Challenges to Endosymbiotic Theory

On the Origin of Mitochondria: Reasons for Skepticism on the Endosymbiotic Story 

https://reasonandscience.catsboard.com/t1303-challenges-to-endosymbiotic-theory

The origin of eukaryotes is one of the hardest and most intriguing problems in the study of the evolution of life, and arguably, in the whole of biology. On average, the volume of eukaryotic cells is about 15,000 times larger than that of prokaryotic cells. 4 A major problem faced by this scenario (and symbiogenetic scenarios in general) is the mechanistic difficulty of the engulfment of one prokaryotic cell by another.  The origin of eukaryotes is a fundamental, forbidding evolutionary puzzle. The scenario of eukaryogenesis, and in particular the relationship between endosymbiosis and the origin of eukaryotes, is far from being clear.  Compared to archaea and bacteria (collectively, prokaryotes), eukaryotic cells are three to four orders of magnitude larger in volume and display a qualitatively higher level of complexity of intracellular organization. Eukaryotic cells function on different physical principles compared to prokaryotic cells, which is directly due to their (comparatively) enormous size. The gulf between the cellular organizations of eukaryotes and prokaryotes is all the more striking because no intermediates have been found. So intimidating is the challenge of eukaryogenesis that the infamous notion of irreducible complexity’ has sneaked into serious scientific debate 2 . The diversity of the outcomes of phylogenetic analysis, with the origin of eukaryotes scattered around the archaeal diversity, has led to considerable frustration and suggested that a ‘phylogenomic impasse’ has been reached, owing to the inadequacy of the available phylogenetic methods for disambiguating deep relationships. The evolutionary trajectory of modern eukaryotes is  distinct from that of prokaryotes. Data from many sources give no direct evidence that eukaryotes evolved by genome fusion between archaea and bacteria. Nuclei, nucleoli, Golgi apparatus, centrioles, and endoplasmic reticulum are examples of cellular signature structures (CSSs) that distinguish eukaryote cells from archaea and bacteria.

Christopher S. Winefield: Evolutionary Analysis of Aspartate Aminotransferases : 22 June 1994
It is widely believed that mitochondria are derived from an endosymbiotic organism that colonized the ancestral eukaryote and that chloroplasts arose from a similar, but the distinct, event (Gray 1989). At the time of colonization, a complete but distinct alternative genome was present in the protomitochondria (and protochloroplast). The mitochondrial genome is now compressed and codes for only a few of the genes that are necessary for, and specific to, mitochondrial function--the balance of these genes now being present within the nuclear genome. This tendency to condense the mitochondrial genome is less marked for green plants where the mitochondrial genome is typically 200 kb, at an intermediate stage in the yeast mitochondrial genome (78 kb), and most pronounced in vertebrate mitochondria where the genome is about 16kb. There are at least two possible mechanisms that could account for the transfer of genetic information from the mitochondrial to nuclear genomes. In one scheme, it is proposed that genes may have duplicated in the nuclear genome and mutated until they were able to take over the role of the mitochondrial genes and subsequently the mitochondrial genes were lost. An alternative proposal is that the genes themselves were "copied" or transferred from the mitochondrial to the nuclear genome and were then deleted from the mitochondria. These mechanisms are not mutually exclusive and it is possible that both mechanisms have operated to produce the current observed state. A further mechanism that may account for different forms of an enzyme being present in the mitochondria and cytosol is differential splicing of a precursor mRNA. Such a mechanism is apparently responsible for the mitochondrial and cytosolic forms of fumarase found in vertebrates
https://pubmed.ncbi.nlm.nih.gov/7769621/


Intracellular Calcium, page 578
Most biology textbooks now tell us that organelles such as chloroplasts and mitochondria, both of which have circular DNA, evolved through endosymbiosis – a hypothesis promoted by Lynn Margulis (Margulis, 1991). However, the evidence for this is weak, and a much more likely origin of organelles such as the ER, mitochondria, lysosomes, peroxisomes, secretory vesicles, chloroplasts and the tonoplast in plants is invagination of membranes. Comparisons of mitochondrial DNA throughout animals, plants and eukaryotic microbes supports the hypothesis that mitochondria arose only once in evolution, and were from a proto-bacterial cell (Lang et al., 1999). But, the genomes of mitochondria and chloroplasts are too small to code for the genes necessary for a complete organism. 

Human mitochondrial DNA has just 16 569 base pairs, coding for only 37 genes, which are all essential for mitochondrial function, but far too little for a cell to survive. Thirteen of these genes produce proteins essential for ATP synthesis by oxidative phosphorylation, the other 25 coding for tRNA and rRNA, necessary for mitochondrial protein synthesis. Mitochondrial ribosomes are like bacterial ribosomes. None are involved in Ca2+ signaling. Yet, E. coli has some 300 essential genes which cannot be knocked-out without killing the cell, yet there are some 1500 proteins found inside a mitochondrion, several of which are involved in transporting Ca2+ in and out or responding to a rise in intra-mitochondrial free Ca2+. Mitochondrial divide, make proteins, make ATP, and carry out several other biochemical pathways, such as fatty acid oxidation. So if mitochondria originated from an endosymbiont such as Rickettsia there are three problems:

1. How did the endocytosed bacterium survive and multiply if its internal environment was oxidizing? The cytosol of all cells is reducing, preventing the formation of S–S bonds and damaging oxidative reactions involving reactive oxygen species. But, remember the first eukaryotes formed before there was significant oxygen in the atmosphere. So oxidative phosphorylation in mitochondria must have evolved after photosynthesis, some 2000 million years ago.
2. Since cells need at least 1200 proteins to survive, replicate, and synthesize their own building blocks, what happened to the proteins essential for nucleotide and nucleic acid, and protein synthesis, and the reactions necessary for ATP synthesis, e.g. glycolysis?
3. How did the 1500 or so mitochondrial proteins in the main genome become targeted to the mitochondria, if they were lost by the initial endosymbiont?
 
For the endosymbiotic hypothesis to work, the primitive bacterium engulfed by the eukaryote precursor must have lost over 90% of its genes, these being taken up by the nuclear genome. Furthermore, most of the proteins involved in Ca2+ signaling must have come from another source.

Plastid DNA contains just 60–100 genes, whereas a typical cyanobacterium DNA codes for 1500. The rest of the chloroplast proteins, like mitochondria, are coded for by the nuclear genome. A chloroplast genome of around 140 kb is comparable to a large bacteriophage, such as T4 whose genome is about 65 kb.

The start of the sequence for the origin of a mitochondrion or chloroplast would have to be a bacterium or cyanobacterium being taken up by the eukaryotic precursor. Then this has to lose genes, which are captured by the main genome. Then some of these genes have to have targeting sequences added them and they have to change several codons so that their genetic code matches the main genome, and not the proteins that remain in the mitochondrial or chloroplast genome, since there are differences in the genetic code between non-plant mitochondria and nuclear DNA, and losses of anticodons in chloroplasts. This is too many ‘ifs’ and ‘buts’ to be credible!

Did eukaryotes evolve from prokaryotic cells?
https://reasonandscience.catsboard.com/t1568-did-eukaryotes-evolve-from-prokaryotic-cells

1. There are no true intermediates in the prokaryote-to-eukaryote transition.  More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. The origin of eukaryotes is certainly one of early evolution's most important topics. “Throughout 150 years of the science of bacteriology, there is no evidence that one species of bacteria has changed into another... Since there is no evidence for species changes between the simplest forms of unicellular life, it is not surprising that there is no evidence for evolution from prokaryotic [i.e., bacterial] to eukaryotic [i.e., plant and animal] cells, let alone throughout the whole array of higher multicellular organisms.” The organizational complexity of the eukaryotes is so much greater than that of the prokaryotes that it is difficult to visualize how a eukaryote could have arisen from any known prokaryote (Hickman et al., 1997, p. 39). In eukaryotes the mitochondria produce most of the cell’s ATP (anaerobic glycolysis also produces some) and in plants the chloroplasts can also service this function. The mitochondria produce ATP in their internal membrane system called the cristae. Since bacteria lack mitochondria, as well as an internal membrane system, they must produce ATP in their cell membrane which they do by two basic steps. The bacterial cell membrane contains a unique structure designed to produce ATP and no comparable structure has been found in any eukaryotic cell (Jensen, Wright, and Robinson, 1997).

2.  The Darwinian Basis of the Prokaryote-to-Eukaryote Transition Collapses
https://reasonandscience.catsboard.com/t1568-did-eukaryotes-evolve-from-prokaryotic-cells#3782
Mitochondria has a different genetic code, and there is no viable route for the evolution of the genetic code. Mitochondria use a slight variation on the conventional genetic code (for example, the codon UGA is a stop codon in the conventional code, but encodes for Tryptophan in mitochondria). This implicates that the genes of the ingested prokaryotes would need to have been recoded on their way to the nucleus. The situation becomes even worse when one considers that, in eukaryotic cells, a mitochondrial protein is coded with an extra length of polypeptide which acts as a "tag" to ensure that the relevant protein is recognised as being mitochondrial and dispatched accordingly. The significant number of specific co-ordinated modifications which would be required to facilitate such a transition, therefore, arguably make it exhibitive of irreducible complexity.

The different genetic codes
https://reasonandscience.catsboard.com/t2277-the-different-genetic-codeses
The National Center for Biotechnology Information (NCBI), currently acknowledges nineteen different coding languages for DNA. And i list 31 different ones.
If the mitochondria in invertebrates use a different genetic code from the mitochondria in vertebrates, and both of those codes are different from the “universal” genetic code, what does that tell us? It means that the eukaryotic cells that eventually evolved into invertebrates must have formed when a cell that used the “universal” code engulfed a cell that used a different code. However, the eukaryotic cells that eventually evolved into vertebrates must have formed when a cell that used the “universal” code engulfed a cell that used yet another different code. As a result, invertebrates must have evolved from one line of eukaryotic cells, while vertebrates must have evolved from a completely separate line of eukaryotic cells. But this isn’t possible, since evolution depends on vertebrates evolving from invertebrates. Now, of course, this serious problem can be solved by assuming that while invertebrates evolved into vertebrates, their mitochondria also evolved to use a different genetic code. However, I am not really sure how that would be possible. After all, the invertebrates spent millions of years evolving, and through all those years, their mitochondrial DNA was set up based on one code. How could the code change without destroying the function of the mitochondria? At minimum, this adds another task to the long, long list of unfinished tasks necessary to explain how evolution could possibly work. Along with explaining how nuclear DNA can evolve to produce the new structures needed to change invertebrates into vertebrates, proponents of evolution must also explain how, at the same time, mitochondria can evolve to use a different genetic code!

3. Membranes of dauther cells are only inherited by membranes of mother cells through fission.

Intracellular Compartments and Protein Sorting
https://reasonandscience.catsboard.com/t3048-intracellular-compartments-and-protein-sorting
Unlike a bacterium, which generally consists of a single intracellular compartment surrounded by a plasma membrane, a eukaryotic cell is elaborately subdivided into functionally distinct, membrane-enclosed compartments. Each compartment, or organelle, contains its own characteristic set of enzymes and other specialized molecules, and complex distribution systems transport-specific products from one compartment to another. To understand the eukaryotic cell, it is essential to know how the cell creates and maintains these compartments, what occurs in each of them, and how molecules move between them. Proteins confer upon each compartment its characteristic structural and functional properties. They catalyze the reactions that occur there and selectively transport small molecules into and out of the compartment. For membrane-enclosed organelles in the cytoplasm, proteins also serve as organelle-specific surface markers that direct new deliveries of proteins and lipids to the appropriate organelle. An animal cell contains about 10 billion (10^10) protein molecules of perhaps 10,000 kinds, and the synthesis of almost all of them begins in the cytosol, the space of the cytoplasm outside the membrane-enclosed organelles. Each newly synthesized protein is then delivered specifically to the organelle that requires it. . By tracing the protein traffic from one compartment to another, one can begin to make sense of the otherwise bewildering maze of intracellular membranes

4. Mitochondrial membrane biogenesis: phospholipids and proteins go hand in hand 1
https://reasonandscience.catsboard.com/t2128-cell-membranes-origins-through-natural-mechanisms-or-design
Mitochondrial membrane biogenesis requires the import and synthesis of proteins as well as phospholipids.The biochemical approach of Kutik et al. (2008) uncovered an unexpected role of the mitochondrial translocator assembly and maintenance protein, Tam41, in the biosynthesis of cardiolipin (CL), the signature phospholipid of mitochondria. The genetic analyses of Osman et al. (2009) led to the discovery of a new class of mitochondrial proteins that coordinately regulate CL and phosphatidylethanolamine, another key mitochondrial phospholipid. These elegant studies highlight overlapping functions and interdependent roles of mitochondrial phospholipid biosynthesis and protein import and assembly

5. The mitochondrial inner membrane has a unique composition of proteins and phospholipids, whose interdependence is crucial for mitochondrial function.

6. Most Organelles Cannot Be Constructed De Novo: They Require Information in the Organelle Itself 
https://reasonandscience.catsboard.com/t2122-most-organelles-cannot-be-constructed-de-novo-they-require-information-in-the-organelle-itself
7. The Interdependency of Lipid Membranes and Membrane Proteins
https://reasonandscience.catsboard.com/t2397-the-interdependency-of-lipid-membranes-and-membrane-proteins
A cell cannot produce the cell membrane de novo from scratch. It inherits it. Daughter cell membranes come only from mother cell membranes.






Functional Anatomy Of Prokaryotic And Eukaryotic Cells


With regret, ENV recently noted the passing of biologist Lynn Margulis. Margulis, a scientist whom I admired greatly, was never a stranger to controversy, going so far as to call neo-Darwinism "a complete funk" and asserting that "The critics, including the creationist critics, are right about their criticism. It's just that they've got nothing to offer by intelligent design or 'God did it.' They have no alternatives that are scientific." She was a scientist who wasn't afraid to think creatively, disregarding the scorn of her colleagues. According to the Telegraph, a response to one grant application she made said: "Your research is crap. Don't ever bother to apply again."
Lynn Margulis took a controversial view on how evolution works, stressing the importance of symbiotic and co-operative relationships over competition. This concept of evolution inspired what is now recognized as her most notable idea, the notion that the eukaryotic mitochondrion -- the power plant of the cell -- was acquired by virtue of an endosymbiotic event. Endosymbiotic theory essentially maintains that mitochondria arose by virtue of a symbiotic union of prokaryote cells. The nearest living relative to the mitochondrion is thought to be the alpha-proteobacteria Rickettsia (Emelyanov, 2000; Andersson et al., 1998). Chloroplasts are also thought to have arisen in a similar manner from the photosynthetic cyanobacteria.
In November 2010, I drew attention to a paper in Nature by Nick Lane and Bill Martin, who showed that the prokaryote-to-eukaryote transition was effectively impossible without the energy demands, pertinent to the biggest event of gene manufacture in the history of life on earth, being met by the mitochondrial processes of oxidative phosphorylation and the electron transport chain. The bacterial cell alone could not meet these energy demands. The evidence that is typically offered for endosymbiotic theory includes the following:

Mitochondria possess a circular genome (lacking in introns and independent from the nuclear DNA) in which transcription is coupled to translation, characteristic of bacterial DNA. There are also some other notable similarities. For example, in both mitochondria and Mycoplasma, the codon UGA specifies the amino acid Tryptophan , whereas in the conventional code it serves as a stop codon. Mitochondria divide and replicate independently of host cell division and do so in a manner akin to binary fission, possessing homologues of the bacterial division protein FtsZ .They are enclosed by a double-membrane.Mitochondria and bacteria are of a similar size and shape.Circular Mitochondrial Genome. As noted, one of the core arguments for endosymbiosis points to its circular genome. What is often not noted, however, are the cases where eukaryotic mitochondria have linear genomes with eukaryotic telomeres. Indeed, two strains of the same species of yeast differ with respect to the linearity or circularity of their mitochondrial genome .In the case of linear chromosomes, the DNA polymerase enzymes are unable to replicate right to the end of the chromosome. This is because the enzymes are unable to replace the lagging strand's terminal RNA primer. Unless there is a mechanism for circumventing this, it will result in the chromosomes shortening after each round of replication (in eukaryotes, the enzyme telomerase attaches extra DNA to the chromosomal ends).

 This means that the transition from genome circularity to linearity -- a fete in itself given the changes that have to be made to the mode of replication -- must happen in concert with the evolution of a mechanism to prevent progressive chromosomal shortening. 

In order to have a transition from prokaryotic to eukaryotic dna replication, telomerase enzymes must arise simultaniously, to prevent the shortening of the telomere region after every replication. 

Telomerase, also called telomere terminal transferase,^[1] is a ribonucleoprotein that adds the polynucleotide "TTAGGG" to the 3' end of telomeres, which are found at the ends of eukaryotic chromosomes. A telomere is a region of repetitive sequences at each end of a chromatid, which protects the end of the chromosome from deterioration or from fusion with neighbouring chromosomes. 5

Such an evolutionary transition is far from trivial. Biologist Albert de Roos writes,n linear mitochondrial chromosomes various different mechanisms to "prevent" shortening exist, ranging from hairpin loops and self-priming to protein-assisted primer synthesis (see here). The telomeric regions of mitochondrial chromosomes do not seem to have a direct phylogenetic relation since they use other proteins and mechanisms than nuclear telomeres. Thus, it is difficult to deduce evolutionary pathways purely based on phylogenetic data on telomeres and mechanisms for end replication.Furthermore, mitochondrial genes often do possess introns . These are particularly prevalent in the mtDNA of fungi and plants. The mitochondrial genetic code may also be slightly different from that of bacteria .Mitochondrial DNA Replication: 

The claim one often hears is that circular mitochondrial DNA replication resembles bacterial binary fission. While this is true, in at least some respects, there are also important differences. For example, many of the key components are of eukaryotic origin and replication beginning at the Displacement (D-) loop (Fish et al., 2004; Clayton, 1996) is not the same as bacterial DNA replication.
Double Membrane
It is frequently asserted that the double membrane of mitochondria provides evidence for its endosymbiotic origin. There are, however, important differences between bacterial and mitochondrial membranes. Albert de Roos observes,

The bacterial membrane is one of the basic characteristics that distinguish bacteria from eukaryotes, see some examples here. In order for mitochondria to resemble bacterial membranes, they should share characteristics such as a cell wall with peptidoglycan and lipopolysaccharides, gram-staining and antibiotic sensitivity. Some effects of antibiotics have been seen with both bacteria and mitochondria, but the effect is minor while the use of antibiotics is based on the principle that they distinguish between bacteria and eukarytes, including the mitochondrion (here). Until then, the selection of a few apparent similarities while ignoring the many differences does not indicate a bacterial origin for mitochondria. On the contrary, the fact that their membranes are so different as well as the fact that nearly all genes are encoded by the nucleus is primarily evidence against a bacterial origin.
Even though some shared characteristics may be found, we have to realize that bacterial and eukaryotic membranes are fundamentally different. It seems virtually impossible to change all fundamental bacterial membrane characteristics and replace them with a eukaryotic counterpart without loosing membrane integrity. The differences between the membranes of mitochondria and the cell walls of bacteria make the endosymbiotic theory mechanistically difficult. It seems quite clear that bacterial membranes do not change easily into other membranes, and frankly I don't see any scenarios in which to change all these membrane components without drastically affecting fitness.

The Size and Shape of Mitochondria
The argument based on the size and shape of mitochondria is one that has been turned on its head in recent years, being transformed from an argument forendosymbiosis to one against it. These organelles are now acknowledged in the literature to be better understood as dynamic reticular structures (see this linkfor references).
Electron micrographs displaying cross-sections of mitochondria portrayed the mitochondrion as a sphere. However, when one looks at 3D models of the organelle, the reality is somewhat different. You can take a look at some of these images by going here, here, here, or here.
The Lack of a Mechanism
By far the most potent challenge to the endosymbiotic origin of eukaryotic mitochondria is the lack of a viable mechanism, perhaps most particularly with respect to the transfer of genes from the mitochondrion to the nucleus.
For one thing, there are the variants on the conventional genetic code. This means that, over the course of their transfer to the nucleus, the genes would need to be "recoded" so as to comply with the conventional genetic code. For example, recognizing UGA as a stop codon instead of the codon for Tryptophan(or vice versa) would cause cellular mayhem.
Secondly, mitochondrial proteins made at the ribosomes in the eukaryote cytoplasm need to be identified as such to ensure that they are properly dispatched (this is normally done by attaching a "label" in the form of an extra length of polypeptide to the protein). This would require a coincidental modification of the correct structural gene (which seems unlikely). Biologist Timothy G. Standish [url=http://www.google.co.uk/url?sa=t&rct=j&q=if genes were to move from the mitochondria to the nucleus they would have to somehow pick up the leader sequences necessary to signal for transport before they could be]notes[/url],

• If genes were to move from the mitochondria to the nucleus they would have to somehow pick up the leader sequences necessary to signal for transport before they could be functional.
  
  
  
• While leader sequences seem to have meaningful portions on them, according to Lewin (1997, p251) sequence homology between different sequences is not evident, thus there could be no standard sequence that was tacked on as genes were moved from mitochondria to nucleus.
  
  
  
• Alternatively, if genes for mitochondrial proteins existed in the nucleus prior to loss of genes in the mitochondria, the problem remains, where did the signal sequences come from? And where did the mechanism to move proteins with signal sequences on them come from?
  
  
  

Albert de Roos explains,

All evolutionary theories must offer an explanation in mechanistic terms of how it should or could have happened in order to be tested. The difficult thing with the endosymbiotic theory is that it proposes no real mechanism and most textbooks show the simplistic picture of a cell that swallows another cell that becomes a mitochondrion. Unfortunately, it is not so simple as that. There is a difference between the process of endosymbiosis and its incorporation in the germline, necessitating genetic changes. What were those changes? What was the host? Was it a fusion, was it engulfment, how did the mitochondrion get its second membrane, how did two genomes in one cell integrate and coordinate? The theory is also strongly teleological, illustrated by the widely used term 'enslavement'. But how do you enslave another cell, how do you replace its proteins and genes without affecting existing functions? The existence of obligate bacterial endosymbionts in some present eukaryotes is often presented as a substitute for a mechanism, but they remain bacteria and give not rise to new organelles. So, before we can speak of the endosymbiotic as a testable scientific theory, we need a mechanistic scenario which is lacking at the moment.
When we do try to envision a mechanistic scenario based on the endosymbiotic theory, we quickly run into problems. Genetic mutations that allow bacteria to thrive in the cytoplasm would not be strategic for survival. Anaerobic cells normally do not survive in environment that contains oxygen, while the endosymbiont would need oxygen in order to present fitness advantage. The two organisms would initially compete for energy sources since bacteria are users of ATP and do not export it. The extensive gene transfer that is needed in the endosymbiotic theory would wreak havoc in a complex genome since frequent insertion of random pieces of mitochondrial DNA would disrupt existing functions. Furthermore, gene transfer is a multi-step process were genes need to be moved to the nucleus, the different genetic code of mitochondria needs to be circumvented, the genes need to be expressed correctly, as well as imported back into the mitochondria in order to be functional. All in all, mechanistic scenarios for the endosymbiotic theory imply many non-functional intermediates or would just be plain harmful to an organism. Therefore, the endosymbiotic theory is in contrast with the concept of gradualism that forms the basis of modern evolutionary theory.

Furthermore, this gene transfer must have taken place at a time extremely early in the history of eukaryotes, substantially reducing the window of time in which gene transfer could have occurred.
Summary and Conclusion
While we find examples of similarity between eukaryotic mitochondria and bacterial cells, other cases also reveal stark differences. In addition, the sheer lack of a mechanistic basis for mitochondrial endosymbiotic assimilation ought to -- at the very least -- give us reason for caution and the expectation of some fairly spectacular evidence for the claim being made. At present, however, such evidence does not exist -- and justifiably gives one cause for skepticism.

Graham JonesSaturday:
I read Nick Lanes's The Vital Question, and there's something that puzzles me, which relates to this article: The energetics of genome complexity, Nick Lane & William Martin, 2010. (http://www.nature.com/nature/journal/v467/n7318/abs/nature09486.html)

They do a back-of-the-envelope calculation. Prokaryotes metabolise about 3x faster than eukaryotes in terms of watts/g, but eukaryotic cells are 15,000x bigger, so eukaryotic cells have 5000x times as much power in terms of watts/cell. Eukaryotes have 4x as many genes as prokaryotes, so they have 1200x as much power per gene.

About 80% of a cell's power is used for making proteins, so it seems eukaryotes make about 1200x as much protein per gene. I started wondering about the mechanics of that. Transcription rates and translation rates per nucleotide are at least as fast in prokaryotes, and there's no introns to transcribe or splice out, so you'd think prokaryotes would make more protein per gene, not 1200x less.

Eukaryotic cells cells live longer of course, and most are diploid, so have two copies of each gene. Some are polyploid or multinucleated - and I think Lane and Martin's "15,000x bigger" includes some eukaryotic cells with lots of nuclei. A fair comparison would be of (rate of protein synthesis)/(gene copy).

So I got some numbers, from the book Cell Biology by the Numbers, and the biomumbers site (http://bionumbers.hms.harvard.edu/). I found it easiest to get figures for cell volumes. I assume (protein weight)/volume is similar for these cells. They are for organisms when they are growing fast (but not as fast as possible). V = volume in um^3 per gene copy per hour.

E Coli: volume 0.7 um^3, #gene copies 4400, doubling time 1h. V = 1.6e-4
Budding yeast: volume 37 um^3, #gene copies 6000, doubling time 3h. V = 20e-4
C Elegans: volume 1800 um^3, #gene copies 40000, doubling time 10h. V = 45e-4
Euglena gracilis: volume 3700 um^3, #gene copies 60000, doubling time 12h. V = 50e-4

It seems that these eukaryotic cells make protein 10-30x faster than E Coli, per gene copy. Looks very odd to me. Presumably the limiting factor for prokaryotes is power, as Lane and Martin suggest. Apparently prokaryotes have the molecular machinery to make protien faster than eukaryotes, but they hardly ever have enough energy to run the machinery anywhere near full speed.



A simplified view of the endosymbiosis theory. 
(a) According to this concept, modern mitochondria were derived from purple bacteria, also called α-proteobacteria. Over the course of evolution, their characteristics changed into those found in mitochondria today. 
(b) A similar phenomenon occurred for chloroplasts, which were derived from cyanobacteria (blue-green bacteria), a bacterium that is capable of photosynthesis.

Because mitochondria look like prokaryotes, it was assumed that eukaryotic cells came into existence when one prokaryote swallowed another prokaryote.  A problem with this idea is that prokaryotic cells lack the ability to swallow other cells. Eukaryotic cells can, but every eukaryote we know about has mitochondria. It's not clear which came first: the ability to swallow other cells, or the mitochondria.

Examples of ecological circumstances driving genome reduction are seen in many intracellular endosymbionts and parasites, which gain few genes but lose many genes responsible formetabolic flexibility The mitochondrion is even more extreme in its reductive evolution; its ancestral bacterial genome has been reduced to a vestigial microgenome supported by a predominantly eukaryote proteome. Genomes of modern mitochondria encode between 3 and 67 proteins, whereas the smallest known free-living a-proteobacterium (Bartonella quintana) encodes 1100 proteins. Taking Bartonella as a minimal genome for the freeliving ancestor of mitochondria, nearly all of the bacterial coding sequences have been lost from the organelle, though not necessarily from the eukaryote cell. The mitochondrial genome of the protist Reclinomonas americana is the largest known but has still lost more than 95% of its original coding capacity. This abbreviated account of genome reduction illustrates the Darwinian view of evolution as a reversible process in the sense that ‘‘eyes can be acquired and eyes can be lost.’’ Genome evolution is a two-way street. This bidirectional sense of reversibility is important as an alternative to the view of evolution as a rigidly monotonic progression from simple to more complex states, a view with roots in the 18th-century theory of orthogenesis. Unfortunately, such a model has been tacitly favored by molecular biologists who appeared to view evolution as an irreversible march from simple prokaryotes to complex eukaryotes, from unicellular to multicellular. The many well documented instances of genome reduction provide a necessary corrective measure to the often-unstated assumption that eukaryotes must have originated from prokaryotes.


1) https://en.wikipedia.org/wiki/Pre-replication_complex
2) http://nar.oxfordjournals.org/content/27/17/3389.full
3) http://www.answersingenesis.org/articles/2006/10/11/endosymbiotic-theory
4) http://www.evolutionnews.org/2012/01/on_the_origin_o054891.html
5) https://en.wikipedia.org/wiki/Telomerase
6) Intracellular Calcium, page 578

PROTEIN IMPORT INTO CHLOROPLASTS



Endosymbiosis was accompanied by massive gene transfer from the endosymbiont to the host nucleus.

Begging the question at its best........

However, before genes could be eliminated from the endosymbiont genome, a system to import the now nuclear-encoded gene products into the new organelle had to be established. Although the endosymbiotic bacterium had several systems to export (or secrete) proteins across the membranes, the organelle now had to import proteins (see figure above).

Most striking is the homology of the translocon of the outer-chloroplast-envelope subunit TOC75 to bacterial outer-membrane proteins that are involved in the transport
of polypeptides across the outer membrane of Gram-negative bacteria95,96.This conserved β-barrel, bacterial-type channel now forms the outer-chloroplast-envelope import channel. The TOC75 homologue in cyanobacteria, SynToc75 seems to be indispensable for growth67,68. A β-barrel ion channel has, in most cases, no strong preference for the direction of ion permeation and therefore represents an ideal starting point to build a translocon.

Subunits that convey the specificity and directionality of transport are eukaryotic additions, for example, the TOC34 receptor and the TOC159 motor.

But, what formed the translocon of the inner chloroplast envelope (TIC)? There is no detectable homologue for the putative TIC110 channel, and the putative second channel
subunit TIC20 shows only a low homology to bacterial proteins.

The homology is most striking, except when it isnt..... and when it isnt, its time to make things up, and just assert that the subunits were  " eukaryotic additions ". Homology can well be explained through common design. Thats common practice. Examples which seem to fit evolutionary assumptions are  cited, while the  examples that do not fit are ignored, or baseless assertions are made, as above shows.

The many similarities that exist among members of the animal kingdom is the result of the fact that a single designer created the basic kinds of living 'systems', then specially modified each type of life to enable it to survive in its unique environmental niche.

Structural similarities among automobiles, however, even similarities between older and newer models  are due to construction according to pre-existing patterns, i.e., to design. Ironically, even striking similarities are not sufficient to exclude design-based explanations. In order to demonstrate naturalistic evolution, it is necessary to show that the mechanism by which organisms are constructed (unlike the mechanism by which automobiles are constructed) does not involve design.

Maybe the early endosymbiont continued to use bacterial export systems in reverse, such as the secretory pathway (SEC), the twin-arginine translocon (TAT) system or the albino3 (ALB3) homologue YIDC8,10.Therefore, the TIC translocon — including the adaptation of chaperones in the stroma to provide the driving force for import — could have been an invention of the endosymbiont

Its remarkable how proponents of evolution frequently borrow a vocabulary from where they are not aloud to. Evolution has no intelligence to invent things.....

Molecular Cell biology, Lodish, 5th edition, pg. 691

At least three chloroplast outer-membrane proteins, including a receptor that binds the stromal-import sequence and a translocation channel protein, and five inner-membrane proteins are known to be essential for directing proteins to the stroma. Although these proteins are functionally analogous to the receptor and channel proteins in the mitochondrial membrane, they are not structurally homologous.The lack of homology between these chloroplast and mitochondrial proteins suggests that they may have arisen independently


Or they were designed.......

1) http://ecoserver.imbb.forth.gr/documents/Soll04.pdf

Did DNA replication evolve twice independently? 1

DNA replication is central to all extant cellular organisms. There are substantial functional similarities between the bacterial and the archaeal/eukaryotic replication machineries, including but not limited to defined origins, replication bidirectionality, RNA primers and leading and lagging strand synthesis. However, several core components of the bacterial replication machinery are unrelated or only distantly related to the functionally equivalent components of the archaeal/eukaryotic replication apparatus.
Consequently, the modern-type system for double-stranded DNA replication likely evolved independently in the bacterial and archaeal/eukaryotic lineages.

This should be one more reason to doubt of the endosymbiotic theory

At the Dawn of Life, a Mystery 2

Stories exist for how mitochondria and chloroplasts came to be present in eukaryotic cells -- they mainly involve the incorporation of ancient bacteria into the incipient eukaryotic cell. The proposed process has been given the name endosymbiosis. There is no single proposed mechanism for the evolution of the nucleus or the other structures I have named. I deliberately call such evolutionary accounts "stories." To become a eukaryote like C. reinhardtii involves enormous changes in cell organization that affect every aspect of cellular life. Most of these structures are common to eukaryotic cells, and most are membrane-bound. Membranes mean there must be transport mechanisms in or out of each compartment. DNA replication and division becomes more complicated because the nuclear membrane must break down and reform at each division. Nuclear genes have somehow come to specify proteins necessary for mitochondrial function; they must be transcribed, the RNA exported to the cytoplasm, made into protein, and then the proteins must be transported into the mitochondrion. Specific problems associated with the replication of chromosomes versus circular DNA as in bacteria have to solved. There are more differences to be dealt with than I can cover -- exons and introns, and the separation of mRNA production in the nucleus from protein synthesis in the cytoplasm, just to name two. All of these problems must be solved somehow if the story of undirected evolution is true.

The PsbO protein is common among all oxygenic photosynthetic organisms, but the number of copies associated with each PSII complex differs between plants and cyanobacteria. The PsbR protein is unique to plants. Both plants and cyanobacteria contain the PsbP and PsbQ proteins, but their roles and modes of association with PSII complexes differ significantly between the two systems.

Co-evolution of primordial membranes and membrane proteins

The chemical compositions and biogenesis pathways of archaeal and bacterial membranes are fundamentally different [4,5,12].The glycerol moieties of the membrane phospholipids in all archaea and bacteria are of the opposite chiralities. With a few exceptions, the hydrophobic chains differ as well, being based on fatty acids in bacteria and on isoprenoids in archaea; furthermore, in bacterial lipids, the hydrophobic tails are usually linked to the glycerol moiety by ester bonds whereas archaeal lipids contain ether bonds [4,5,12]. The difference extends beyond the chemical structures of the phospholipids, to the evolutionary provenance of the enzymes involved in membrane biogenesis that are either non-homologous or distantly related but not orthologous in bacteria and archaea [4,5,12,13]. 



1) http://nar.oxfordjournals.org/content/27/17/3389.full
2) http://www.evolutionnews.org/2015/05/at_the_dawn_of095801.html
3) http://newunderthesunblog.wordpress.com/my-research/
4) Co-evolution of primordial membranes and membrane proteins

http://reasonandscience.heavenforum.org/t2277-the-different-genetic-codes

if the mitochondria in invertebrates use a different genetic code from the mitochondria in vertebrates, and both of those codes are different from the “universal” genetic code, what does that tell us? It means that the eukaryotic cells that eventually evolved into invertebrates must have formed when a cell that used the “universal” code engulfed a cell that used a different code. However, the eukaryotic cells that eventually evolved into vertebrates must have formed when a cell that used the “universal” code engulfed a cell that used yet another different code. As a result, invertebrates must have evolved from one line of eukaryotic cells, while vertebrates must have evolved from a completely separate line of eukaryotic cells. But this isn’t possible, since evolution depends on vertebrates evolving from invertebrates.
Now, of course, this serious problem can be solved by assuming that while invertebrates evolved into vertebrates, their mitochondria also evolved to use a different genetic code. However, I am not really sure how that would be possible. After all, the invertebrates spent millions of years evolving, and through all those years, their mitochondrial DNA was set up based on one code. How could the code change without destroying the function of the mitochondria? At minimum, this adds another task to the long, long list of unfinished tasks necessary to explain how evolution could possibly work. Along with explaining how nuclear DNA can evolve to produce the new structures needed to change invertebrates into vertebrates, proponents of evolution must also explain how, at the same time, mitochondria can evolve to use a different genetic code!


  In Current Biology,⁵ Paul Jarvis wrote about the “backchat” that goes on between chloroplasts and the nucleus in plant cells.  He assumed that chloroplasts evolved as once free-living cells that were engulfed by an ancestral prokaryote, and that their separate genomes were partitioned, most of the DNA going to the nucleus of the host.  Still, a remarkable degree of communication is required to ensure the proper amounts of chloroplast proteins are produced in the nucleus: “To ensure the correct, stoichiometric assembly of these complexes, and to enable their rapid reorganization in response to developmental or environmental cues, the activities of the nuclear and chloroplast genomes must be synchronized through intracellular signalling,” he said.  Each protein must then traverse the inner and outer membranes of the chloroplast, assisted by complexes of molecular machines.  Jarvis presented one example of the complexity involved in signalling:

A particularly nice example is provided by the plastid protein import 1 (ppi1) mutant, which lacks the chloroplast protein import receptor atToc33.  This is actually one of two similar receptors in Arabidopsis, the other being atToc34, which are thought to have distinct substrate preferences: atToc33 mediating the import of the highly abundant precursors of the photosynthetic apparatus, and atToc34 the import of‘housekeeping’ proteins (for example, components of the plastid’s genetic system, or enzymes of non-photosynthetic metabolism).  Remarkably, the ppi1 mutation triggers the specific down-regulation of photosynthesis-related genes (Figure 2), suggesting that retrograde signalling mechanisms exist to prevent the futile expression of proteins not able to reach their final, organellar destination.  Clearly, such exquisite regulation specificity could not be achieved were all plastid signalling pathways to converge and control gene expression through a common process.

He did not elaborate on how all this “organellar repartee” could have evolved, though.  He just ended on the note, “Observations such as these suggest that a great deal remains to be learnt concerning plastid-to-nucleus signalling.”


http://creationsafaris.com/crev200707.htm

The origin of mitochondria in light of a fluid prokaryotic chromosome model

http://rsbl.royalsocietypublishing.org/content/3/2/180

On the basis of sequence similarity to α-proteobacterial homologues, it has been estimated that 630 eukaryotic genes trace to α-proteobacteria (Gabaldon & Huynen 2003). But there are thousands of eukaryotic nuclear genes that are clearly eubacterial, but not specifically α-proteobacterial, in terms of their patterns of sequence similarity (Esser et al. 2004; Rivera & Lake 2004; Embley & Martin 2006). Finding a eukaryotic gene that branches with a group other than α-proteobacteria is often taken as evidence for an origin from that group (for example, Baughn & Malamy 2002), the methodological problems of deep phylogenetic trees notwithstanding (Susko et al. 2006). But if we let go of the static prokaryotic chromosome model and assume a fluid chromosome model for prokaryotes, then the expected phylogeny for a gene acquired from the mitochondrion would be common ancestry for all eukaryotes, but not necessarily tracing to α-proteobacteria, because the ancestor of mitochondria possessed an as yet unknown collection of genes. A previous investigation of genome evolution in α-proteobacteria considered the genome size and functional classes (Boussau et al. 2004), but not sequence similarities. Hence, we wished to know how many of the α-proteobacterial genes pass the test of being α-proteobacterial by the nearest-neighbour criterion.
The answer, based upon the current sample, ranges from approximately 97% for Sinorhizobium to approximately 33% for Magnetococcus sp. The mitochondrial genomes studied (figure 1d) did not differ in terms of the nearest-neighbour composition from α-proteobacterial genomes.


An Overview of Endosymbiotic Models for the Origins of Eukaryotes, Their ATP-Producing Organelles (Mitochondria and Hydrogenosomes), and Their Heterotrophic Lifestyle

http://www.molevol.de/molevol2/publications/98.pdf

The evolutionary processes underlying the differentness of prokaryotic and eukaryotic cells and the origin of the latter’s organelles are still poorly understood. For about 100 years, the principle of endosymbiosis has figured into thoughts as to how these processes might have occurred. A number of models that have been discussed in the literature and that are designed to explain this difference are summarized. The evolutionary histories of the enzymes of anaerobic energy metabolism (oxygen-independent ATP synthesis) in
the three basic types of heterotrophic eukaryotes – those that lack organelles of ATP synthesis, those that possess mitochondria and those that possess hydrogenosomes
– play an important role in this issue. Traditional endosymbiotic models generally do not address the origin of the heterotrophic lifestyle and anaerobic energy metabolism in eukaryotes. Rather they take it as a given, a direct inheritance from the host that acquired mitochondria. Traditional models are contrasted to an alternative endosymbiotic model (the hydrogen hypothesis), which addresses the origin of heterotrophy and the origin of compartmentalized energy metabolism in eukaryotes

The evolution of cardiolipin biosynthesis and maturation pathways and its implications for the evolution of eukaryotes
Cardiolipin (CL) is an important component in mitochondrial inner and bacterial membranes. Its appearance in these two biomembranes has been considered as evidence of the endosymbiotic origin of mitochondria. But CL was reported to be synthesized through two distinct enzymes--CLS_cap and CLS_pld in eukaryotes and bacteria. Therefore, how the CL biosynthesis pathway evolved is an interesting question.



Its not just an interesting question. Its evidence against the endosymbiotic theory. 

Bacterial Origins for Mitochondria and Primary Plastids
Mitochondria and primary plastids almost certainly evolved from α-proteobacteria and cyanobacteria, respectively (Figure 2), but a more exact determination of their bacterial sources remains elusive. In the case of mitochondria, it has been suggested they evolved from Rickettsia-, Pelagibacter-, or Rhodospirillum-like species. A similar uncertainty concerns primary plastids, with proposal of an Anabena-like species versus an unknown ancient cyanobacterial lineage. A major to resolving these controversies is the chimerism of bacterial genomes; it probably results from horizontal gene transfers between distinct bacterial lineages on the one hand, and lineage-specific gene duplications and losses on the other hand.


Evolution of primary plastids.
Primary plastids evolved from cyanobacteria that were engulfed by phagotrophic protozoans. Driving forces behind the establishment of primary plastids could have been photosynthesis, as well as nonphotosythetic functions such as nitrogen fixation or fatty acid synthesis. Numerous genes of the cyanobacterial endosymbiont have moved to the host nucleus via the endosymbiotic gene transfer (EGT). Almost all their protein products are equipped with transit peptides (TPs) and imported into the plastid with the help of Toc and Tic translocons; however, some plastid pre-proteins carry signal peptides (SPs)(e.g., α-carbonic anhydrase) that deliver them to primary plastids via the endoplasmic reticulum and/or the Golgi apparatus. The inner membrane of primary plastids is certainly derived from the endosymbiont plasma membrane, but the origin of their outer membrane is still controversial. This membrane could have come from the host phagosomal membrane (as demonstrated by the endomembrane system-mediated targeting of some proteins) or the outer membrane of the cyanobacterial endosymbiont (the membrane contains porin-like proteins such as the Toc75 channel). It is possible, however, that the outer membrane of primary plastids has a chimeric eukaryotic–bacterial origin, because the engulfed cyanobacteria (with their outer and plasma membranes) must initially have been surrounded by a phagosomal membrane, and, after its disruption, their outer membrane could have acquired features of the host phagosomal membrane.

α-Proteobacteria and cyanobacteria are Gram-negative bacteria, which means the ancestors of mitochondria and primary plastids were very likely surrounded by two membranes, a plasma membrane and an outer membrane, with a peptidoglycan wall between them.

Chloroplasts in red and green algae (and their descendants, the land plants) are surrounded by two membranes. The outer membrane is synthesized by the surrounding cytoplasm, following genetic instructions issued from the nucleus. In contrast, the inner membrane is made by the chloroplast itself. Moreover, the outer chloroplast membrane is part of an extensive membrane system that includes the cell’s bounding membrane, the nuclear membrane, and an internal membrane system that permeates the cytoplasm. These membranes are in dynamic continuity, which means that while they may be distinct and unconnected at any one moment, they occasionally combine to form a complex and nearly continuous surface. The significance of this seemingly arcane detail is that the nucleus and cytoplasm lie within this membranous boundary, whereas the chloroplast and its inner membrane lie outside it

W. F. Martin (2015): For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. ( Maybe there was never a transition, to begin with) Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. 

The origin of eukaryotes is certainly one of the most important topics. There are various perspectives from which eukaryote origins can be viewed, including palaeontological evidence, energetics, the origin of eukaryote-specific traits or the relationships of the different eukaryotic groups to one another. The concept of symbiosis (Latin, ‘living together’), that two different organisms can stably coexist and even give rise to a new type of organism, traces to Simon Schwendener, a Swiss botanist who discovered that lichens consist of a fungus and a photosynthesizer. The German botanist Heinrich Anton de Bary (1878) coined the term ‘Symbiose’ to designate this type of coexistence.

Many biologists still have a problem with the notion of endosymbiosis and hence prefer to envisage the origin of eukaryotes as the product of gene duplication, point mutation and micromutational processes. Besides energy,there is the origin of the nucleus to deal with, and the role that gene phylogenies have come to play in the issues. In addition, there is the full suite of characters that distinguish eukaryotes from prokaryotes to consider (meiosis, mitosis, cell cycle, membrane traffic, endoplasmic reticulum (ER), Golgi, flagella and all the other eukaryote-specific attributes, including a full-blown cytoskeleton—not just a spattering of prokaryotic homologues for cytoskeletal proteins).

To get a fuller picture of eukaryote origins, we have to incorporate lateral gene transfer (LGT) among prokaryotes, endosymbiosis and gene transfer from organelles to the nucleus into the picture. That is not as simple as it might seem, because it has become apparent that individual genes have individual and differing histories. Thus, in order to get the big picture, we would have to integrate all individual gene trees into one summary diagram in such a way as to take the evolutionary affinities of the plastid (a cyanobacterium), the mitochondrion (a proteobacterium) and the host (an archaeon) into account. Nobody has done that yet, although there are some attempts in that direction. In 2015, our typical picture of eukaryotic origins entails either a phylogenetic tree based on one gene or, more commonly now, a concatenated analysis of a small sample of genes (say 30 or so from each genome), which generates a tree, the hope being that the tree so obtained will be representative for the genome as a whole and thus will have some predictive character for what we might observe in phylogenies beyond the 30 or so genes used to make the tree. The 30 or so genes commonly used for such concatenated phylogenies are mostly ribosomal proteins or other proteins involved in information processing, genes that Jim Lake called informational genes in 1998.

But because of the role of endosymbiosis in eukaryote cell evolution, eukaryotes tend to have two evolutionarily distinct sets of ribosomes (archaeal ribosomes in the cytosol and bacterial ribosomes in the mitochondrion), or sometimes three (an additional bacterial set in the plastid) and in rare cases four sets of active ribosomes (yet one more set in algae that possess nucleomorphs). The ‘core set of genes’ approach, in all of its manifestations so far, only queried cytosolic ribosomes for eukaryotes, and thus only looked at the archaeal component of eukaryotic cell history. Some of us have been worried that by looking only at genes that reflect the archaeal component of eukaryotic cells we might be missing a lot, because it was apparent early on that many genes in eukaryotes do not stem from archaea, but from bacteria instead and, most reasonably under endosymbiotic theory, from organelles.

William F. Martin: Endosymbiotic theories for eukaryote origin 2015 Sep 26

Mitochondrial Genetic Code Differences

1. The mitochondrial genetic code differs from the universal genetic code in several ways, but these differences are not uniform across all organisms. Key differences include:

  - In vertebrate mitochondria:
    - AUA codes for methionine instead of isoleucine
    - UGA codes for tryptophan instead of being a stop codon
    - AGA and AGG are stop codons instead of coding for arginine

  - In invertebrate mitochondria:
    - The differences vary, but often include AUA coding for methionine and UGA for tryptophan

These differences are not present in all mitochondria. Plant and fungal mitochondria, for example, often use a genetic code more similar to the universal code. The genetic code of the supposed host cell that received the proto-mitochondrion would likely have used the universal genetic code, as do most modern eukaryotic nuclear genomes.

Hypotheses and Explanations

1. Gradual Evolution: Some scientists propose that the mitochondrial genetic code evolved gradually after the endosymbiotic event. This could have occurred through a process of genetic drift in the small mitochondrial genome, followed by adaptation.

I'll explain the differences in genetic codes and expand on the related hypotheses:

Mitochondrial Genetic Code Differences:

1. The mitochondrial genetic code differs from the universal genetic code in several ways, but these differences are not uniform across all organisms. Key differences include:

   - In vertebrate mitochondria:
     - AUA codes for methionine instead of isoleucine
     - UGA codes for tryptophan instead of being a stop codon
     - AGA and AGG are stop codons instead of coding for arginine

   - In invertebrate mitochondria:
     - The differences vary, but often include AUA coding for methionine and UGA for tryptophan

2. These differences are not present in all mitochondria. Plant and fungal mitochondria, for example, often use a genetic code more similar to the universal code.

3. The genetic code of the supposed host cell that received the proto-mitochondrion would likely have used the universal genetic code, as do most modern eukaryotic nuclear genomes.

Hypotheses and Explanations:

Gradual Evolution 
Some scientists propose that the mitochondrial genetic code evolved gradually after the endosymbiotic event. This could have occurred through a process of genetic drift in the small mitochondrial genome, followed by adaptation.

Codon Reassignment: The "codon capture" hypothesis suggests that certain codons became rare or absent in the mitochondrial genome due to mutational pressures, allowing them to be reassigned to new amino acids or functions without disrupting existing proteins.
Selective Pressure: The unique environment inside mitochondria may have created selective pressures favoring a modified genetic code. For example, the high oxidative stress in mitochondria might have favored certain amino acid substitutions.
Import Mechanisms: The development of protein import mechanisms from the cytoplasm to mitochondria may have allowed some mitochondrial genes to be transferred to the nucleus, reducing constraints on the mitochondrial genetic code.
Compensatory Evolution: As changes in the genetic code occurred, compensatory mutations in tRNAs, release factors, and other components of the translation machinery may have co-evolved to maintain functionality.
Multiple Origins: Some researchers suggest that mitochondria may have multiple origins, which could explain the diversity of mitochondrial genetic codes across different taxonomic groups.
Reversion Prevention: Once established, the modified genetic code might prevent the reversion of mitochondrial genes to the nucleus, as they would be mistranslated.

These hypotheses are still subjects of ongoing research and debate in the scientific community. The evolution of the mitochondrial genetic code remains a complex issue, intertwined with questions about the origin of eukaryotes and the mechanisms of genome evolution.

Codon Reassignment
 The "codon capture" hypothesis suggests that certain codons became rare or absent in the mitochondrial genome due to mutational pressures, allowing them to be reassigned to new amino acids or functions without disrupting existing proteins.

Selective Pressure 
The unique environment inside mitochondria may have created selective pressures favoring a modified genetic code. For example, the high oxidative stress in mitochondria might have favored certain amino acid substitutions.

Import Mechanisms 
The development of protein import mechanisms from the cytoplasm to mitochondria may have allowed some mitochondrial genes to be transferred to the nucleus, reducing constraints on the mitochondrial genetic code.

Compensatory Evolution 
As changes in the genetic code occurred, compensatory mutations in tRNAs, release factors, and other components of the translation machinery may have co-evolved to maintain functionality.

Multiple Origins 
Some researchers suggest that mitochondria may have multiple origins, which could explain the diversity of mitochondrial genetic codes across different taxonomic groups.

Reversion Prevention 
Once established, the modified genetic code might prevent the reversion of mitochondrial genes to the nucleus, as they would be mistranslated.

These hypotheses are still subjects of ongoing research and debate in the scientific community. The evolution of the mitochondrial genetic code remains a complex issue, intertwined with questions about the origin of eukaryotes and the mechanisms of genome evolution.

The Genetic Code Conundrum: Challenges to the Endosymbiotic Theory of Mitochondrial Origin

The endosymbiotic theory, which proposes that mitochondria originated from free-living bacteria engulfed by early eukaryotic cells, has become a cornerstone of our understanding of cellular evolution. However, this theory faces a significant challenge when confronted with the reality of differing genetic codes between the host cell and the mitochondria. The universal genetic code, once thought to be invariant across all life forms, shows variations in mitochondrial genomes across different species. This discrepancy raises questions about the mechanisms and plausibility of such a fundamental change occurring during the transition from a free-living organism to a cellular organelle. The process of altering a genetic code is not trivial; it involves restructuring the very language of life at a molecular level. This transition becomes even more perplexing when considered within the framework of the endosymbiotic theory, as it requires an explanation not just of code evolution, but of how this evolution occurred in tandem with the complex process of endosymbiosis. The challenges inherent in this transition are numerous and multifaceted, touching upon issues of evolutionary probability, functional maintenance, and cellular compatibility. Transitioning from a universal genetic code to a different mitochondrial code in the context of the endosymbiotic theory presents several significant challenges:

1. Rarity of code changes: Alterations to the genetic code are extremely rare events in evolutionary history, making such a transition highly improbable.
2. Deleterious mutations: Changing a genetic code would likely involve numerous deleterious mutations before a new functional code is established, potentially compromising the organism's viability.
3. Codon disappearance: Some models of code evolution require the complete disappearance of certain codons, which is highly unlikely in a functional genome.
4. Optimization of the canonical code: The standard genetic code is well-optimized for most organisms. Deviating from this optimized code would likely lead to reduced fitness in most cases.
5. Complexity of the transition: Changing the genetic code involves altering the entire translation machinery of the cell, including tRNAs, release factors, and potentially ribosomes. This is a complex and coordinated process.
6. Maintaining functionality: During the transition, the endosymbiont/proto-mitochondrion would need to maintain its essential functions while fundamentally altering its protein synthesis mechanism.
7. Compatibility issues: The endosymbiont/proto-mitochondrion would need to maintain compatibility with the host cell's systems during and after the transition, as it's dependent on nuclear-encoded proteins.
8. Evolutionary pressure: There would need to be a significant selective advantage to changing the code to overcome the inherent risks and costs of such a transition.
9. Coordinated changes: Multiple components of the translation system would need to change in a coordinated manner to maintain functionality throughout the transition.
10. Time frame: The transition would need to occur within a timeframe that allows for the endosymbiont to become fully integrated as a mitochondrion, while also allowing for such a fundamental change to take place.

Even with a reduced genome, these challenges would still apply to mitochondria. The complexity of changing a genetic code, combined with the need to maintain functionality and compatibility with the host cell, makes this transition a significant hurdle for the endosymbiotic theory to explain. While smaller genomes might theoretically make code evolution easier, the fundamental problems of altering such a crucial cellular system remain formidable.

Several different proposals, while attempting to address the challenges of genetic code changes in mitochondria, all have significant shortcomings:

1. Codon Reassignment: Shortfall: This hypothesis requires the complete disappearance of a codon, which is highly improbable in a functional genome. Even if a codon becomes rare, it's unlikely to vanish entirely without causing issues in existing proteins.
2. Selective Pressure: Shortfall: While selective pressures might favor certain amino acid substitutions, changing the genetic code itself is a drastic solution. It's unclear why adapting protein sequences wouldn't be sufficient, rather than altering the fundamental translation system.
3. Import Mechanisms: Shortfall: While this explains how some genes could be transferred to the nucleus, it doesn't address why the genetic code itself needed to change. Moreover, it doesn't explain how the transition period was managed when genes were split between two different genetic codes.
4. Compensatory Evolution: Shortfall: This assumes that the system could tolerate significant disruption while waiting for compensatory mutations. The likelihood of multiple, coordinated changes occurring simultaneously to maintain functionality is extremely low.
5. Multiple Origins: Shortfall: This hypothesis doesn't solve the fundamental problem; it merely suggests that the improbable event of code change happened multiple times, which is even less likely than a single occurrence.
6. Reversion Prevention: Shortfall: This explains why the changed code might persist, but not how it arose in the first place. It also doesn't address how the initial transition occurred without causing massive disruption to cellular function.

Overall, these proposals face several common issues:

1. Transition Period: None adequately explain how the organism survived the transition period when the code was changing, which would cause widespread mistranslation and protein malfunction.
2. Coordination Problem: Changing the genetic code requires simultaneous alterations in multiple cellular components (tRNAs, release factors, etc.). The proposals don't satisfactorily explain how this coordination occurred.
3. Fitness Valley: Most of these changes would need to pass through a stage of reduced fitness before reaching a new, functional state. It's unclear how natural selection would favor this process.
4. Complexity vs. Simplicity: In many cases, simpler solutions (like changing protein sequences or gene regulation) would seem more probable than altering the fundamental genetic code.
5. Universality: These proposals struggle to explain why, if code changes are advantageous or easily achieved, we don't see more variation in genetic codes across all forms of life.
6. Reverse Engineering: Many of these proposals seem to reverse-engineer explanations from the observed end state, rather than providing a plausible evolutionary pathway.

While these hypotheses offer interesting perspectives, they all fall short of providing a fully satisfying explanation for the complex transition from a universal genetic code to variant mitochondrial codes within the framework of the endosymbiotic theory. The challenges involved in such a fundamental change remain significant hurdles for these explanatory models.

References: 

1. Lucas, S., Solowiej-Wedderburn, J., Bonforti, A., Libby, E. (2024). Modeling endosymbioses: Insights and hypotheses from theoretical approaches. PLOS Biology, 22(1), e3002583. Link. (This paper reviews theoretical approaches to modeling endosymbioses, providing insights into the evolution of these intimate partnerships.)

2. Siozios, S., Nadal-Jimenez, P., Azagi, T., Sprong, H., Frost, C.L., Parratt, S.R., ... Darby, A.C. (2023). Genome dynamics across the evolutionary transition to endosymbiosis. bioRxiv. Link. (This preprint examines genome dynamics during the transition to endosymbiosis, offering insights into the evolutionary processes involved.)

3. von der Dunk, S.H.A., Hogeweg, P., Snel, B. (2022). Obligate Endosymbiosis Explains Genome Expansion During Eukaryogenesis. bioRxiv. Link. (This preprint proposes that obligate endosymbiosis can explain genome expansion during the evolution of eukaryotes.)

4. Shimpi, G.G., Bentlage, B. (2022). Ancient endosymbiont‐mediated transmission of a selfish gene provides a model for overcoming barriers to gene transfer into animal mitochondrial genomes. BioEssays, 44(11), 2200190. Link. (This paper presents a model for gene transfer into animal mitochondrial genomes based on ancient endosymbiont-mediated transmission.)

5. Libby, E., Kempes, C.P., Okie, J.G. (2022). Metabolic compatibility and the rarity of prokaryote endosymbioses. Proceedings of the National Academy of Sciences, 119(41), e2206527120. Link. (This study explores the rarity of prokaryote endosymbioses, focusing on metabolic compatibility as a key factor.)

The origin of eukaryotic cells through endosymbiosis is an area of study in evolutionary biology. Several proposals have been made to explain the origin of eukaryotes through endosymbiotic events. Here's a list of the major endosymbiotic proposals:

1. Mitochondrial Endosymbiosis:
- This is the most widely accepted endosymbiotic event.
- Proposes that mitochondria originated from an alpha-proteobacterium engulfed by a host cell.
- Proposed by Lynn Margulis in the 1960s, building on earlier ideas.

2. Chloroplast Endosymbiosis:
- Explains the origin of chloroplasts in plant and algal cells.
- Suggests that chloroplasts originated from a cyanobacterium engulfed by a eukaryotic cell that already had mitochondria.

3. Secondary and Tertiary Endosymbiosis:
- Explains the complex plastids found in some algal groups.
- Involves the engulfment of a eukaryotic alga by another eukaryote.

4. Hydrogen Hypothesis:
- Proposed by William Martin and Miklós Müller.
- Suggests that the host was a hydrogen-dependent archaeon that engulfed a hydrogen-producing alpha-proteobacterium.

5. Syntrophy Hypothesis:
- A variation of the hydrogen hypothesis.
- Proposes a symbiotic relationship between a methanogenic archaeon and a bacterial partner.

6. Phagotrophy Hypothesis:
- Suggests that the ability to perform phagocytosis was a prerequisite for the engulfment of the mitochondrial ancestor.

7. Archaeal Host Hypothesis:
- Proposes that the host cell was an archaeon, specifically from the Asgard superphylum.

8. Viral Eukaryogenesis Hypothesis:
- Proposed by Philip Bell.
- Suggests that the nucleus evolved from a complex DNA virus.

9. Serial Endosymbiosis Theory (SET):
- Developed by Lynn Margulis.
- Proposes that eukaryotic organelles evolved through a series of endosymbiotic events.

10. Endosymbiotic Theory of Peroxisome Origin:
- Suggests that peroxisomes originated from an endosymbiotic bacterium.
- This idea is now largely discounted in favor of peroxisomes evolving from the endomembrane system.

11. Endosymbiotic Theory of Nucleus Origin:
- Proposes that the nucleus originated from an endosymbiotic bacterium.
- Less widely accepted than mitochondrial or chloroplast endosymbiosis.

12. Chronocyte Hypothesis:
- Suggests that the last common ancestor of eukaryotes had a symbiotic algal partner that was subsequently lost in many lineages.

These proposals are not mutually exclusive, and the current understanding of eukaryotic origins often involves elements from multiple hypotheses. The field continues to evolve as new genomic and cellular evidence comes to light.