, 2002), and have been lost subsequently in some members (notably Giardia and its relatives), or are characteristic of only some groups of eukaryotes. It is even more uncertain whether the "host" organism was a stem or crown group archaebacterium (A), an unusual primitive bacterium (B; Philippe 2002), or a member of an extinct lineage (C) that diverged prior to the universal last common ancestor of all life on Earth (Hartman and Federov 2002). When these relationships are better understood, it should be possible to use the major amalgamation events as timelines across the tree of life. For example, if all mitochondria are derived from a unique obligate endosymbiosis between a Rickettsia-like a proteobacterium, and if mitochondria were acquired by stem group eukaryotes, then the last common ancestor of all living eukaryotes must postdate the differentiation of the proteobacteria (Gray et al. 1999). Combining this approach with molecular clock estimates of divergence times and evidence from the fossil record will ultimately lead to an understanding of the early history of eukaryotes in a planetary context. 4.2.1 Investigating the prokaryotic sources of eukaryotic genes The increasing availability of eukaryotic and prokaryotic genomes, and of new phylogenetic methods, promises to help understand which prokaryotic groups are the antecedents of eukaryotes. Knowledge of eukaryotic ancestors and their metabolisms, may in turn tell us about the environment in which eukaryotes arose and suggest what conditions on Earth facilitated their evolution. Furthermore, knowing whether eukaryotes are descended from anaerobes or from aerobes may greatly constrain possible scenarios for their evolution. At present, there is increasing interest in proposals that eukaryotes are chimeric and have multiple origins (Gupta et al. 1994; Lake and Rivera 1994; Martin and Müller 1998). Evidence from whole-genome analyses indicates that those eukaryotic genes coding for translation and transcription have come from archaebacterial hyperthermophiles whereas genes used for amino acid biosynthesis and other cellular processes have come from several different groups of eubacteria (Koonin et al. 1997; Rivera et al. 1998; Martin et al. 2002). Johnson and her collaborators will focus on the steps by which eukaryotes have obtained and lost their genes and organelles (mitochondria and hydrogenosomes). Using methods developed previously, Lake and Rivera will search for those prokaryotic "phyla" (Garity and Holt 2001) that have made the largest genetic contributions to the eukaryotic genome. In preliminary studies, it was found that several prokaryotic genomes have made significant contributions to eukaryotes. However, by using representative members of the ~80 currently available prokaryotic genomes and procedures similar to those used for studies of horizontal gene transfer (§3.6.1; Jain et al. 1999), they expect to determine which of the major bacterial groups have been the sources of the eukaryotic genes. The plan then is to identify which classes of genes (e.g., amino acid biosynthesis genes, cell envelope protein genes, energy metabolism genes) have come from which prokaryotes. These will then be compared with the metabolic properties of the contributing prokaryotes in order to test existing hypotheses for the origins of eukaryotes. In a complementary study, Fitz-Gibbon and House will carry out additional whole genome comparisons in order to obtain more fully resolved phylogenetic trees. They score individual genes as being either present or absent, just as any particular nucleotide is scored as being in one of four possible states (A, G, C or T) in DNA sequence comparisons. This method of whole genome comparison has been successfully applied previously (House and Fitz-Gibbon 2002; House et al. submitted). Since the method of whole genome analysis is in its infancy, Lake and Rivera propose to investigate some of the potential problems. When genomes differ considerably in size, whole genome analyses tend to put adjacent taxa together even if the organisms are not closely related. This effect, "big genome attraction", is similar to the long branch attraction artefact of gene and protein sequence comparisons (Philippe 2002). It can become important because eukaryotic genomes differ so greatly in size. For example, trees were constructed using the following eukaryotes, listed according to increasing genome size: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophilia melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Homo sapiens. Invariably, the taxa clustered together according to the size of their genomes as expected from the "big genome attraction" concept, with further exploration of this problem. 4.2.2 Origin and evolution of eukaryotic respiratory organelles Modern eukaryotes are viewed as a consortium of ancient associations between the nucleus plus cytoplasmic "host" and endosymbiotic bacteria that gave rise to energy-generating organelles, the mitochondria, chloroplasts, and hydrogenosomes. Competing hypotheses speculate about the role these ancient endosymbioses had in shaping the early eukaryotic cell. One view, the "hydrogen hypothesis" (Martin and Müller 1998), suggests that it was an association between a methanogenic host and a hydrogen-producing bacterial symbiont that led to the establishment of mitochondria in extant eukaryotes. Other theories hold that the mitochondrial symbiosis occurred substantially after the origin of the eukaryotic nucleus, pointing to the existence of ancestral amitochondriate eukaryotes (Andersson and Kurland 1999). Each of these hypotheses implies a different time of origin and selective force for the establishment of symbiont-derived organelles and has implications for the origins of anaerobic and aerobic eukaryotes and their metabolic pathways. The credibility of various hypotheses concerning eukaryotic organellar origins depends upon knowledge of hydrogenosomes (complex organelles involved in anaerobic carbohydrate metabolism) and mitochondria, the relationships between these organelles, and the origin of their protein components (Roger 1999; Dyall and Johnson 2000). To better understand the origin and nature of the hydrogenosomes, Johnson and her collaborators will probe the evolutionary and biochemical properties of this organelle in the anaerobic protist Trichomonas vaginalis, using a combination of genomics, proteomics and phylogenetic approaches. They will identify all genes encoding hydrogenosomal proteins by scanning the soon-to-be-completed genome using a highly conserved part of all matrix protein-coding genes. A proteomics approach, using mass spectrometry and genome sequence mining, will also be used to directly identify organellar proteins. Proteins identified by both methods will be subjected to phylogenetic analyses and their presence in the hydrogenosome will be confirmed using a variety of cell biological techniques. By combining genomics, proteomics, molecular evolutionary and cell biological approaches, they will attempt to elucidate the ancestry of the trichomonad hydrogenosome, an organelle that is central to energy metabolism in diverse anaerobic, and possibly other primitive, eukaryotes. The data generated will be used to assess alternative hypotheses for the origin and evolution of energy-generating organelles in early eukaryotes. Only six proteins from hydrogenosomes have been subjected to phylogenetic analyses and the results are ambiguous (Dyall and Johnson 2000; Horner et al. 2000). While the analyses of four of these genes are consistent with a common ancestry for hydrogenosomes and mitochondria, the other two genes suggest an ancestry from certain kinds of anaerobic bacteria. Whether any or all of these genes arose via an endosymbiotic event or via lateral gene transfer is unclear. The central aim of the work is to identify most, if not all, proteins that are targeted for import to the hydrogenosomes. In brief: (1) to identify genes encoding hydrogenosomal matrix proteins as described above; (2) to identify all proteins in the hydrogenosome; (3) to test whether the putative hydrogenosomal proteins found are localized to the organelle in vivo; and (4) conduct phylogenetic analyses on newly-identifed hydrogenosomal proteins. Johnson’s laboratory is currently collaborating with TIGR (The Institute of Genomic Research) to obtain the complete genome of Trichomonas vaginalis. These studies, which directly test whether hydrogenosomes and mitochondria arose from a common endosymbiont, will improve understanding of how organelles and eukaryotic cells first arose, and shed light on ancestral metabolic associations that led the evolutionary specialization and complexification of eukaryotic cells. They will explore the ancestry of organelles which are central to energy metabolism in eukaryotes, and will highlight processes by which cells gain complexity and evolve. If hydrogenosomes arose from an ancestor different from the mitochondrion, then hypotheses concerning the nature of living eukaryotes will be narrowed to those postulating endosymbiotic associations among a consortium of eubacterial and eukaryotic progenitors. The combination of phylogenetics, genomics and proteomics to assess homologies among organelles sets this part of the researchl apart. However, Johnson’s work and the work of Lake and Rivera (§4.2.1) are intimately related as they both address the potential contribution of bacterial consortia in the formation of the eukaryotic cell. 4.2.3 The fossil record of eukaryotic diversification The diversification of eukaryotes coincided with dramatic changes in Earth’s climate, ocean chemistry, atmospheric oxygen levels, and tectonic configuration. Mechanisms that link eukaryotic diversification to changes in the physical environment have recently been proposed (e.g., Brasier and Lindsay 1998; Anbar and Knoll 2002), but, with a few notable exceptions (Javaux et al. 2001), the stratigraphic and paleontologic datasets needed to test these ideas are not available. Recent work in well preserved, widely separated successions in western North America offers an unparallelled opportunity to gain insight into the interaction between biology and environmental change during the early to middle Neoproterozoic interval of Earth history (1,000 to ~600 Myr ago). Sequence stratigraphic, chemostratigraphic, geochronologic, and tectonic studies are in progress or have already been completed for two basins: the ~770-742 Myr-old Chuar Group, Grand Canyon, and its likely correlative, the Uinta Mountain Group, Utah (Weil et al. 1999; Karlstrom et al. 2000; Dehler et al. 2001; Timmons et al. 2001; Dehler et al. 2002; C. Dehler and L. Crossey, pers. comm. 2002). These studies provide a detailed record of environmental change with which paleobiological studies can be integrated. Initial work in the Chuar Group indicates a tantalizing connection between increasing carbon isotope variability and the first appearance of heterotrophic protists within the basin (Porter and Knoll 2000; Dehler et al. 2001; Porter et al. 2003). This possible causal connection will be explored using studies that investigate the abundance, diversity, and paleoenvironmental distribution of organic walled microfossils (acritarchs), and also the relationship of these variables to environmental parameters. In addition, ultrastructural and ion microprobe analyses of individual acritarchs will help elucidate the affinities of these early eukaryotic fossils, aiding in the calibration of branch points in the eukaryotic tree. Ion microprobe carbon isotope analysis of individual acritarchs may also prove useful for inferring paleoecological or paleoclimatic information; these possibilities and others will be explored. Chuar Group samples have already been collected and processed; we will conduct additional fieldwork in the Uinta Mountain Group to collect samples for processing.
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