Intestinal parasites don’t normally make it into science’s hall of fame. But Giardia, notorious for causing stomach cramps and diarrhoea, is fast proving the exception. This single-celled organism has always defied the norms of conventional classification. Now biologists think they know why. Giardia, they say, could be a ‘missing link’ in one of the biggest upheavals in the history of early life – the evolution of complex cells with nuclei. Without Giardia, or cells like it, plants, animals and fungi might never have evolved.
This is just one of many findings that are beginning to revitalise research into ‘early evolution’ after decades of neglect. Armed with DNA techniques, biologists are seeking to turn what was once a fringe pursuit into something far more concrete. It is early days, of course: most textbook charts of the history of evolution still show the first two billion years of life on Earth as a featureless expanse of white. Science has yet to tell us much about the first bacteria-like cells on Earth and how they evolved into cells which store their DNA in nuclei, the kind from which plants and animals are made.
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There are good reasons for this hole in the data – not least of which is the fact that if you go back more than two billion years, the conventional fossil record begins to peter out. What is changing, however, is that biologists no longer see this as a reason for shrugging their shoulders in despair. Increasingly, they are turning to an alternative source of clues about early evolution: the kind of history that is ‘written’ in the genes and cellular structures of organisms like Giardia.
But how far can you get with this approach? Judging from biologists’ efforts to ‘debrief’ Giardia, and recent research into a class of heat-loving bacteria known as ‘eocytes’, broad insights are certainly possible. Until recently, for example, some theories held that cells with nuclei (eukaryotes) first evolved from cells without nuclei (prokaryotes) some two billion years ago. Now, the gene sequences of Giardia suggest otherwise. Single-celled eukaryotes may have swum in primordial seas over three billion years ago, more than two billion years before the first multicellular organisms evolved.
As for eocytes: there is growing evidence that they may have a starring role in two evolutionary stories. First, the signs are that life could well have begun in the pressure-cooker conditions that eocytes enjoy. And looking at a later stage of evolution – the emergence of eukaryotes – some researchers believe eocytes may be the eukaryotes’ closest bacterial relatives.
The flood of new data on early evolution springs largely from genetic and molecular techniques. Yet it is still true to say that the estimates for dating the evolution of prokaryotes and eukaryotes comes from fossil studies. One major finding, made in Michigan in 1992, includes the oldest known eukaryotes, fossilised algal cells discovered in rocks dated at 2.1 billion years. Since algae are near the top of the eukaryotic tree of life, all the lower branches of the tree, including Giardia, must have emerged before then.
Life within rocks
The oldest known fossils? These are in Australian rocks that formed about 3.5 billion years ago, trapping what seem to be wide variety of bacteria in the process. At that stage, evidently, prokaryotes had already become quite diverse. Yet no such diversity is apparent in the oldest known rocks, dated at 3.8 billion years and found in Greenland. These harbour only chemical signs that suggest the evolution of life was under way but give no clues about the kinds of organisms that existed. By extracting and analysing organic polymers from these Greenland rocks, chemists detected an unusually high ratio of carbon-12 to carbon-13. An important find, because biochemical reactions, of the kind carried out by enzymes, show a preference for carbon-12.
Fascinating though such observations are, they provide only the crudest sketch of early evolution. If (as the evidence suggests) life arose as long as 3.8 billion years ago, just a few hundred million years after the Earth’s surface cooled sufficiently for oceans to form, how did it do so? If the rocks are telling truth and the first prokaryotes had evolved by 3.5 billion years ago, what were these cells like? And if organisms like Giardia did indeed evolve from prokaryotes between 2 and 3 billion years ago, can we say how?
Enter DNA and cell biology. Drawing on DNA sequences of genes, biologists are busy piecing together the most detailed evolutionary trees ever envisioned. All the major taxonomic groups of living things are under the microscope. And as the relationships between these groups become clearer, researchers hope to be able to do more than just speculate about the life forms that inhabited the early Earth.
The principle behind the approach has been known for years. Genes from closely related species tend, on the whole, to have similar DNA sequences. By comparing genes from different species, researchers can build a family tree showing the likely relationships within groups. Of course, if you want to compare species that diverged early in evolution, you have to pick genes that date that far back. It is unlikely, for example, that cells living three billion years ago, before life was dominated by oxygen, would have had genes for oxygen-carrying proteins such as haemoglobin. But if the cells bore any resemblance to modern organisms, they must have contained at least some proteins.
That is why research into early evolution relies heavily on genes encoding molecules known as ribosomal RNAs. Cells produce proteins on particles called ribosomes and these are themselves built from a mixture of proteins and RNA molecules. Since all known cells – prokaryotic or eukaryotic – build proteins on ribosomes, then ribosomal RNA molecules must have been around at the beginning of life. Or so the argument goes.
Giardia has ribosomes. It also has genes encoding ribosomal RNA molecules. By analysing these genes in 1989, Mitchell Sogin, a molecular biologist then at the National Jewish Center for Immunology and Respiratory Medicine in Denver, Colorado, and his colleagues could begin to show where Giardia lies on the evolutionary tree of eukaryotes.
Why did they bother? Giardia and its relatives, known collectively as ‘diplomonads’, had long puzzled biologists. Unlike other eukaryotes, diplomonads have two nuclei rather than one and they are devoid of oxygen burning organ-elles, or mitochondria, usually found in eukary-otic cells. To produce energy in the form of ATP, diplomonads rely instead on anaerobic metabolism such as glycolosis. Another hallmark of diplomonads is they all have unusually simple skeletons (protein scaffolds which prevent eukaryotic cells from collapsing or bursting).
There seemed to be two possible explanations for these peculiarities. One was that diplomonads are relics from an earlier stage of evolution featuring cells which had yet to evolve mitochondria. In the 1960s, American biologist Lynn Margulis, now at the University of Massachusetts, Amherst, proposed that cells acquired organelles such as mitochondria by the process of symbiosis. Organelles, she argued, were once free-living organisms that somehow forged a mutal dependence with their future hosts and then, over time, became absorbed within their hosts’ cytoplasm. Cell nuclei may have evolved in a broadly similar way. If so, it is conceivable that the earliest eukaryotic cells had only ‘partially evolved’ cell nuclei and no mitochondria – like Giardia.
An alternative explanation is that the diplomonads are descended from highly conventional (and evolved) eukaryotic cells which proceeded to lose features in the course of adapting to new environments. An analogy could be made with blind cave-dwelling fish that we now know are descended from conventionally sighted ancestors.
Blind fish
To distinguish between these two alternatives, Sogin’s team needed an independent method of estimating when the diplomonads first branched off from the main evolutionary line of the eukaryotes. If these organisms did so very early on, it would – by Occam’s razor – lend weight to the idea that they are evolutionary relics rather than the cellular equivalents of cave-dwelling fish.
So the researchers studied a gene for a ribosomal RNA molecule in Giardia and its relatives and compared it with similar genes from species as diverse as humans, maize and the gut-dwelling bacterium Escherichia coli. The DNA analysis puts Giardia on the lowest branch of the eukaryotic family tree, and two of its relatives on the next two branches up. In other words, the diplomonads are out on a biological limb. They have the hallmarks of an evolutionary relic.
But that is not to say that the diplomonads stopped evolving when their lines branched off. Indeed Sogin, who is now at the Marine Biological Laboratory in Woods Hole, Massachusetts, argues against viewing Giardia as a living fossil. ‘To think that an organism which exists today is primitive is a misconception,’ he says. ‘Giardia is as evolved as you or I’ But equally, the organism’s unique position on the evolutionary tree of eukaryotes makes it a treasure trove of clues about early evolution.
Take the organism’s curious ‘twin’ nuclei. According to biologists Karen Kabnick and Debra Peattie at the Harvard School of Public Health in Massachusetts, these could be a sign that Giardia is a kind of halfway-house on the road between bacteria-like cells and conventional eukaryotes.
The argument goes like this. At some stage in their life cycle, conventional eukaryotes have a single nucleus containing two complete sets of genes. They are called ‘diploid’. In contrast, a pro-karyotic cell usually has only a single set of genes. It is ‘haploid’. Giardia seems to be midway between the two. Kabnick and Peattie have shown that each of Giardia’s two nuclei probably contains just one set of genes. Based on this, the researchers suggest that the first eukaryotes on Earth each had a single haploid nucleus. Then some kind of doubling-up ‘error’ occurred during cell division, resulting in the evolution of cells with two haploid nuclei, like Giardia. Conventional eukaryotes may have arisen later following a further upset in which the two haploid nuclei joined to produce a single diploid nucleus.
Research into Giardia’s evolutionary heritage has also shaken established beliefs about when eukaryotes evolved. This is because genes, or more specifically their relative variabilities, contain information about time as well as genetic distance. This so-called ‘molecular clock’ approach is not going to win any awards for accuracy, as genes in different species can mutate at different rates. But the approach does allow researchers to write rough dates next to the branches of evolutionary trees. ‘There is no reason why Giardia could not have diverged three billion years ago,’ says Sogin commenting on mutations in ribosomal RNA sequences. ‘I think that eukaryotes are older than people think.’
This could explain why Giardia and its relatives lack mitochondria. Mitochondria evolved from bacteria living as symbionts within primitive eukaryotic cells and they generate energy by burning up oxygen. Three billion years ago, around the time Giardia may have diverged from other eukaryotes, there were only traces of oxygen in both the atmosphere and the oceans. Mitochondria would have no place in such an environment.
Indeed, the research by Sogin’s group confirms the belief that changes in the amount of oxygen in the atmosphere have shaped the overall structure of the euka-ryotic evolutionary tree. The chemical composition of ancient rocks suggests that atmospheric oxygen increased in relatively distinct steps separated by very long periods of stability. This pattern is seen in the branches of the eukaryotic tree: higher branches lead to organisms that need more and more oxygen.
On branches in the middle of the tree lie single-celled organisms with mitochondria that require little oxygen. These groups may have emerged when atmospheric oxygen reached about 1 per cent of its present level. At the top of the tree, several branches appear almost simultaneously and include familiar groups such as plants, animals and fungi. This sudden burst of diversity could have been triggered when oxygen reached about 10 per cent of its current level, say some biologists.
The eukaryotic tree is far from complete. As researchers analyse genes from more and more species they will inevitably add more branches. Will they also succeed in tracing the evolutionary roots of eukary-otes all the way back to proka-ryotes? Everyone assumes that eukaryotes, from Giardia to humans, evolved from prokaryotes. Yet the debate still rages as to where to find our closest relatives in the world of bacteria.
The conventional view comes from the work of Carl Woese at the University of Illinois, Urbana, and his colleagues. By analysing genes of ribosomal RNA molecules, Woese’s group produced a universal tree of life with three main branches (see ‘Kingdoms in turmoil’, ¿ìè¶ÌÊÓƵ, 23 March 1991). One branch leads to the eukaryotes and the other two branches to groups of prokaryotes. Woese’s tree splits the prokaryotes into ‘eubacteria’: organisms like E. coli, most of which live under relatively normal conditions; and ‘archaebacteria’, which often have more extreme haunts such as places saturated with salt or that are extremely hot. A key distinction between the two groups is that all archaebacteria have certain fat molecules in their cell membranes which neither eukaryotes nor eubacteria can produce.
This pattern implies that the archaebacteria are our closest relatives. But a rival theory goes further. In the late 1980s, James Lake of the University of California at Los Angeles suggested splitting off one group of archaebacteria, the eocytes, and placing them closer to the eukaryotes. Eocytes have a distinctly alien feel. They live above volcanic springs and at the openings of volcanic vents in the mid-ocean. They can thrive at temperatures of up to 110 °C. Yet Lake’s tree would make them our closest relatives in the world of bacteria.
Then there were five
Who is right? This is contentious ground. Lake claims that the archaebacterial tree is a mirage produced by the way Woese and his colleagues analyse gene sequences. Woese says the same about Lake’s evolutionary tree. The arguments on both sides are esoteric and mathematical, but so far most molecular biologists have remained in Woese’s camp. Others have no truck with either camp. Margulis, for example, favours a classification system based on five kingdoms – plants, animals, fungi, prokaryotes and protoctists – and is deeply sceptical about what gene sequences can reveal about evolutionary history.
One criticism is that genes may mutate at irregular rates or may even ‘jump’ from one organism to another, giving a false reading of the organism’s genetic relatedness to other organisms. Haemoglobin-like genes, for example, have turned up in plants. Advocates of the molecular genetic approach say these problems pale to insignificance if you look at enough genes in an organism. Arguments based on individual genes, however, remain open to attack.
For example, Lake has recently found what he believes to be a trump card. With Maria Rivera, he compared the sequences of amino acids in different forms of a naturally occurring molecule called EF-tu, which cells use to synthesise proteins. In eukaryotes and eocytes, the researchers found a distinctive segment containing 11 amino acids in EF-tu’s structure. In eubacteria and other types of archaebacteria, this segment is replaced by a group of four amino acids. This certainly fits Lake’s theory that eukaryotes and eocytes share a special relationship. But Woese is more than a little sceptical. ‘Anecdotal’ is how he describes the EF-tu evidence: ‘You can always find one or two pieces to support any theory you want.’
Whatever happens in this debate, eocytes are sure to continue to attract attention. For many biologists (Lake and Woese included) believe they resemble the very first living cells. Lake himself coined the name ‘eocyte’ which means ‘dawn cell’. At the core of this belief lies a simple fact. The genes for ribosomal RNA in eocytes appear to have accumulated far fewer mutations than the equivalent genes in any other group of organisms. Indeed, these genes may have changed little from those in the universal ancestor at the ‘root’ of the tree of life. If the same is true for other genes in the eocytes, then the entire physiology of this universal ancestor may have been similar to that of eocytes living in volcanic vents today.
This would imply that life evolved at very high temperatures. Which is entirely possible. If time travel is ever invented, the early Earth will only attract the very hardiest tourists. Four billion years ago, give or take, an atmosphere virtually devoid of oxygen would have provided no ozone layer to filter out UV-radiation. There would have been periods of massive volcanic activity. And the oceans could have been near to boiling point (although some researchers have suggested a more swimming pool-like 30 °C). In short, it would have been heaven for eocytes, which gain their energy not from oxygen but by converting sulphur to hydrogen sulphide. Eocytes are not the only bacteria that like it hot. Other heat-lovers are to be found among the archaebacteria and eubacteria. Furthermore, the bacteria that enjoy the highest temperatures are mostly found in the ‘lower’ branches of their respective groups, strengthening the belief that life began in the heat. The search is now on for heat-loving eukaryotes. For the moment eukaryotes living at temperatures above 55 °C appear to be rare. Maybe Giardia-like cells did not appear until life had spread to cooler places.
But even if DNA techniques succeed in tracing the evolutionary path between early bacteria and modern eukaryotes, one period of evolution remains tantalisingly out of reach. By studying genes biologists can, with some provisos, try to trace evolution back to the last common ancestor of all living things. That ancestor was probably an eocyte-like cell that lived between 3.5 billion and 4.0 billion years ago. Yet the last common ancestor was not the first living thing on Earth: it was simply the only organism alive at its time whose descendants still survive. Genes can say nothing about the trillions of other cells with which the ancestor must have shared the planet. The variety of ancient genes passed to us by the last common ancestor shows that it was far from primitive. ‘(That) ancestor was already a very complex, modern sort of organism,’ says Lakes. ‘It was not something that was just barely surviving.’
Stephen Day is a freelance science writer.
