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  The archezoa—eukaryotes without mitochondria

  According to the theory put forward by Cavalier-Smith as long ago as 1983, some of the simple single-celled eukaryotes living today do still resemble the earliest eukaryotes. More than a thousand species of primitive eukaryotes do not possess mitochondria. While many of these probably lost their mitochondria later, simply because they didn’t need them (evolution is always quick to jettison unnecessary traits), Cavalier-Smith argued that at least a few of these species were probably ‘primitively amitochondriate’—in other words, they never did have any mitochondria, but were instead primitive relics of the age before the eukaryotic merger. To generate their energy, most of these cells depend on fermentations in the same way as yeast. While a few of them tolerate the presence of oxygen, most grow best at very low levels or even in the complete absence of the gas, and thrive today in low-oxygen environments. Cavalier-Smith named this hypothetical group the ‘archezoa’ in deference to their ancient roots and their animal-like, scavenging mode of living, as well as their similarities to the archaea. The name ‘archezoa’ is unfortunate, in that it is confusingly similar to ‘archaea’. I can only apologize for this confusion. The archaea are prokaryotes (without a nucleus), one of the three domains of life, while the archezoa are eukaryotes (with a nucleus) that never had any mitochondria.

  Like any good hypothesis, Cavalier-Smith’s was eminently testable by the genetic sequencing technologies then reaching fruition—the capacity to work out the precise sequence of letters in the code of genes. By comparing the gene sequences of different eukaryotes, it is possible to determine how closely related different species are to each other—or conversely, how remote the archezoa are from more ‘modern’ eukaryotes. The reasoning is simple. Gene sequences consist of thousands of ‘letters’. For any gene, the sequence of these letters drifts slowly over time as a result of mutations, in which particular letters are lost or gained, or substituted one for another. Thus, if two different species have copies of the same gene, then the exact sequence of letters is likely to be slightly different in the two different species. These changes accumulate very slowly over millions of years. Other factors need to be considered, but to a point the number of changes in the sequence of letters gives an indication of the time elapsed since the two versions diverged from a common ancestor. These data can be used to build a branching tree of evolutionary relationships—the universal tree of life.

  If the archezoa really could be shown to be among the oldest of eukaryotes, then Cavalier-Smith would have found his missing link—a primitive eukaryotic cell, that had never possessed any mitochondria, but which did have a nucleus and a dynamic cytoskeleton, enabling it to change shape and feed by phagocytosis. The first answers became available within a few years of Cavalier-Smith’s hypothesis, and apparently satisfied his predictions in full. Four groups of primitive-looking eukaryotes, which not only lacked mitochondria but also most other organelles, were confirmed by genetic analysis to be amongst the oldest of the eukaryotes.

  The first genes to be sequenced, by Woese’s group in 1987, belonged to a tiny parasite, no larger than a bacterium, which lives inside other cells—indeed, can only live inside other cells. This was the microsporidium V. necatrix. As a group, the microsporidia are named after their infective spores, all of which come replete with a projecting coiled tube, through which spores extrude their contents into a host cell, then multiply to begin their life cycle afresh, ultimately producing more infective spores. Perhaps the best-known representative of the microsporidia is Nosema, which is notorious for causing epidemics in honeybees and silkworms. When feeding inside the host cell, Nosema behaves like a minute amoeba, moving around and engulfing food by phagocytosis. It has a nucleus, a cytoskeleton and small bacterial-style ribosomes, but has no mitochondria or any other organelles. As a group, the microsporidia infect a wide variety of cells from many branches of the eukaryote tree-of-life, including vertebrates, insects, worms, and even single-celled ciliates (cells named after their tiny hair-like ‘cilia’, used for feeding and locomotion). As all microsporidia are parasites that can survive only inside other eukaryotic cells, they can’t truly represent the first eukaryotes (because they would have had nothing to infect) but the diverse range of organisms that they do infect suggests that they have ancient origins, going back to the roots of the eukaryotic tree. This assumption seemed to be confirmed by genetic analysis, but there was a catch, as we shall see in a moment.

  Over the next few years, the ancient status of the three other groups of primitive eukaryotes was confirmed by genetic analyses—the archamoebae, the metamonads, and the parabasalia. All three groups are best known as parasites, but free-living forms do also exist, perhaps fitting them better than the microsporidia as the earliest eukaryotes. As parasites, these three groups occasion much misery, illness, and death; how ironic that these repellent and life-threatening cells should be singled out as our own early ancestors. The archamoeba are best represented by Entamoeba histolytica, which causes amoebic dysentery, with symptoms ranging from diarrhoea to intestinal bleeding and peritonitis. The parasites burrow through the wall of the intestine to gain access to the bloodstream, from where they infect other organs, including the liver, lungs, and brain. In the long term, they may form enormous cysts on these organs, especially the liver, causing up to 100 000 deaths worldwide each year. The other two groups are less deadly but no less smelly. The best-known metamonad is Giardia lamblia, another intestinal parasite. Giardia does not invade the intestinal walls or enter the bloodstream, but the infection is still thoroughly unpleasant, as any travellers who have incautiously drunk water from infected streams know to their cost. Watery diarrhoea and ‘eggy’ flatulence may persist for weeks or months. Turning to the third group, the parabasalia, the best known is Trichomonas vaginalis, which is among the most prevalent, albeit least menacing, of the microbes that cause sexually transmitted diseases (though the inflammation it produces may increase the risk of contracting other diseases such as AIDS). T. vaginalis is transmitted mainly by vaginal intercourse but can also infect the urethra in men. In women, it causes vaginal inflammation and the discharge of a malodorous yellowish-green fluid. All in all, this portfolio of foul ancestors just goes to prove that we can choose our friends but not our relatives.

  The eukaryote’s progress

  For all their unpleasantness, the archezoa nonetheless fitted the bill as primitive eukaryotes, survivors from the earliest days before the acquisition of mitochondria. Genetic analysis confirmed that they did branch away from more modern eukaryotes at an early stage of evolution, some two thousand million years ago, while their uncluttered morphology was compatible with a simple early lifestyle as scavengers that engulfed their food whole by phagocytosis. Presumably, one fine morning, two thousand million years ago, a cousin of these simple cells engulfed a bacterium, and for some reason failed to digest it. The bacterium lived on and divided inside the archezoon. Whatever the original benefit might have been to either party the intimate association was eventually so successful that the chimeric cell gave rise to all modern eukaryotes with mitochondria—all the familiar plants, animals, and fungi.

  According to this reconstruction, the original benefit of the merger was probably related to oxygen. Presumably it was not a coincidence that the merger took place at a time when oxygen levels were rising in the air and the oceans. A great surge in atmospheric oxygen levels certainly occurred around two billion years ago, probably in the wake of a global glaciation, or ‘snowball earth’. This timing corresponds closely to that of the eukaryotic merger. Modern mitochondria make use of oxygen to burn sugars and fats in cell respiration, so it is not surprising that mitochondria should have become established at a time when oxygen levels were rising. As a form of energy-generation, oxygen respiration is much more efficient than other forms of respiration, which generate energy in the absence of oxygen (anaerobic respiration). All that said, it is unlikely that superior energy generation could have b
een the original advantage. There is no reason why a bacterium living inside another cell should pass on its energy to the host. Modern bacteria keep all their energy for themselves, and the last thing they do is export it benevolently to their neighbouring cells. Thus while there is a clear advantage for the ancestors of the mitochondria, which had intimate access to any of the host’s nutrients, there is no apparent advantage to the host cell itself.

  Perhaps the initial relationship was actually parasitic—a possibility first suggested by Lynn Margulis. Important work from Siv Andersson’s laboratory at the University of Uppsala in Sweden, published in Nature in 1998, showed that the genes of the parasitic bacterium Rickettsia prowazekii, the cause of typhus, correspond closely with those of human mitochondria, raising the possibility that the original bacterium might have been a parasite not unlike Rickettsia. Even if the original invading bacterium was a parasite, the unbalanced ‘partnership’ may have survived, as long as its unwelcome guest did not fatally weaken the host cell. Many infections today become less virulent over time, as parasites also benefit from keeping their host alive—they do not have to search for a new home every time their host dies. Diseases like syphilis have become much less virulent over the centuries, and there are hints that a similar attenuation is already underway with AIDS. Interestingly, such attenuation over generations also takes place in amoebae such as proteus. In this case, the infecting bacteria initially often kill the host amoebae, but eventually become necessary for their survival. The nuclei of infected amoebae become incompatible with the original amoebae, and ultimately lethal to them, effectively forcing the origin of a new species.

  In the case of the eukaryotic cell, the host is good at ‘eating’ and through its predatory lifestyle provides its guest with a continuous supply of food. We are told that there is no such thing as a free lunch, but the parasite might simply burn up the metabolic waste-products of the host without weakening it much at all, which is not far short of a free lunch. Over time the host learned to tap into the energy-generating capacity of its guest, by inserting membrane channels, or ‘taps’. The relationship reversed. The guest had been the parasite of the host, but now it became the slave, its energy drained off to serve the host.

  This scenario is only one of several possibilities, and perhaps the timing holds the key. Even if energy was not the basis of the relationship, the rise in oxygen levels might still explain the initial benefits. Oxygen is toxic to anaerobic (oxygen-hating) organisms—it ‘corrodes’ unprotected cells in the same way that it rusts iron nails. If the guest was an aerobic bacterium, using oxygen to generate its energy, while the host was an anaerobic cell (generating energy by fermentation), then the aerobic bacterium may have protected its host against toxic oxygen—it could have worked as an internally fitted ‘catalytic converter’, guzzling up oxygen from the surroundings and converting it into harmless water. Siv Andersson calls this the ‘Ox-Tox’ hypothesis.

  Let’s recapitulate the argument. A bacterium loses its cell wall but survives because it has an internal cytoskeleton, which it had made use of before to keep in shape. It now resembles a modern archaeon. With a few modifications to its cytoskeleton, the wall-less archaeon learns to eat food by phagocytosis. As it grows larger it wraps its genes in a membrane and develops a nucleus. It has now turned into an archezoon, perhaps resembling cells like Giardia. One such hungry archezoon happens to engulf a smaller aerobic bacterium but fails to digest it, let’s say because the bacterium is a parasite like the modern Rickettsia, and has learned to evade the defences of its host. The two get along together in a benign parasitic relationship, but as atmospheric oxygen levels rise, the relationship begins to pay dividends to both the host and parasite: the parasite still gets its free lunch, but the host is now getting a better deal—it’s protected from toxic oxygen from within by its catalytic converter. Then, finally, in an act of breathtaking ingratitude, the host plugs a ‘tap’ into the membrane of its guest and drains off its energy. The modern eukaryotic cell is born, and never looks back.

  This long chain of reasoning is a good example of how science can piece together a plausible story and back it up with evidence at almost every point. To me there is a feeling of inevitability about the whole process: it could happen here and it could happen anywhere else in the universe—no single step is particularly improbable. There is simply a bottleneck, as postulated by Christian de Duve, in which the evolution of the eukaryotes is unlikely when there is not much oxygen around, but almost inevitable as soon as the oxygen levels rise. While everybody agrees this story is broadly speculative, it was widely believed to be plausible, and made use of most of the known facts. Nothing prepared the field for the reversal that was to follow in the late 1990s. As sometimes happens to the ‘good’ stories in science, virtually the entire edifice collapsed in the space of just five years. Nearly every point has now been contradicted. But perhaps the writing was on the wall. If the eukaryotes only evolved once, then a plausible story may be exactly the wrong kind of story.

  Reversal of a paradigm

  The first stone to crumble was the ‘primitively amitochondriate’ status of the archezoa. This term, if you recall, means that the archezoa never did have any mitochondria. But when more genes from different archezoa were sequenced, it began to look as if postulated progenitors of eukaryotic cells, such as Entamoeba histolytica (the cause of amoebic dysentery), were not the earliest representatives of their group after all. Other types of cell in the same group appeared to be even older—but did have mitochondria. Unfortunately, the genetic dating techniques were approximate and liable to error, and so the results were controversial. But if the estimated dates were correct, then the results could only mean that Entamoeba histolytica did have ancestors that had once possessed mitochondria, and so must have lost its own, rather than never having had any at all. If the archezoa are defined as a group of primitive eukaryotes that never had mitochondria, then E. histolytica could not be an archezoon.

  In 1995, Graham Clark at the National Institutes of Health in the United States, and Andrew Roger at Dalhousie University in Canada, went back to look more closely at E. histolytica to see if there were any traces that it had formerly possessed mitochondria. There were. Hidden away in the nuclear genome were two genes that, from their DNA sequences, almost certainly derived from the original mitochondrial merger. They were presumably transferred from the early mitochondria to the host cell nucleus, and the cell later lost all physical traces that it had ever had any mitochondria. We should note that the transfer of genes from the mitochondria to the host is quite normal, for reasons that we’ll consider in Part 3. Modern mitochondria have retained only a handful of genes, and the rest were either lost altogether or transferred across to the nucleus. The proteins encoded by these nuclear genes are often targeted back to the mitochondria. Interestingly E. histolytica does actually possess some oval organelles that might be the corrupt remains of mitochondria; they resemble mitochondria in their size and shape, and several of the proteins that have been isolated from them have also been found in the mitochondria in other organisms.

  Not surprisingly, the burning question transferred to the other supposedly primitively amitochondriate groups. Had they, too, once possessed mitochondria? Similar studies were carried out, and so far all the ‘archezoa’ that have been tested turn out to have once possessed mitochondria, and lost them later on. For example, not only did Giardia apparently once have mitochondria, but it, too, may still preserve relics, in the form of tiny organelles called mitosomes, which continue to carry out some of the functions of mitochondria (if not the best known, aerobic respiration). Perhaps the most surprising results concerned the microsporidia. This supposedly ancient group not only did possess mitochondria in the past, but now turns out not to be an ancient group at all—they are most closely related to the higher fungi, a relatively recent group of eukaryotes. The apparent antiquity of the microsporidia is merely an artefact of their parasitic lifestyle inside other cells. And t
he fact that they infect so many different groups is but a testament to their success.

  While it remains possible that the real archezoa are still out there, just waiting to be found, the consensus view today is that the entire group is a mirage—every single eukaryote that has ever been examined either has, or once had, mitochondria. If we believe the evidence, then there never were any primitive archezoa. And if this is true, then the mitochondrial merger took place at the very beginning of the eukaryotic line, and was perhaps inseparable from it: the merger was the unique event that gave rise to the eukaryotes.

  If the prototype eukaryote was not an archezoon—in other words, not a simple cell that made its living by engulfing its food by phagocytosis—then what did it look like? The answer might possibly lie in the detailed DNA sequences of eukaryotes living today. We have seen it is possible to identify ex-mitochondrial genes by comparing their gene sequences; perhaps we can do the same with those genes inherited from the original host. The idea is simple. Because we know that mitochondria are related to a particular group of bacteria, the α-proteobacteria, we can exclude any genes that seem to derive from this source, and look to see where the rest come from. Of the rest, we can assume that some are unique to eukaryotes—they evolved in the last two thousand million years since the merger—while some might have been transferred from elsewhere. Even so, at least a few ought to line up with the original host. These genes should have been inherited by all the descendents of the original merger, and gradually accumulated modifications ever since; but they should still bear some resemblance to the original host cell.