THE EVOLUTION OF LIFE FROM OTHER PLANETS Part 2 CONSERVED GENES, WHOLE GENOME DUPLICATION INTRONS, EXONS, TRANSPONSONS, REGULATORY GENES RETRO-VIRUSES Rhawn Joseph, Ph.D. THE ORIGIN OF EARTHLY LIFE Life had taken root and repeatedly arrived on this planet between 3.8 to 4.2 BYA, a time period during which Earth was undergoing continual pummeling from the remnants and debris produced by the exploding parent star and its planetary system. Although microfossils resembling yeast cells and fungi were discovered in 3.8 BY old quartz (Pflug 1978), the nature of the first and earliest Earthlings can only be inferred indirectly based on the residue of photosynthesis, oxygen secretion, carbon isotopes, the structure of banded iron formations and high concentrations of carbon 12, or “light carbon;” all of which are typically associated with microbial life (Manning et al. 2006; Mojzsis et al. 1996; Nemchin et al. 2008; O'Neil et al. 2008; Rosing, 1999, Rosing and Frei, 2004; Schoenberg et al. 2002). However, if simple eukaryotes such as fungi and yeast cells had arrived on Earth by 3.8 BYA, then we can certainly assume that those sojourners from the stars who had arrived hundreds of millions of years earlier, included bacteria, archae, and viruses--and this has been demonstrated by geo-physical and biochemical analysis (Manning et al. 2006; Mojzsis et al. 1996; Nemchin et al. 2008; O'Neil et al. 2008; Rosing, 1999, Rosing and Frei, 2004; Schoenberg et al. 2002). THE FIRST EUKARYOTES It is generally assumed based on genomic analysis, that the first Earthly unicellular eukaryotes were fashioned when genes from archae and bacteria combined thereby inducing eukaryogenesis and giving rise to the eukaryote genome. These genes subsequently underwent repeated single gene and whole genome duplications, perhaps in response to regulatory signals or environmental triggers, and unicellular eukaryotes became multicellular and then increasingly complex and intelligent. However, the possibility that the first eukaryotes also arrived on Earth contained in jettisoned planetary debris and ejecta from the shattered remnants of the parent star's solar system, cannot be ruled out. Many species of bacteria form spores (Marquis and Shin 2006) and some survive in a state of suspended animation for hundreds of millions of years (Satterfield et al. 2005; Vreeland et al. 2000). Simple eukaryotic organisms, including yeast and fungi (Botts et al., 2009), also produce spores, often for reproductive purposes, but also in response to adverse, life threatening conditions. Therefore, it is possible that some simple eukaryotic organisms, and their descendants, along with trillions of other microbes, may have survived the destruction of the parent star system only to be hurled upon the newly forming Earth hundreds of millions of years later. This would account for the presence of microfossils resembling yeast cells and fungi, discovered in 3.8 BY old quartz (Pflug 1978). Once on Earth, these simplified eukaryotes may have phagatocized archae and bacteria (Kurland et al., 2006; Poole and Penny, 2007) and incorporated their genes, or were infiltrated by parasitic prokaryotes which donated genes to the eurkayotic genome. Woese, (2004) has proposed that these initial bacteria, archaea and eukaryotes may have lived together and repeatedly swapped and shared genes. "Eventually this collection of eclectic and changeable cells coalesced into the three basic domains known today. These domains become recognisable because much (though by no means all) of the gene transfer that occurs these days goes on within domains" (Woese, 2004). A second possbility is that the first Earthly eukaryotic cells were created by the genetic fusion of bacteria and archae, and possibly the injection of viral genes. Thus, hundreds of millions of years after arriving on Earth, archae, bacteria, and a virus may have joined together, combining their genomes, and in so doing, created the first eukaryotes, which, nearly 4 billion years later, would give rise to humans. The activity of photosynthesizing organisms and prokaryotic genes altered the environment via the liberation, secretion, and synthesis of a variety of chemicals and enzymes including oxygen (Buick 1992, 2008; Falkowski and Godfrey 2008; Holland 2006; Olson 2006; Williams and Fraústo da Silva 2006). The changed environment acted on gene selection, activating genes contributed by bacteria and archae, giving rise to new traits and new species perfectly adapted for a world that had been prepared for them. CONSERVED GENES & GENE EXPRESSION Thousands of orthologous genes and hundreds of conserved genes can be traced back to the last common ancestor for eukaryotes (Snel et al., 2002; Mirkin et al., 2003; Kunin and Ouzounis 2003; Koonin 2003; Mushegian 2008; Bejerano et al., 2004). Almost all underwent duplication at the onset of eukaryotic evolution (Makarova et al., 2005). These genes then continued to undergo repeated episodes of single gene and whole genome duplication such that the eukaryotic genome increased in size. However, these duplications were often coupled with gene deletions, obscuring their original relationship with prokaryotes. Almost all of the genes donated by prokaryotes, including those subsequently deleted from the eukaryotic genome, performed crucial functions that would guide the future or evolution. These included regulatory genes and genes controlling core cellular activities and the capacity to make duplicates of individual genes and the entire genome. Genome sequencing has revealed an extensive conservation of the same repertoire of genes coding for core cellular functions in the genomes of prokaryotes and eukaryotes (Koonin et al., 2004; Koonin and Wolf 2008). A core set of approximately 70 genes contributed by archae and bacteria have been conserved and passed down, without deletion, for billions of years, and which make up around between 1% to 10% of the genes in the genomes of all multicellular life (Koonin 2003; Koonin and Wolf, 2008; Harris et al., 2003; Charlebois and Doolittle 2004). These conserved genes, proteins, (Koonin 2002) and gene sequences (Koonin 2009b), include those governing translation, the core transcription systems, and several central metabolic pathways, such as those for purine and pyrimidine nucleotide biosynthesis (Koonin 2003). Moreover, protein sequence conservation extends from mammals to bacteria thus demonstrating their great antiquity (Dayhoff et al., 1974; Eck and Dayhoff 1966; Dayhoff et al., 1983). Between 2150 to 4137 orthologous gene sets are highly conserved and can be traced back to the last common ancestor for eukaryotes (Makarova et al., 2005). And often these orthologs express or perform the same function regardless of species. In yet other instances, these conserved genes had not been expressed in ancestral species and were activated only after hundreds of millions of years had passed; activated in response to changing environmental or regulatory conditions. These genes generally have numerous interaction partners indicating they can exert widespread effects across networks of genes. Consider, the evolution of the eye. It has been claimed that the chief components of the eye, such as photoreceptors must have evolved essentially de novo 40–65 times independently according to Darwinian principles (Salvini-Plawen & Mayr 1977). However, genes do not evolve de novo or ex nihilo; they are transferred from another species, inherited from an ancestral species, or they are produced by exon shuffling, whole gene duplication, and numerous other replicative mechanisms. Genes involved in eye development, known as Pax, "Pax-6" and opsin in vertebrates and "eyeless" in fruit flies, are homologous between diverse phyla (Quiring et al., 1994; Gehring & Ikeo 1999). Pax genes ("Pax-6"). They have also been found in the genomes of ancient species such as the sea urchin and trichoplax, both of which have no eyes and cannot see (Sodergren et al., 2007; Callaerts et al. 1997; Hadrys et al., 2005). Pax-6 serves as a master regulator of a network of genes that can give rise to a variety of different types of eyes that utilize the same visual pigment genes. That is, Pax-6 appears to act on different genes to produce the different structures on which the pigment cells are mounted in different creatures giving rise to a variety of eyes (Sheng et al., 1997; Gehring and Ikeo, 1999; Davidson, 2001). Moreover, Pax 6 proteins show an 90-90% identity between vertebrate and invetebrates (e.g. squid) as well as insects (Drosophila) and marine worms (Tomarev, 1997). These genes also utilize identical homologous Pax-6 proteins during eye development (Gehring & Ikeo 1999). As the common ancestors for vertebrates and invetebrates diverged between 600 mya to 1.6 bya (Ayala et al., 1998; Wray et al., 1996; Gu, 1998; Cutler, 2000), this is an indication of the great antiquity of Pax genes--many of which can be traced to ancestral species who had no eyes and were unable to see. Those ancestors could include prokaryotes. Consider, for example, vitamin-A-related chromophores in the visual pigment and which is the single most prerequisite for vision in the vertebrate or invertebrate genome. Vitamin-A-related chromophores are also found in bacteria as well as algae (Seki and Vogt 1998; von Lintig, J., Vogt 2004). These highly conserved genes were then passed down, through numerous diverging ancestral species until activated in the period leading up to and including the Cambrian Explosion. Over 1000 genes involved in visual functioning, including ancestral Pax-6 genes, were inherited and are homologous between phyla (Quiring et al., 1994; Gehring and Ikeo, 1999),(Tomarev et al. 1997), and have been isolated from several invertebrate and vertebrate species, including squid, flatworm, ribbonworm, ascidian, sea urchin, nematode, and fruit flies (Callaerts et. al., 1997; Tomarev, 1997). Be it vertebrate, flatworm or insect, and in spite of the large differences in eye morphology and mode of development (Gehring 1996), the same genes and same gene products related to the visual system are under the same genetic control (Quiring et al., 1994). Thus, regardless of species some parts of eyes are homologous because they are coded by the same genes and the same proteins. Between 70% to 80% of these genes are common and evolutionary conserved in the genomes of mammals, squid, octopus, flatworm, ribbonworm, ascidian, and nematode mosquitos, flies, tunicates, and vertebrate genomes including humans (Ogura et al., 2004). The common ancestors for these species diverged anywhere from 1.2 bya to 830 million years ago (Ma) (e.g., Wray et al., 1996; Peterson et al., 2004, Nei et al., 2001; Gu 1998). As there is no evidence for visual functioning in any creature before 550 mya, these genes were therefore inherited, in silent form, from ancestral species which could not see. However, regardless of their activity, genes that are highly conserved over the course of eukaryotic evolution not only remain in the same location but accumulate fewer substitutions in their protein sequences. Therefore the conservation of a gene and the fact that it is passed down vertically to subsequent species and is maintained unchanged in the same position, indicates biological importance and the identical roles it plays, almost regardless of species, over the course of evolution. That importance may also have more to do with the future of evolution rather than the survival of the species possessing that gene. Therefore, some highly conserved genes can be removed (knocked out) of various genomes without having any noticeable impact on the viability of the organism or its ability to function (Koonin 2000). In fact, hundreds of genes have been knocked out, or stripped from various species which remained viable (Glass et al., 2006; Koonin 2000). Mycoplasma genitalium, for example, has one of the smallest genome of any organism but remained viable even after 100 of its 482 genes were removed (Glass et al., 2006). However, 28% of the minimal set of genes coded for unknown functions (Glass et al., 2006). Moreover, 80 genes of the original minimal gene set were represented by orthologs in all forms of life and many of these coded for unknown functions (Koonin 2000). Therefore, not all highly conserved genes are related to the viability of the organism, but instead serve the future evolution of new functions, new structures, and new species. Mycoplasma genitalium Likewise, features of gene architecture that are not necessarily directly relevant to gene function are highly conserved across lengthy periods of evolutionary history. This includes the positions of a large fraction of introns with 25–30% conservation in orthologs from plants and chordates (Fedorov et al., 2002; Rogozin et al., 2003; Roy and Gilbert 2006). In the human genome, these ultraconserved elements often overlap introns or nearby genes involved in the regulation of transcription and development. Highly conserved genes are also located adjacent to exons involved in RNA processing (Bejerano et al., 2004). In addition, the positions of a large number of introns are conserved between plants and vertebrates (Fedorov, et al., 2002; Rogozin et al., 2003; Roy and Gilbert 2006) and between mammals and "living fossils" such as as Trichoplax and the sea anemone (Putnam et al., 2007; Srivastava et al., 2008); species which diverged over a billion years ago. Introns play a major role in the regulation of gene expression and transcription and creation of new genes from old genes. Thus, genes involved in transcription regulation and which were donated by prokaryotes to eukaryotes interact with and overlap genes and introns also contributed by prokarayotes to eukaryotes. Moreover, these same genes were repeatedly duplicated and dispersed to a wide range of divergent species, and when activated gave rise to identical or similar evolutionarily advanced characteristics such as the eye and brain. GENE REPLICATION & WHOLE GENOME DUPLICATION Some of these highly conserved genes act as a genetic mechanism through which prokaryote genes, gene sequences, and proteins, could be repeatedly duplicated within the eurkaryotic genome. For example, a variety of regulatory genes and proteins were donated which insure that specific genes and the functions they code for remained inhibited, while guaranteeing these same genes could be repeatedly duplicated and their functions preserved even as they grew in number and were passed down to subsequent species over hundreds of millions of years. Nevertheless, many of these genes were suppressed and remained silent. For example, archae and bacteria contributed three subunits of the core DNA-dependent RNA polymerase (Iwabe et al. 1991; Klenk et al. 1993) and two enzymes of DNA metabolism, RecA and Pol1A to the eukaryotic genome (Eisen and Hanawalt 1999; Harris et al., 2003) . These enzymes and the core RNA polymerase subunits serve many regulatory and replicative functions. For example, both RecA and Pol1A contributed to genetic continuity by gene conversion after recombination. They also insure the integrity and maintenance of genetic information as the lengths of DNA strands increase and the genome grows larger in size (Eisen and Hanawalt 1999). The replicative DNA polymerase, DnaN (COG0592), and the gene for the “sliding clamp” were also donated. This gene and proteins are necessary for the high degree of processivity of DNA polymerase during replication (Kuriyan and O'Donnell 1993;Hingorani and O'Donnell 2000). This enables the accurate replication of linked genes and the preservation of the information they encode. Many of the proteins that regulate eukaryotic signal transduction networks, including those involved in programmed cell death, are also derived from the prokaryotic genome (Aravind et al., 1999; Koonin and Aravind 2002; Bidle and Falkowski 2004). These signaling molecules are common in bacteria, cyanobacteria, and archae and include proteases from the AP-ATPase family. These proteases perform catalytic functions, and are found in the plant and animal genome (Koonin and Aravind 2002; Bidle and Falkowski 2004) and are utilized by mitochondria. Replication is a universal feature of cellular organisms, and eurkaryotes and prokaryotes share many genes and characteristics involved in replication, including the production of RNA primers, replication bidirectionality, strand synthesis, and the utilization of the same principal proteins involved in transcription and translation. That these genes were transferred from prokaryotes to eukaryotes is demonstrated by their commonality. Prokaryotic genes which guide replication and duplication contributed to the expanding size of the eukaryotic genome. Indeed, the number of signal transduction and regulatory proteins that are encoded parallel the increasing size of the genome. Thus, the larger the genome, the greater the number of genes dedicated to signal transduction (van Nimwegen 2003; Konstantinidis Tiedje 2004; Galperin 2005). Some of these genes that can be traced to a common ancestor also perform functions that involve the transfer of genetic information (Harris et al., 2003). Some interact with ribosomes and those ribosomal RNA genes which play fundamenal roles in cellular functioning and DNA translation and transcription (Harris et al., 2003). Ribosome and ribosomal RNA genes were also likely transferred from prokaryotes to eukaryotes (Lake et al. 1984; Lake 1988; 1998; Rivera and Lake 1992; Rivera and Lake 2004; Vishwanath et al. 2004). Thus, the ability to replicate and duplicate genes, and to transfer genes and to express these genes can be traced backwards in time to prokaryotes and to the direct descendants of the first creatures to arrive on Earth. Moreover, the donation of these genes and proteins was not random but under extreme regulatory control, performing essential functions related to the metamorphosis and evolution of future eukaryotic species; and this is why they are highly conserved across diverse species. These functions include gene and whole genome duplications (Dehal and Boore 2005; Lynch and Conery 2000; Lynch et al., 2001; McLysaght et al., 2002). Repeated replication, including whole genome duplication, freed up duplicated genes from regulatory restraint. Thus pre-coded genetic instructions were expressed giving rise to advanced traits which had been suppressed. Gene duplication is a major evolutionary mechanism (Ohno 1970). However, with each duplication, genes were also deleted, often the original prokaryotic insert. For example, a comparison of the numbers of ancestral gene clusters with those of extant animals such as the nematode, fly, mouse and human, established that extant bilaterian animals have retained more than 3500 gene clusters of the ancestral gene set, and have lost more than 1600 gene clusters (Ogura et al., 2004). Following duplication the originals or the copies were moved to new locations within the eukaryotic genome. Therefore, most of the genes which originated in the prokaryote genome can no longer be traced back to their prokaryotic source. After they had been donated and transferred to the eurkaryotic genome, many of these genes were simultaneously deleted from the prokaryotic gene pool thus insuring they would not affect prokaryote evolution. In prokaryotes, gene loss is one of the two major evolutionary processes, along with horizontal gene transfer (HGT), that contribute to the intensive “gene flux” that seems to have shaped the genomes of these organisms. Those donated genes included those regulating whole genome duplication (WGD). Thus, it appear that these genes underwent WGD only after they had been acquired by eukaryotes as there is little evidence of WGD in prokaryotes. There have been several whole gene duplications during the early evolution of eukaryotes and which date back to the emergence of the first eukaryotic cells or their ancestors (Makarova et al., 2005). Reconstruction of ancestral gene repertoires has identified 4137 orthologous gene sets in the last multicellular eukaryotic common ancestor, and 2150 orthologous sets in the hypothetical first unicellular eukaryotic common ancestor, which is indicative of WGD coupled with deletions. (Makarova et al., 2005). There is evidence to suggest that the genome may be duplicated at least every 100 million years (Lynch et al., 2001; Lynch and Conery 2000). Therefore majority of the genes in most genomes of cellular life underwent at least one duplication at some point during evolution (Lynch 2007; Koonin et al., 1996) and many genes belong to large families of paralogs. The number of ancestral gene sets at the time of the split of plant–animal–fungi and the divergence of bilaterian animals, is estimated to be 2469 and 6577, respectively (Ogura et al., 2004). There is a 2.7-fold increase in the number of gene clusters during the period from the evolutionary split of plant–animal–fungi to the divergence of bilaterian animals (Ogura et al., 2004). This indicates that at least one and possibly two whole genome duplications must have occurred coupled with massive deletions. Whole genome duplications have occurred in almost all lineages, including yeast (Wong et al., 2002; Vision et al., 2000; Kellis et al., 2004; Dietrich et al., 2004), fish (Van de Peer et al., 2003; Jaillon et al., 2004; Taylor et al., 2001), frogs (Tymowska et al., 1977; Jeffreys et al., 1980) and plants (Blanc and Wolfe 2004). The relatively large and complex vertebrate genome appears to have been duplicated at least twice (McLysaght et al., 2002; Dehal and Boore 2005). Whole genome duplication played a central role in the primary radiation of chordates (Dehal and Boore 2005) during the Cambrian explosion over 500 million years ago. There followed additional duplications during chordate evolution, thereby forming many of the gene families of vertebrates (McLysaght et al., 2002). Dehal and Boore (2005 reconstructed the evolutionary relationships of all gene families from the complete gene sets of a tunicate, fish, mouse, and human, and then determined when each gene underwent duplication relative to the evolutionary tree of the organism. An analysis of the global physical organization and genomic map positions of paralogous genes indicates these specific genes were duplicated prior to the fish–tetrapod split, some 400 million years ago. This was followed by two distinct genome duplication events early in vertebrate evolution as indicated by clear patterns of four-way paralogous regions covering a large part of the human genome (Dehal and Boore 2005). Large-scale genomic events marked the transition and divergence between yeast and fungi (Liti and Louis, 2005) chordates and non-chordates (McLysaght et al., 2002), fish and tetrapods (Dehal and Boore 2005), and then once or twice more after vertebrates began to colonize the surface of Earth (Dehal and Boore 2005). There is evidence to suggest that the genome may have been duplicated dozens of times over the course of evolutionary history (Lynch and Conery 2000; Lynch et al., 2001) thereby triggering the transition and divergence between numerous species, ranging from yeast and fungi (Liti and Louis, 2005) to chordates and non-chordates (Dehal and Boore 2005; McLysaght et al., 2002). Gene and whole genome duplication are crucial mechanisms of evolutionary innovation and when coupled with regulatory genes contributed by prokaryotes, enabled the genomes of eukaryotes to become increasingly complex as well as larger in size. This also allowed for multiple copies of the same genes to appear in divergent species and to be passed down until a regulatory or environmental signal triggered their activation. Gene duplication appears to provide t he raw material for major evolutionary transitions and triggering the emergence of new species in the absence of obvious intermediaries. The duplication of all genes at the same time could possibly induce rapid and extensive evolutionary change; i.e. the emergence of new species from old in the absence of obvious transitional species. Whole genome duplication also enabled the entire expanded gene repertoire to evolve together and reach a greater level of interaction and complexity as compared to single gene duplications. Duplication is often followed by accelerated sequence evolution as well as rearrangement of a gene, an evolutionary mode that obliterates detectable connections to the original gene source. Moreover, although numerous genes might be retained, other duplicated genes or the original might be quickly eradicated (Wolfe 2001) thus erasing the genetic footprints that would lead back to the prokaryotic source. This would make it appear that a new gene has emerged because its origins are no longer apparent. In fact, the vast majority of duplicated genes are subsequently deleted (Dehal and Boore 2005); an event which may also lead to freeing the original, or the duplicate, from inhibitory restraint, and which can erase all evidence of genome duplication (Dehal and Boore 2005). GENE LOSS & GENE EXPRESSION Lineage-specific gene loss is one of the major evolutionary processes that have been brought to light by comparative analyses of gene sets from completely sequenced genomes (Aravind et al. 2000; Moran 2002). Genome analysis has revealed the extensive loss of genes after WGD, in yeasts (Katinka et al. 2001; Scannell et al., 2007; Wolfe and Shields 1997), plants (Soltis et al., 2008; Tuskan et al., 2006), and chordates (Dehal and Boore 2005; Durand 2003; McLysaght et al., 2002). Gene loss without replacement is a common phenomenon in many genomes and appears to play an important role in shaping genome content (Snel et al. 2002). The extent of gene loss can be dramatic, and it can occur relatively rapidly under a strong selective pressure (Baumann et al. 1995). Although genomes of parasites expose the most striking cases of massive gene loss, a possible function of deletion following transfer, the fact is: substantial gene loss has occurred in all phylogenetic lineages (Snel et al. 2002; Mirkin et al. 2003). The eradication of the original gene may also play a role in the expression of the duplicate. Some of these duplicate genes appeared to have been freed from inhibitory restraint and were able to undergo an accelerated rate of sequence change thereby inducing the rapid evolution of new characteristics and abilities (Seoighe et al., 2003) Thus after duplication followed by deletion, the duplicate or original genes, now freed of the constraints, could express an already encoded function (“neofunctionalization”) which had been repressed (Conant and Wolf 2008). In many cases the 'new' function of one gene copy is a secondary property, or subfunction, that was always present, but which may have been suppressed, or which only came to be expressed when other more dominant functional capabilities were inhibited, suppressed or deleted. Therefore, old functions might be fractionated giving rise to new subfunctions (“subfunctionalization”). That is, the new function was not really "new" but had always been a property of a specific gene that could only be expressed following duplication, or duplication coupled with deletion. Thus, it is not uncommon for the new paralogs to retain or express distinct subsets of the original functions of the ancestral gene whereas the rest of the functions differentially deteriorate (Lynch and Force 2000; Lynch and Katju 2004) Duplication and the lessening of regulatory restraints might also make the gene more susceptible to environmental triggers. ARCHAE VS BACTERIA: GENE TRANSFER Numerous species of bacteria act as endosymbionts or endoparasites (Dyall et al., 2004; Poole and Penny 2007). Viruses are parasitic by nature. Archaea do not generally serve in this capacity--though there are exceptions. Bacteria, of course, are not uniform and there may be innumerable species (Nakabachi et al., 2006; Ranea et al., 2005; Schulz and Jorgensen 2001; Schneiker et al., 2007) . Considered in the broadest terms, archaea are highly distinct from bacteria, particularly in regard to the size of their genomes and cell membranes. For example, archaean membranes are made of ether lipids where as bacterial cell membranes are created from phosphoglycerides with ester bonds (De Rosa et al., 1986). Like bacteria, archae can live in the most extreme environments (Kimura et al, 2006, 2007; Leininger et al., 2006; Robertson et al., 2005). However, whereas bacteria are usually (but not always, e.g. Leininger et al., 2006) the most common form of life in the soil, archaeota are the most common form of life in the ocean, dominating ecosystems below 150 m in depth (Karner et al., 2001; Robertson et al., 2005). The genomes of archae are rather uniform and compact in size ranging from 0.5 Mb in the parasite Nanoarchaeum equitans (Waters et al., 2003) to 5.5 Mb in Methanosarcina barkeri (Maeder et al., 2006). Bacterial genomes can vary by two orders of magnitudes, from 180 kb in an intracellular symbiont, Carsonella rudii (Nakabachi et al., 2006), to 13 Mb in Sorangium cellulosum which dwells in soil (Schneiker et al., 2007). Although there are bacterial genomes of intermediate size, the vast majority of bacteria so far sequenced show a clear-cut bimodal distribution of genomes; i.e. large vs small, suggesting the existence of two distinct classes of bacteria: those with ‘small’ genomes (Ranea et al., 2005) with the highest peak at 2 Mb and those with "large" genomes at about 5 Mb (Schulz and Jorgensen 2001). By contrast, eukaryotic genomes range wildly in size and are generally several magnitudes larger than those of prokaryotes. However, the genomes of some eukaryotic species, such as microsporidian Encephalitozoon cuniculi (Katinka et al., 2001) are substantially smaller than many bacteria and archaeal genomes. Encephalitozoon cuniculi is also a parasite and may serve as a genetic messenger. Likewise, those bacteria and archae with the smallest genomes share a significant behavioral feature with Encephalitozoon cuniculi: they too are parasites and they prey upon other prokaryotes as well as eukaryotes (Waters et al., 2003; Huber et al., 2002). It is these parasitic behaviors which may explain their small genomes, and the presence of prokaryotic genes in the eukaryotic genome. These prokaryotes may have donated their genes to a eukaryotic host billions of years ago. Once donated many of these genes were not replaced. For example, prokaryotes with the smallest genomes, i.e. parasitic and symbiotic bacteria and archaeal parasites (e.g., N. equitans) no longer encode or express a variety of protein regulators, indicating the responsible genes have been transferred to the genome of the eurkaryotic host. With the donation of these regulatory genes, the genomes of these parasitic and symbiotic prokaryotes decreased in size. However, in addition to genes, many species of parasitic bacteria/archae may have taken up residence inside a eukaryotic host after which they continued to transfer and donate genes (Dyall et al., 2004; Margulis et al., 1997). Hundreds of specialized prokaryotic genes have been donated to the genomes of their hosts, possibly by horizontal gene transfer (Yutin et al., 2008) and were then preserved, unchanged, often in the same position even after hundreds of millions and, perhaps, even after billions of years of evolution. Some of these donated genes, or the complete engulfment of a bacterial parasite by eurkayotes, appear to to be responsible for the metamorphosis of mitochondria which also donated genes to the eukarayote genome (Margulis et al., 1997). These prokaryotic genes and bacteria/archae symbionts, enabled eukaryotes to become increasingly complex and to colonize and conquer new environments which were being genetically engineered by prokaryotic genes. PHOTOSYNTHESIS & OXYGENATION Initially Earth was devoid of a significant atmosphere, and lacked free oxygen, as the oceans were anoxic and possibly sulphidic (Barleya et al., 2005; Canfield 2005; Holland 2006; Mentel and Martin 2008). Only anerobic organisms, and those adapted to breathing hydrogen or methane, or feasting on iron and sulphites and other minerals and metals in the absence of oxygen, were able to thrive (Barleya et al., 2005; Olson 2006; Rosing and Frei 2004; Sleep and Bird 2008)--as is the case with many modern day species of bacteria and archae (Richardson 2000). Photosynthesizing cyanobacteria contributed genes to the eukaryotic genome (Howe et al., 2008), possibly at the initial stages of eukaryotic evolution. Gene transfer may have taken place secondary to endosymbiont engulfment by non-photosynthetic eukaryotic hosts (Howe et al., 2008). The genes donated by these cyanobacteria enabled some eukaryotes to develop pigmented plastids which engaged in photosynthesis. Plastid formed the major organelles which are now found in plants and algae and are responsible for the synthesis of fatty acids and the storage of starch. Plastid DNA exists as large protein-DNA complexes, each containing at least 10 copies of the plastid DNA. Plastids also possess numerous internal membrane layers which raises the possibility that plastids are stripped down photosynthetic prokaryotic endosymbionts (Howe et al., 2008). Thus some eurkayotes began to engage in photosynthesis in an oxygen free environment, and to secrete oxygen as a waste product (Buick 1992; Holland 2006). In addition to photosynthesis, some prokaryotes including aerobic photoautrophic marine plankton were producing oxygen via the photobiologically catalysed oxidation of water (Buick 2008; Falkowski and Godfrey 2008) and were engaging in oxygen metabolism as demonstrated by U–Pb data from metasediments, and the creation of thick kerogenous shales dated to 3.8  bya to 3.2 bya respectively (Buick 2008). The excretion of "waste" products, such as oxygen, over hundreds of millions of years, directly altered the environment (Barleya et al., 2005; Buick 1992; Canfield 2005; Holland 2006; Rosing and Frei 2004), and the altered environment acted on gene selection, activating genes that had been donated to the eukaryotic genome by prokaryotes and viruses. The buildup of free molecular oxygen resulted in nitrate being oxidized from ammonium and subsequently denitrified. Increased production of oxygen led to decreased fixed inorganic nitrogen in the oceans--as is evident from isotopic analyses of fixed nitrogen in sedimentary rocks from the Late Archaean (Falkowski and Godfrey 2008). The interaction between the oxygen and nitrogen cycles and the continued buildup of oxygen in Earth's atmosphere allowed nitrification to become dominant over denitrification (Falkowski and Godfrey 2008), such that oxygenic photosynthesis and aerobic respiration became the preferred mode of energy acquisition within eukaryotic host cells-- a function of the activation of gene selection and possibly the horizontal transfer of these activated genes from prokaryotes to eukaryotes (Falkowski and Godfrey 2008). As certain elements, gasses, and minerals built up as waste, they acted on gene selection (Williams and Fraústo da Silva 1996, 2006), giving rise to metabolic processes that enabled these creatures to biologically catalyse electron transfer (redox) reactions, beginning with H, C, N, and then O and S (Falkowski and Godfrey 2008). This sequence of changing environments acting on gene selection, led to the production of oxygen via the photobiologically catalysed oxidation of water and photosynthesis. As atmospheric oxygen levels continued to build up, this resulted in the surface weathering of soil-bound sulphides which were reduced to sulphates which drained into the oceans as sulphate (Mentel and Martin 2008). Under anaerobic conditions, chemolithotrophic microbes break down and convert ferric iron which is employed a oxidant to decompose other minerals, and producing sulfate and ferrous iron as waste products (Fernandez-Remolar et al., 2008). Thus, in addition to oxygen soil weathering, innumerable bacteria and archae were also acting on soils, such that ferrous iron and sulphates were being liberated and draining into the oceans. In consequence, sulphate reducers and anaerobic, hydrogen sulphide-producing prokaryotes, as well as ferrous iron producing bacteria, began to proliferate on a global scale (Mentel and Martin 2008; Sleep and Bird 2008). The continued production and buildup of sulphide and ferrous iron, were eventually incorporated within eukaryotic cells and protein-bound and served as oxygen acceptors (Sleep and Bird 2008). Thus what had been oxygen-independent ATP-generating pathways, became oxygen-dependent. In fact, cyanobacteria may have begun to use ferrous iron as reductant as early as 3.0 bya (Olson 2006). However, as based on an analysis of microfossils, stromatolites, and chemical biomarkers in Australia and South Africa, chlorophyll containing cyanobacteria had switched to oxygenic photosynthesis by 2.8 Ga (Olson 2006). Thus, a complex genetic-environmental feedback system was established, with genes acting on the environment and the biologically altered environment acting on gene selection which gave rise to species which utilized these "wastes" and rejected those which were no longer or not as useful (Richardson 2000; Williams 2007). As summarized by Williams (2007), "in essence, organisms at all times had to accumulate certain elements while rejecting others. Central to accumulation were C, N, H, P, S, K, Mg and Fe while, as ions, Na, Cl, Ca and other heavy metals were largely rejected." One step leads to the next, beginning with the use of hydrogen, methane, Fe, sulphur, and nitrates by bacteria and archae (Berks et al., 1995; Bult et al., 1996; Gold, 1992; Lonergan et al., 1996; Lovley, 1991; Richardson 2000; Vargas et al., 1998), followed by oxygenic photosynthesis (Castresana & Saraste, 1995; Castresana & Moreira, 1999; Falkowski and Godfrey 2008; Schafer et al., 1996; Schwartzman et al., 2008; Sleep and Bird 2008). Thus, the evolution of subsequent species utilized new byproducts, such as sulphides, ferrous iron, glucose, pyruvate, and NADH, which provided new and additional sources of energy and nourishment, and which acted on gene selection (Williams and Fraústo da Silva 2006). The liberation and incorporation of these chemical substances led to increasing cellular complexity, a function of cells acting on the environment which acts on gene selection which acts on the environment, creating a complex feedback system which promotes the evolution of increasingly complex creatures. For example, "in order to form the vital biopolymers, C and H, from CO2 and H2O, had to be combined generating oxygen. The oxygen then slowly oxidized the environment over long periods of time. These environmental changes were relatively rapid, unconstrained and continuous, and they imposed a necessary sequential adaptation by organisms while increasing the use of energy. Then, evolution has a chemical direction in a combined organism/environment ecosystem. Joint organization of the initial reductive chemistry of cells and the later need to handle oxidative chemistry has also forced the complexity of chemistry of organism in compartments. The complexity increased to take full advantage of the environment from bacteria to humans in a logical, physical, compartmental and chemical sequence of the whole system" (Williams 2007). OXYGENATION & MITOCHONDRIA When the environment had become sufficiently oxygenated and enriched with sulphide and ferrous iron which could served as oxygen acceptors (Sleep and Bird 2008) oxygen-dependent ATP-generating pathways replaced the less efficient oxygen-independent pathways and eukaryotic cells underwent a significant alteration and began breathing oxygen via the metamorphosis of mitochondria (Schafer et al., 1996). The genomes of all extant eukaryotes contain genes which can be traced to ancestors that possessed the -proteobacterial endosymbiont that gave rise to the mitochondria (van der Giezen and Tovar 2005; Embley 2006) Presumably, the genes of this α-proteobacterium symbiont underwent transformation in response to the increasing levels of oxygen in the atmosphere, becoming a mitochondria. Mitochondria serves as the powerhouse of the eukaryote cell and are located outside the nucleus. Mitochondria generate most of the cell's supply of adenosine triphosphate (ATP) which is used as a source of chemical energy (Akao et al., 2001; Dahout-Gonzalez et al., 2006; Garlid et al., 2003; Margulis et al., 1997). The production of ATP is accomplished by oxidizing the major products of glucose, pyruvate, and NADH, which are produced in the cytosol (Akao et al., 2001; Dahout-Gonzalez et al., 2006; Garlid et al., 2003; Herrmann and Neupert 2000) and by bacteria and archae (Richardson 2000). Many cells have only a single mitochondrion, whereas others contain several thousand. Mitochondria have their own independent genomes and their DNA shows substantial similarity to bacterial genomes (Pace 2006; Woese 1994). Mitochondria are enclosed in their own inner and outer membrane, play a significant role in signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth (Anderson et al., 1981; Chipuk et al., 2006; Mannella 2006; Rappaport et al., 1998). Thus, mitochondria are essential to the functioning of the eukaryote cell (Margulis et al., 1997). The DNA of multicellular eukaryotes is contained within the nucleus of every cell and mitochondria sit adjacent to the nucleus. It appear that the eurkaryotic nucleus was fashioned hundreds of millions of years after phogotrophy and hundreds of millions of years before the metamorphosis of mitochondria (Margulis et al., 1997). The nucleus which protects the eukaryotic genome, and the establishment of compartments, may have originally consisted of stripped down bacteria/archae. The nucleus and compartmentalization made it possible for predatory eukaryotes to ingest and phagotocize other creatures while minimizing the risk of random gene mixing and the unregulated incorporation of foreign DNA. Some researchers believe that the nucleus, the organelles, as well as mitochondria may have been created from bacterial and archae genes (Pace 2006; Woese 1994; Embley and Martin, 2006; Martin and Koonin, 2006; Martin and Muller 1998). Some believe the nucleus may be a derived endosymbiont, a descendant of an archaeon that invaded a bacterial host, or a bacteria and archae which invaded or was engulfed and phagotocyzed by the first eukaryotes (Lake and Rivera 1994; Horiike et al. 2004; Hartman and Fedorov 2002). Many researchers also believe that the mitochondria are directly linked to the engulfment of an anaerobic symbiont α-proteobacterium (reviewed by Gray et al., 1999) or a free-living photo-synthesizing bacteria by a methanogenic archaeon. Presumably this bacterium supplied hydrogen to the host (Martin W, Muller, 1998) which then engaged in anaerobic respiration to metabolize glycolytic products and turn them into energy; releasing oxygen as waste. Hundreds of millions of years would pass before eukarayotes began breathing oxygen (Schafer et al., 1996). Once the environment became sufficiently oxygenated, the α-proteobacterium either underwent metamorphosis to become a mitochondria, and/or contributed genes which gave rise to aerobic mitochondria (Embley and Martin, 2006; Gray et al., 1999; Martin and Koonin, 2006; Martin and Muller 1998; Rivera and Lake 2004; Martin and Koonin 2006). The activation of these genes, and the metamorphosis of mitochondria enabled eukaryotes to colonize emerging aerobic environments. Others have argued that mitochondria arose when the first multi-cellular eukaryotes internalized and formed a symbiogenetic relationship with a free-living proto-mitochondria or an α-proteobacterium symbiont (Cavalier-Smith, 2009). This could explain why a few groups of unicellular eukaryotes lack mitochondria: the microsporidians, metamonads, and archamoebae. As based on phylogenetic trees constructed using rRNA information, these unicellular eukaryotes appeared before the origin of mitochondria. Thus, the endosymbiont may have been incorporated only after larger, more complex multicellular eukaryotes evolved. However, unicellular eukaryotes who are without mitochondria nevertheless, possess organelles of -proteobacterial descent (Gray et al., 1999). This has led to the possibility that the genes giving rise to mitochondria, organelles, and the nuclear compartment originated at the same time in the common ancestor of all extant eukaryotes rather than in separate, subsequent events (Gray et al., 1999). The mitosome, for example, is an organelle found in some unicellular eukaryotic organisms and is related to mitochondria (Bakatselou et al., 2003; Tovar et al., 1999; Williams et al., 2002). Like mitochondria, they have a double membrane. The mitosome, however, has been detected only in anaerobic or microaerophilic parasitic organisms that do not have mitochondria (Bakatselou et al., 2003; Mentel and Martin 2008; Tovar et al., 1999; Williams et al., 2002). Nevertheless, the organelles of most unicellular eukaryotes have also been shown to be of -proteobacterial descent (Gray et al., 1999). Mitosomes therefore, may also be related to to an -proteobacteria which gave rise to mitochondria, or they may be derived from mitochondrial genes (Mentel and Martin 2008). However, unlike mitochondria, mitosomes genes are contained in the nuclear genome of the eukaryotic host (Bakatselou et al., 2003; Tovar et al., 1999). Thus mitosomes appear to be mini-mitochondria albeit stripped of its genes ( Williams et al., 2002). The existence of the mitosome does not appear to be compatible with endosymbiotic theory that postulates that mitochondria arose following the phagocytosis of a mitocondria-like organisms by a multi-cellular eukaryote (Mentel and Martin 2008). Unlike mitochondria mitosomes do not have the capability of gaining energy from oxidative phosphorylation (Mentel and Martin 2008) and t his may be due anaerobic environments in which they dwell (Tovar et al., 1999). The existence of the mitosome in anaerobic unicellular eukaryotes, and the link to an -proteobacteria and mitochondria, suggests that mitosomes and mitochondria are derived from the genes that gave rise to the first eukaryotes, and that the metamorphosis of mitochondria was in response to increased levels of oxygen, sulphur and ferrous iron, and other gasses, ions and minerals; a consequence of the environment acting on gene selection. Mitochondria, as a distinct entity within eukaryotic cells, did not arise until between 2.3 to 1.8 BYA (Mentel and Martin 2008). It was during this time that oxygen, produced by photosynthetic bacteria, had begun to enrich the atmosphere (Barleya et al., 2005; Eigenbrode and Freeman 2006), and during which Earth had became glaciated, fueled by oxygenic photosynthesis (Eigenbrode and Freeman 2006; Evans et al., 1997; Kirschvink, et al. 2000). This rise in oxygen has been referred to as the Paleoproterozoic "Great Oxidation Event" (~2.2 to 2.0 Ga), when atmospheric oxygen may have risen to >1% of modern levels, a byproduct of oxygenic photosynthesis (Buick 2008; Canfield 2005; Holland 2006; Nisbett and Nisbett 2008; Olson 2006). Initially, those α-proteobacterium genes which contained the DNA instructions for the metamorphosis of mitochondria, remained suppressed and were not activated, as the environment and atmosphere of Earth lacked oxygen and other chemicals such as NADH and other oxidases. In the absence of an oxygen rich atmsophere, eurkaryotes had no need for a mitochondria, and instead use alternate energy sources. Therefore, the first eurkaytoes probably did not posses mitochondria but mitosomes, as is also exemplified by many unicellular eukaryotes (Bakatselou et al., 2003; Tovar et al., 1999; Williams et al., 2002). Thus, mitochondria may have also evolved from prokaryotic genes, around 2.3 - 1.8 bya, when increasing levels of oxygen acted on gene selection. Using sulfur isotopes to determine the oxygen content of ~2.3 billion year-old rocks, Guo and colleagues (2009) found that "the Archean-Proterozoic transition is characterized by the widespread deposition of organic-rich shale, sedimentary iron formation, glacial diamictite, and marine carbonates recording profound carbon isotope anomalies." This includes the first known anomaly in the carbon cycle indicative of a sudden increase in atmospheric oxygen. "All deposits reflect environmental changes in oceanic and atmospheric redox states, in part associated with Earth's earliest ice ages...a rise in atmospheric oxygen... and the Great Oxidation Event (ca. 2.3 Ga)." Thus not just the metamorphosis of mitochondria but Earth's earliest ice age are linked to the rise of oxygen in Earth's atmosphere. During this same time period, when oxygen levels increased, organisms with more than 2-3 cell types appeared (Hedges et al., 2004). This increase in energy availability (oxygen) and the ability to extract it (mitochondria) conferred major advantages for the eukaryotic host which became increasingly complex and expanded in size. By 1.5 BYA, eukaryotes expanded to approximately 10 cell types (Hedges et al., 2004). This increase in size and complexity was made possible, in part, by the energy provided by mitochondria which used oxygen as an energy source. A billion years later, and by the onset of the Cambrian Explosion, so much oxygen had been released into the atmosphere that ozone was established which blocked out life-neutralizing UV rays. With the establishment of ozone, innumerable creatures could emerge from the sea or from beneath the soil and exploit new environments; environments which acted on gene selection giving rise to new capabilities and new species. Those who breathed oxygen were at a signficiant advantage, increasing the number of environments they could invade and conquer. The activity of photosynthesizing organisms and prokaryotic genes altered the environment via the liberation, secretion, and synthesis of a variety of chemicals and enzymes including oxygen (Buick 1992, 2008; Falkowski and Godfrey 2008; Holland 2006; Olson 2006; Williams and Fraústo da Silva 2006). The changed environment acted on gene selection, activating genes contributed by bacteria and archae, giving rise to new traits and new species perfectly adapted for a world that had been prepared for them. OYXGEN ENVIRONMENT, MITOCHONDRIA & ENDOSYMBIOTIC GENE TRANSFER Although the genes necessary for creating a mitochondria may have been present when Earthly eukaryotes were first fashioned, it was not until the planet became sufficiently oxygenated that the metamorphosis of mitochondria ensued via the transformation and activation of genes provided by α-proteobacterium, and triggered by a signfiicant increase in oxygen levels (Hedges et al., 2004) and other essential elements necessary for the functioning of mitchondria such as NADH. However, the rise of oxygen was also a function of biological activity ( (Buick 1992, 2008; Castresana and Moreira 1999; Castresana and Saraste 1995; Falkowski and Godfrey 2008; Holland 2006; Olson 2006; Schafer, et al., 1996). Thus once altered by photosynthetic organisms the environment acted on gene selection, and the rise in oxygen resulted in the diversification and increased complexity of the photosynthetic life that produced the oxygen that changed the atmosphere (Guo et al., 2009). As genes act on the environment which acts on gene selection, additional genes were activated, and new functions, characteristics, and species began to appear. However, not just the eukaryotic genome was impacted, but the mitochondria genome. Mitchondria subsequently donated numerous genes which were integrated into the eukaryotic genome (Rogers et al., 2007). These included genes coding for organelles and the endoplasmic reticulum, as well as genes contributing to the nucleus, and the bacterial-type plasma membrane that displaced the original archaeal membrane (Esser et al., 2004; Rivera andLake 2004); a process Andersson (2005) refers to as “endosymbiotic gene transfer." Endosymbiotic gene transfers are a common and ongoing process in diverse eukaryotes (Bensasson et al. 2001; Leister 2003; Timmis et al. 2004). Further endosymbiotic gene transfer from mitochondria may have facilitated the invasion of group II introns into host genes (Martin and Koon, 2006) which served as the precursors of spliceosomal introns (Cavalier-Smith, 2009). This invasion of introns exerted a profound effect on the regulation of gene expression (e.g. Brietbart et al., 1985; Leff et al., 1986; Yoshihama et al., 2007), the expansion and duplication of the eurkayotic genome, and the evolution and metamorphosis of increasingly complex creatures.