Eukaryotes possess complex intracellular organelles, such as mitochondria and chloroplasts (such as the spiral chloroplast in the alga Spirogyra).   How did these structures evolve?

     Endosymbiosis refers to a condition in which one cell lives inside another cell for the benefit of both.  Is this possible? Yes.  There are hundreds of known examples of endosymbionts such as bacteria living inside of protists, algae living inside corals, worms, clams, even mollusks called nautiloids.  For example, there are a large number of species in the protozoan family Trypanosomatidae, many of which cause human diseases such as Chaga’s disease and African sleeping sickness. Some members of the family possess endosymbionts (such as Blastocrithidia cullicis, Crithidia deanei, C. desouzai, C. oncopelti, and Heretomonas roitmani; de Souza, 1999).  Not only are endosymbionts known in modern organisms (including modern termites), they have been identified in fossils as well.  Spirochete and protest symbionts of a fossil termite were identified in Miocene amber (Wier, 2002). 

     Bacterial symbionts in the amoeba Pelomyxa palustris are kept in vacuoles.  This species is considered to be one of the most primitive eukaryotes and it lives in very low-oxygen environments.  The eukaryotes Plasmodium falciparum and Toxoplasma gondii retain a vestigial plasmid, the apicoplast which is probably derived from a photosynthetic algae which has lost its photosynthetic pathways.  The parasitic plant Epifagus virginiana possesses vestigial plasmids (Berry, 2003; Gull, 2001).   There are a number of algae which have resulted from endosymbiosis.  Cryptomonads have retained the nucleus of their red algal endosymbiont as a small organelle known as the nucleomorph which contains three small chromosomes.  A second group of organisms, the chorarchniophytes, possess possess a nucleomorph remnant of a green algal symbiont.  The reduced genome of the chloroarachniophyte nucleomorph is the smallest known eukaryotic genome (Zauner, 2000; Gilson, 1996; Gray, 1992).     In addition to the endosymbiotic events which occurred in ancestral eukaryotes, ancestral dinoflagellates absorbed an additional red alga and some lineages of dinoflagellates have acquired another plastid from a tertiary endosymbiotic event as well (Patron, 2006).Giant clams and other mollusks possess endosumbiotic algae (Kutschera, 2005).

     The parasitic trypanosomes seem to be algae which have lost their chloroplasts, given nuclear genes which appear to have originated from an endosymbiont.  Oomycetes appear to be algae which have lost their plastids (Martin, 2003).  Entamoeba possesses a vestige of a mitochondria which no longer functions in ATP production (but which may perform other mitochondrial tasks such as reactions involving iron and sulfur) (Martin, 2003). The amoeba Paulinella chromatophora possesses a cyanobacterial endosymbiont which performs photosynthesis. It is more closely related to free-living cyanobacteria than to chloroplasts (Marin, 2005). The conidial fungus (Scopulariopsis brevicaulis) is an endosymbiote within glands of the American dog tick (Yoder, 2007).

     It is common that genes which were originally present in the endosymbiont eventually are transferred to the host nucleus.  Modern chloroplasts encode between 60 and 200 proteins.  Most of the ancestral genes seem to have been translocated to the nucleus.  About 18% of the nuclear genes of the plant Arabidopsis, seem to have originated from the cyanobacterial endosymbiont (Martin, 2002) and a copy of the mitochondrial genome (99% identical) has been copied to chromosome 2 (Martin, 2003).  The genome of the plastids of dinoflagellates has been greatly reduced, consisting of single-gene minicircles which encode about 15 proteins.  Most of the genes for the photosystems have been translocated to the nucleus (Hackett, 2004). 

     Not only are there a number of endosymbionts evolved recently which still retain most of their ancestral nature, there is also evidence of more ancient endosymbiotic events, including the origin of mitochondria and chloroplasts.  Mitochondria and chloroplasts are eukaryotic organelles which have a number of features which suggest they are derived from eubacterial ancestors.  They are similar in size to bacteria and they possess their own chromosomes which are circular, like those of bacteria.  (As a result, it is incorrect to say that human cells have 46 chromosomes: the mitochondrial chromosome composes a 47^th and it may be present in many copies in any given cell.)  Mitochondria and chloroplasts are also similar to bacteria in their ribosomes, cytochrome c, genetic code, translation initiation (use the tRNA.fMet), translation initiation factors; and internal structure.  Both mitochondria and chloroplasts reproduce by fission as do bacteria and cannot be synthesized by the genes in the nucleus.   If they are removed from a cell, the cell cannot replace them (Gray, 1992).

     Both mitochondria and chloroplasts are sensitive to antibiotics which affect bacteria such as streptomycin, spectinomycin, neomycin, and chloramphenicol while they are unaffected by agents such as cyclohexamide that affect the cytoplasm.  Many of these antibiotics act on bacterial ribosomes.  However, eukaryotic mitochondria possess their own genes which contribute to ribosomes.  There are two rRNAs encoded by the mitochondrial genome: MTRNR1 (nucleotides 648-1601) and MTRNR2 (nucleotides 1671-32229).  Not only are high doses of certain antibiotics potentially dangerous to all humans (because they inhibit mitochondria in addition to inhibiting bacteria) some people possess variations in these mitochondrial rRNA genes which make their mitochondria more “bacteria-like” and thus can cause serious reactions if they take an antibiotic.  Mutations in mitochondrial genes can cause a number of inherited genetic disorders in humans.  For example, mutations in the mitochondrial MTRNR1 gene can cause deafness (OMIM). The apparatus which controls mitochondrial division consists of both eukaryotic and prokaryotic components (Kuriowa, 2006).

     Plasmids are small pieces of DNA which exist outside major chromosomes.  Although virtually all plasmids are known from bacteria, some are known to exist in eukaryotic mitochondria.  A number of linear mitochondrial plasmids are known in fungi and higher plants, some of which require the presence of two plasmids in order to replicate (Chan, 1991; Gray, 1992).

     Are mitochondria as old as eukaryotes?  In comparisons of rRNA molecules, the three groups of eukaryotes which seem to branch off the main ancestral lineage first (microsporidia, metamonada including the diplomonads, and parabasala including the trchomonads) lack mitochondria (Germot, 1997; Gray, 1992).  Although it was originally thought that these primitive eukaryotes were descended from ancestors which diverged after the evolution of the eukaryotic nucleus and before the evolution of the mitochondria, that now no longer seems to be the case.  Microsporidia have a ribosome whose size is similar to that of prokaryotes given that they, alone of the eukaryotes, lack a 5.8 S subunit.  The large subunit rRNA in microsporidia and prokaryotes is homologous to the 5.8S subunit at its 5’end (Vossbrink, 1986; Gray, 1992).   Sequence comparisons of a number of molecules (such as HSP70) suggest that microsporidian nuclei possess genes which seem to have been derived from ancestral mitochodria which were later lost (Germot, 1997). 

    These most primitive eukaryotes retain hints that their ancestors possessed mitochondria, including organelles called hydrogenosomes.  Hydrogenosomes are intracellular organelles found in a number of eukaryotes that lack mitochondria (such as trichomonads) and similar organelles have been observed in some fungi and ciliates.  These organelles ferment pyruvate, producing carbon dioxide and molecular hydrogen and generating ATP through substrate level phosphorylation.  Although they are coated by a double membrane and use succinyl CoA synthtase for ATP production like mitochondria, they lack cristae, cytochromes, FoF1 ATPase, or a pyruvate dehydrognease complex.  The ferredoxin oxodireductase of hydrogenosomes is homologous to the pyruvate dehydrogenase of mitochondria.  Although they lack their own chromosomes, the nuclear genes coding their proteins are similar to mitochondrial genes.  It appears that hydrogenosomes and mitochondria have their origin in the same endosymbiontic organelle and that trichomonads diverged from other eukaryotic lineages before this endosymbiont had given rise to mitochondria (Bui, 1996; Andersson, 1999). 

     As a result, there do not appear to be any modern eukaryotic cells whose lineage diverged from others prior to endosymbiotic event which resulted in mitochondria.  It is possible that these ancient lineages simply have not survived (or have not been discovered) or it is possible that the origin of mitochondria coincided with the origin of the nucleus, and thus, the eukaryotic cell.

MITOCHONDRIA

     It is often observed that cells which live inside other cells have reduced numbers of genes.  Even bacterium Rickettsia prowazekii, which is an intracellular parasite which has a reduced genome with only 834 genes.  The genome size of mitochondria varies from 60 to 200 genes in different organisms (Berry, 2003). Mycoplasma capricolum is a bacterium which has the smallest known number of tRNAs at 29: three for leucine, two for arginine, ileucine, lysine, methinine, serine, threonine, and tryptophan, and one for all other amino acids which is capable of binding to four different codons.  It seems that no tRNA exists which can translate the codon CGG.  There are several characteristics (such as low tRNA diversity) which are shared between mycoplasma and mitochondria suggesting that they experienced similar selective pressures, or perhaps even that mycoplasma-like microbes (which are parasitic in eukaryotes) may have evolved into mitochondria (Andachi, 1989).

      Mitochondrial genomes are typically smaller than 200 kilobase pairs (kbp) in size but they vary from 13.8 kbp in the nematode C. elegans to 2400 kbp in muskmelon Cucumis melo.  These mitochondrial genomes consistently encode proteins required for respiration (although there are a few variations, such as the absence of NADH dehydrogenase in some fugal mitochondria).  The mitochondrial genes of plants and some protists typically possess genes for translational proteins and RNAs which are absent (or virtually absent) in the genomes of fungal and animal mitochondria.  Mitochondrial genomes in animals typically have the same gene order (which is invariant in mammals), lack introns (although some have been found in cnidarians), and possess few non-coding nucleotides (with the least number of noncoding nucleotides being about 100 in some sea urchins) (Gray, 1992).  In contrast, plant mitochondria have very low gene density; less than 10% is coding as opposed to the more than 90% coding in animals (Gray, 1992).  The mitochondrial genes of Reclinomonas americana possesses the same arrangement of ribosomal protein genes as exist in bacteria (Andersson, 1999). There are similarities in the sites where protein complexes are formed between mitochondria and bacteria (Kutschera, 2005).

     The reduced diversity of tRNA types has resulted from a pressure in mitochondria to reduce the sizes of their genomes.  Although many mitochondrial codons depart from the “universal” genetic code, these codon reassignments are specific to different lineages.  In insects, mammals, and amphibians, the reassignment of AUA to encode methionine has increased the percentage of methionine in mitochondrial proteins.  In most eukaryotic mitochondria studied (with the exception of higher plants), UGA has been reassigned to code for tryptophan, the most highly conserved amino acid in mitochondrial proteins, and presumably one with considerable functional importance (Andersson, 1991).

     The outer membrane of the chloroplasts and mitochondria are probably derived from the host cell that engulfed them (Berry, 2003).  The bacterium Paracoccus denitrificans is similar to mitochondria in its membrane organization and its possession of cytochromes b, c, c₁, and aa₃ (Verseveld, 1987).  Sequence comparisons and biochemical analyses both indicate that the ancestor of mitochondria probably arose from the α-subdivision of purple bacteria (Gray, 1992).  The completed genome sequences of Rickettsia prowazekii support that mitochondria evolved from α-proteobaceria (Andersson, 1999).

     Some have suggested that the ancestors of mitochondria which were engulfed by proto-eukaryotes had not yet evolved the ability to utilize oxygen.   It has been proposed that mitochondria evolved from an α bacterium which produced hydrogen and carbon dioxide as wastes.  The first eukaryotes would have lived in anaerobic environments until the endosymbionts adapted to oxygen (Lopez-Garcia, 1999).

     Organelles have kept the major bacterial proteins which performed electron transport, but additional subunits have been added (Berry, 2003).  Modern bacteria, such as Rhodospirillum and Paracoccus possess the respiratory chain found in mitochondria.  Instead of hydrogen ions being pumped outside of an inner membrane, they are pumped outside the cell.  (Baltscheffsky, p. 26; Darnell, p. 595). The mitochondria of higher eukaryotes possess one cytochrome c while many bacteria have multiple electron carriers which function in different physiological conditions (Myllykallio, 1999).

Mitochondria utilize genes which seem to have originated from a bacteriophage rather than the ancestral bacterium (Shutt, 2006). Genetic analysis suggests that some of the proteins present in mitochondria may have originated from viruses which infected the ancestral α proteobacteria (Filee, 2005).

CHLOROPLASTS

     Shortly after the evolution of eukaryotes, two lineages evolved.  The lineage which evolved into animals, fungi, and choanoflagelleates evolved flat  mitochondrial cristae, positioned the ancestral cilium in the posterior, developed chitin and lanosterol.  The lineage which led to plants and most protists began with tubular mitochondrial cristae, an anterior cilium, and developed cellulose and cycloartenol (Cavalier-Smith, 2003).  In the second group, additional endosymbiotic events occurred which resulted in plastids such as chloroplasts.

     Eubacterial endosymbionts provided the basis for eukaryote photosynthesis and no archaea perform this type of photosynthesis. Bacteriochlorophyll g from heliobacteria and cyanobacteria (such as the cyanobacteria in the above photo) is very similar to chlorophyll a from chloroplasts.  Gloeobacter violaceus may be the most primitive cyanobacteria given its lack of thylakoids and the presence of its electron transport system on its cell membrane (Berry, 2003). The cyanobacteria genus Nostoc  includes species which have formed a number of symbioses with other cells.  Nostoc punctiforme is very similar to chloroplasts (Berry, 2003).  Some phototrophic prokaryotes are very similar to chloroplasts and are called prochlorophytes and Prochloron is an endosymbiont of marine worms.   Chloroplasts have 100-200 genes while cyanobacteria possess about 3,000 (McFadden, 1999). 

     While most plastid genomes range in size from 120 to 160 kbp, those in green algae range from 89 kbp to more than 400 kbp.  The smallest genomes (about 70 kbp) are known from nonphotosynthetic algae and plants. These plastid genomes support the idea of a eubacterial endosymbiont since not only homologous genes but also similar gene orders are shared between plastid and eubacterial chromosomes (such as a cluster of 10 ribosomal protein genes in the same order in both plastids and E. coli.)  These plastid genes can be organized into operons and controlled by promoters very similar to those found in eubacteria.  Plastids are similar to eubacteria in their ribosomal subunits,  ribosomal proteins, translation initiation, and antibiotic sensitivity.  Sequence comparisons of plastid rRNA genes and those of the three divisions of life identify the plastid genes as being eubacterial and, more specifically, cyanobacterial.  (Gray, 1992).  Gene comparisons support that there was one single endosymbiotic origin of plastids (Morden, 1992). The similarity between cellulose genes of algae and cyanobacteria suggest that cellulose might have originated in the chloroplast endosymbiont (Kutschera, 2005).

     One of the membrane proteins of chloroplasts, coded by the gene Toc75, had no known homologs until a similar membrane protein was discovered in cyanobacteria (the gene was named SynToc75).  In chloroplasts, the protein helps to import other proteins from the cytoplasm to the chloroplast.  In cyanobacteria, the protein clearly performs an essential function, since null mutants do not survive; perhaps it transports a virulence factor from inside the cell.  SynToc75 was shown to be related to proteins found in all groups of Gram-negative bacteria where most function as prokaryotic secretion channels for virulence factors, such as hemolysins and adhesins (Reumann, 1999).

     The several kinds of plastids in eukaryotes seem to have arisen from separate endosymbiotic events, given the varying pigments and membrane compositions of the plastids in plants and algae.  Some plastids are surrounded by additional membranes which may have originated from the phagosome of their original host.  (Gray, 1992).  In plants, the plastid replication utilizes proteins homologous to those bacteria use in division.  The tubulin-like protein FtsZ is a bacterial division protein which is also required in plant and algae organelles. 

     Comparisons of the genomes of mitochondria and chloroplasts are useful in determing phylogenetic relationships.  The chloroplast genome of grasses possess 4 deletions, an inversion, and in insertion in the rpoC2 gene (Clegg, 1994).  The lineages of angiosperms which can fix nitrogen are not immediately related in that there are many non-nitrogen fixing plants which are more closely related to various members.  However, these nitrogen-fixing groups (and their non-nitrogen fixing relatives) do form a clade based on chloroplast gene sequences suggesting that some preadaptations to nitrogen fixation evolved in the early members of this clade and various descendant lineages were able to take advantage of this mechanism to develop the ability to fix nitrogen (Soltis, 1995).

      The rate of change in chloroplast DNA changes between loci and between groups of plant lineages (Gaut, 1993).  Liverworts lack three mitochondrial introns which are present in all other groups of land plants (including mosses and hornworts), suggesting that liverworts are the earliest land plants and that all other groups share a common ancestry after diverging from the liverwort lineage (Qiu, `1998).  In plants, evolution in nuclear genes tends to occur faster than for chloroplast genes whose evolution is faster than plant mitochondrial genes (Laroche, 1997).  Trypanosomes are considered to be one of the earliest branches of the mitochondria-containing eukaryotes and their mitochondrial sequences support this position (Gray, 1992).  Kinetoplastid protozoa have the most divergent mitochondrial DNA including a chromosome composed of minicricles and a maxicircle (de la Cruz, 1984)

ADDITIONAL ENDOSYBIOTIC EVENTS

Secondary plastids are known in three of the six major groups of eukaryotes: Rhizaria, Excavata and Chromalveolata. The groups Rhizaria and Excavata captured green algae which allowed chlorarachniophytes and euglenids to perform photosynthesis, unlike other members of these groups. In the group Chromalveolata, a red alga was the source of the secondary plastid. These secondary endosymbioses involve a eukaryote inside a second eukaryote (Lane, 2008).

The remnants of the ancestral nuclei are still present as nucleomorphs in the eukaryotic endosymbiots of the Cryptophyte and chlorarachniophyte plastids, originally derived from red and green algae. The malaria parasite Plasmodium falciparum also retains remnants of a plastid which may be derived from either green or red algae (Teich, 2007).

CENTRIOLES

     Other organelles such as peroxisomes, glyoxysomes (plants), glycosomes (trypanosomes), and flagella have been considered for endosymbiotic origin but this has proved more difficult to test (Gray, 1992). The phylum Archaeprotista includes some 28 families, all of which lack mitochondria and exist in anoxic environments.  Most possess a karymastigont system of organelles in which the flagellum (or related structures) are connected to the nucleus.  It may be that the karymastigont is a remnant of the apparatus which first accommodated the structures of the two ancestral cells in the resulting chimeras.  Microtubule-organizing centers and other structures may be remnants of the karymastigont (Margulis, 2000).

    Some have suggested that cilia and flagella were originally derived from spirochete bacteria which colonized the surface of ancestral eukaryotes.  There have been some reports of DNA associated with basal bodies (at the base of flagella) which might support this.  There was a report that green alga Chlamydomonas possesses a group of linked genes on a 6-9 megabase DNA molecule associated with their basal bodies.  Other studies suggested the same for several other protists.  This chromosome was reported in the anterior end of the elongated nucleus and might contact the basal bodies directly. (Hall, 1995; Hall 1989).  Other reports concluded that the basal bodies of Chlamydomonas reinhardtii do not contain immunologically detectable DNA (Johnson, 1990; (Johnson, 1991).

     In most cells, the centrioles (which are similar to basal bodies) are derived from existing centrioles which replicate.  Such a process is reminiscent of the duplication of other endosymbiotic organelles and may support a symbiotic origin for centrioles and basal bodies. There are exceptions to this, however.  Although most centriolar production occurs through replication of existing centrioles, there are examples of the synthesis of basal bodies de novo, as in meiosis in the fern Marsilea and the protist Naegleria.   Mouse embryos lack centrioles until the blastocyst stage, apparently indicating that the nucleus is capable of synthesizing them. (Johnson, 1991; (Hall, 1995)

 The unicellular green alga Ostreococcus tauri possesses the smallest eukaryotic genome and its lineage is thought to have diverged very early in the history of photosynthetic eukaryotes. The genome is 12.56-Mb in size and has a high gene density (Derelle, 2006).