1 The Cell Theory

The ancient philosophers discussed many theories about the world, such as the existence of atoms, chaos and determinism, evolution and relativity, and yet none of them conceived the cell theory, the idea that all living creatures are made of cells. This great generalisation was made possible by the invention of the microscope, but did not appear suddenly. It was the result of a research that lasted more than 200 years.

The microscopists of the seventeenth century were the first men who saw an entirely new world of living creatures that are invisible to the naked eye, the so-called micro-organisms. They saw bacteria, protozoa, blood cells and thousands of other animalcula, and gradually realized that the visible creatures are actually a minority in nature.

Unfortunately, the microscopes of the seventeenth and eighteenth century had a structural defect. All lenses that are made of a single piece of glass cannot focus in one point all the light rays that cross them, and their images are inevitably affected by aberrations. Because of this, people could see only isolated cells, such as bacteria and protozoa, or plant cells, which are separated by thick cellulose walls, but could not see cells in animal tissues. The discovery that cells exist in all living creatures required a new type of microscope, and this came only in the nineteenth century, with the introduction of the achromatic lenses. These are made of two or more pieces arranged in such a way that the aberrations of one piece are compensated by those of the other. The first achromatic microscope was built by Giovanni Battista Amici in 1810 and with this new instrument came a systematic revision of all that the microscope had revealed in the previous centuries.

In 1831 Robert Brown discovered that plant cells contain a round refracting mass that he called nucleus, and inside the nucleus it was often possible to see an even more refracting structure that later on became known as nucleolus. In 1839 Matthias Schleiden and Theodor Schwann compared plant embryos with animal embryos, and discovered that their structures are strikingly alike. They are both made of cells, hence the conclusion that represents the first part of the cell theory: all living creatures are made of cells and of cell products.

This raised the problem of the mechanism that gives origin to cells, and here Schleiden and Schwann made a proposal that turned out to be completely wrong. They suggested that cells originate inside other cells with a mechanism which is similar to crystal growth, and which they called free formation.

The discovery of the true mechanism required many other years of research, and came primarily from embryology. In the earliest stages of development it is often possible to see all the cells of an embryo and realize that their sizes and shapes are practically constant. This means that cells are produced by a process that keeps their basic structure invariant, i.e., by a process of replication.

In 1852 Robert Remak explicitly rejected the free-formation idea and concluded that “cells always come from the division of other cells”. In 1855 Rudolf Virchow reached the same conclusion by studying a great number of normal and pathological adult tissues, and condensed it with the motto “omnis cellula e cellula”. The final version of the cell theory is therefore the combination of Schleiden and Schwann’s first theory with the conclusion of Remak and Virchow: “all living creatures are made of cells and of cell products, and cells are always generated by the division of other cells”.

At the same time, the microscope was making it clear that there are two very different types of cells in life, cells without a nucleus (prokaryotes) and cells with a nucleus (eukaryotes).

In 1866, Ernst Haeckel proposed a tree of life where the first creatures were prokaryotes (that he called monera) and where the eukaryotes (or protista) evolved at a later stage from prokaryotes and gave origin to all multicellular organisms (Haeckel 1866). The greatest divide of the living world, in other words is not between plants and animals, as it has been thought for centuries, but between prokaryotes and eukaryotes.

2 The Energy Revolution

In 1883, Andreas Schimper proposed that the chloroplasts of the plants had once been free-living bacteria that became incorporated, by a kind of internalization, or endosymbiosis, into plant cells (Schimper 1883), and later on this mechanism was also proposed for the origin of mitochondria by Mereschowsky (1910), by Portier (1918) and by Wallin (1927).

The endosymbiosis hypothesis was totally ignored at first but after a few decades it was forcefully re-proposed by Lynn Margulis (1970) and within a few years it received the support of an astonishing number of experimental data. It was found, for example, that mitochondria are still carrying fragments of their ancient circular bacterial DNA, and contain bacterial ribosomes whose molecular weight is about half that of eukaryotic ribosomes.

Today it is universally acknowledged that mitochondria and chloroplasts originated by endosymbiosis, and the analysis of their RNA sequences has shown that mitochondria derived from alfa-proteobacteria whereas chloroplasts are the modified descendants of cyanobacteria (Yang et al. 1985; Woese 1987).

The origin of mitochondria had a massive impact on evolution because it set in motion an energy revolution that profoundly changed the history of life (Lane and Martin 2010; Lane 2011).

The genome of a prokaryote gets all the energy it needs from a single bacterium, whereas the genome of a eukaryote gets its energy from thousands of symbiotic bacteria. This means that endosymbiosis allowed the eukaryotes to have much more energy at their disposal, and it has in fact been calculated that “… the average eukaryote has 1200 times as much energy per gene as the average prokaryote” (Lane 2015).

Cells spend as much as 80% of their total energy budget in protein synthesis, and the higher the number of genes the higher is the cost of protein synthesis. This is why “… there are about 13,000 ribosomes in an average bacterium such as E. coli, and at least 13 million ribosomes in a single eukaryotic cell” (Lane 2015).

The acquisition of mitochondria, in other words, allowed the eukaryotes to enormously increase their energy sources, but how did it take place? What were the cells that engulfed the ancestors of the mitochondria? This is a problem that can be solved only by reconstructing the history of the cells, or, more precisely, their phylogenetic trees.

3 The First Cells

The reconstruction of the phylogenetic trees has traditionally been obtained with the data of comparative anatomy and paleontology, but ever since the pioneering work of Zucherkandl and Pauling (1965) it has become clear that the sequences of genes and proteins provide an additional source of information.

The protein citochrome-c, for example, has sequences of amino acids that are different in many species and the differences are closely similar to the evolutionary distances that separate the species. Between man and monkeys, for example, the differences are small, and so are those between ducks and pigeons, but between man and ducks or between monkeys and pigeons they are much greater. The differences between sequences, in conclusion, can be used to build molecular trees and it turns out that these trees are remarkably similar to the trees obtained from comparative anatomy and from paleontology, thus confirming that molecular sequences contain phylogenetic information.

A major turning point in the reconstruction of the history of the cells came in 1977, when Carl Woese and George Fox obtained phylogenetic trees from the sequences of the ribosomal-RNAs and discovered that these trees divide all living creatures in three groups: two different types of prokaryotes that Woese and Fox (1977) called archaebacteria and eubacteria, and a third group containing the ancestors of the eukaryotic cytoplasm that they called urkaryotes.

This discovery has two outstanding implications:

  1. 1.

    Bacteria do not form a single group but two distinct populations (archaebacteria and eubacteria).

  2. 2.

    The ancestors of the eukaryotes are as old as the ancestors of the two types of bacteria and represent a third type of primitive population.

Later on, Woese renamed the three groups and proposed that all cells belong to three distinct primary kingdoms, or domains, that were called Archaea, Bacteria and Eukarya (Woese 1987, 2000; Woese et al. 1990).

This proposal was strongly opposed by Ernst Mayr who argued that archaebacteria and eubacteria are undoubtedly prokaryotes from a morphological and a physiological point of view and their molecular differences cannot be enough to classify them into two distinct kingdoms.

Woese replied that the history of the cells can only be reconstructed from molecular data and these data tell us that there have been three distinct types of ancestral cells, not two.

In Bacteria, for example, the cell membrane contains phospholipids whereas in Archaea it contains isoprenoid lipids. In Bacteria the cell wall is made of peptidoglycans whereas in Archaea it is made of proteinaceous material. Bacteria move by flagella and these organelles obtain energy from the circulation of protons or sodium ions, whereas Archaea move by totally different organelles that obtain energy from ATP (Harold 2014).

Today it is universally accepted that Archaea and Bacteria are two different primary kingdoms, but there are still different opinions about the third kingdom of the Eukarya.

4 The Common Ancestor

The greatest generalization of biology is the cell theory, the idea that all living organisms are made of cells and that cells derive from preexisting cells. This implies that all cells of the present are related to all cells of the past by an uninterrupted chain of descent that goes all the way back to the first cells that appeared on the primitive Earth.

The greatest discovery of paleontology is that our planet has been inhabited exclusively by free-living cells, or microorganisms, for the first 3000 million years of evolution. For more than 80% of the history of life, in other words, the microorganisms have been the sole living creatures on Earth and the sole protagonists of evolution (Schopf 1999).

The greatest discovery of molecular biology is that all known cells contain a virtually universal genetic code, a fact which implies that that code evolved in populations of primitive systems that preceded the first cells and are collectively known as the common ancestors of all life.

According to Haeckel (1866) the first living systems were bacteria, and this is still a very popular theory, because it is based on an apparently incontrovertible concept: the idea that bacteria are primitive because they are the simplest known cells.

This implies that the common ancestor had bacterial-like characteristics, but in fact this is not a realistic conclusion. More precisely, these are the three characteristics that we can reasonably attribute to the common ancestor.

  1. 1.

    The genome of the bacteria is a single molecule where all genes are arranged one after the other without interruptions and all of them code for functional molecules. Such organization is highly efficient, but precisely for this reason it could not have appeared at the very beginning. A genome made of various pieces of DNA where only a few of them are expressed is definitely more primitive, and it is likely therefore that the common ancestor did have this type of imperfect genomes.

  2. 2.

    The protein synthesis of bacteria is based on short-lived messengers that allow the cells to adapt very rapidly to changing environments. Again, such a fast-reacting system can hardly be primitive and it is likely therefore that the common ancestor was reacting more slowly to the environmental changes and was using therefore more stable long-lived messengers.

  3. 3.

    In bacteria, the transcription of the genes is immediately followed by their translation, to the extent that in most cases protein synthesis starts on messenger-RNAs that are still attached to DNA. Such a fast sequence of precisely coordinated steps is hardly primitive and it is likely therefore that in the common ancestor transcription was not immediately followed by translation.

A genome made of many pieces of DNA, the use of long-lived messengers, and a separation between transcription and translation are intrinsically primitive features that we can attribute to the common ancestor and they are not bacterial characteristics.

The common ancestor, on the other hand, did not have eukaryotic characteristics because it did not have a nucleus, a cytoskeleton, mitochondria, chloroplasts, mitosis and meiosis.

The common ancestor, in conclusion, did not have bacterial characteristics and did not have eukaryotic characteristics and this suggests that it was totally different from those two types of cells but had the potential to evolve into either of them.

5 Consumers and Producers

The molecules that are found in meteorites and those that are formed spontaneously in laboratory experiments suggest that a wide variety of organic molecules were present on the primitive Earth and the ancestral systems could used them as nutrients. Such a food store, however, was inevitably destined to be depleted, and this created the conditions for two different survival strategies. Some cells adapted their metabolism to smaller and smaller nutrients, and eventually learned to rely only on inorganic molecules. In this way they ceased to be consumers, and became producers of organic matter.

Other ancestral systems continued to feed on organic matter, but were forced to use increasingly bigger compounds. A potentially important source of food was provided by the bodies of other cells, especially the dead ones, and some ancestral consumers learned to develop structures that enabled them to ingest increasingly bigger pieces of organic matter.

This tells us that the first dichotomy in cellular evolution was the separation between producers and consumers of organic matter.

The second dichotomy was the split between eubacteria and archaebacteria. The archaebacteria are microorganisms that have been called ‘extremophiles’ because they are adapted to extreme conditions. ‘Thermophiles’ and ‘hyperthermophiles’, for example, grow at temperatures between 80 and 120 degrees centigrades, especially in hydothermal vents. ‘Psycrophiles’ live in extremely cold environments, between 0° and 4°, and even stop reproducing when the temperature rises above 12°. ‘Halophiles’ (salt-loving) grow in highly salty niches, such as salt-evaporation basins. As for pH, there are two different types of extremophiles: ‘basophiles’ prosper in habitats, such as soda lakes, whose pH is greater than 9, while ‘acidophiles’ colonize areas with pH between 1 and 5, like sulphur vents and rot undergrounds.

The extremophiles are an outstanding example of adaptation to exceptional conditions and this implies that their ancestral cells were predisposed to make experiments and were potentially capable to profoundly change their metabolism.

In the early history of life, in other words, there have been evolutionary experiments which ended with the ‘discovery’ of two different bacterial cells, and those experiments have been independent because the gulf that divides eubacteria from archaebacteria is a very deep one.

Eubacteria and archaebacteria are both producers of organic matter, so the divide between them was a divide between ‘normal producers’ and ‘extreme producers’. But what about the ancestral ‘consumers’?

We have seen that the common ancestor was necessarily a consumer of organic matter, so what happen to its descendants? Did they all become bacteria or did some of them maintain the consumer strategy and gave origin to the third type of primitive cells that Woese called ‘urkaryotes’?

6 The Intermediate Ancestors

The fact that all living systems contain a virtually universal genetic code implies that the evolution of that code took place in stages and was completed in what has become known as the last universal common ancestor (LUCA). The first genetic code that appeared on Earth was necessarily ambiguous and its evolution was a process that steadily reduced and finally eliminated its initial ambiguity. When that happened, it became possible to translate genes into specific proteins and life as we know it—life based on biological specificity—came into existence. The origin of the modern genetic code, however, was not enough to produce a modern cell.

The reason is that the descendants of the last common ancestor could produce specific proteins but without a signal transduction code they could not produce specific responses to the environment. They had biological specificity in protein synthesis, but not in their interactions with the environment, and it is for this reason that they had not yet reached the status of modern cells.

As the genetic code marked the transition from statistical to specific proteins, the signal transduction code marked the transition from statistical to specific cell behaviours, and was therefore an equally foundational event in macroevolution.

This is the code theory on the first cells, the idea that the first systems with a modern cell organization came into existence when they acquired a signal transduction code that allowed them to produce specific reactions to the signals from the environment (Barbieri 2016).

As in the case of the genetic code, the signal transduction code could not have appeared fully formed at the beginning, and this means that that code too was initially ambiguous and had to go through a process of evolution.

The ancestral systems where the first signal transduction codes appeared and evolved can be referred to as ‘intermediate ancestors’ in the sense that they came after the last universal common ancestor and before the modern cells. It must be underlined that they were necessarily a plurality of ancestors, because different modern cells have different signal transduction codes and the evolution of these codes took necessarily place in different intermediate ancestors.

The modern eubacteria and archaebacteria are highly streamlined systems because they only contain the components that are strictly necessary to their survival and this means that they evolved from precursors that were capable of streamlining. Those precursors can be referred to as “proto-bacteria” and “proto-archaea” because they were not yet modern bacteria but were already committed to the bacterial way of life.

7 The Phylogenetic Trees

The phylogenetic trees reconstructed by Woese were obtained from ribosomal RNAs, but in principle they could also have been reconstructed from proteins because these molecules too contain phylogenetic information. When the techniques of molecular phylogeny were applied to proteins, however, the results turned out much more complex than expected. Some proteins confirmed the three domains obtained from the ribosomal RNAs, but other proteins led to different phylogenetic trees (Brown and Doolittle 1997).

It turned out that these differences were due to the fact that bacteria are regularly swapping genetic material with the process of horizontal gene transfer (Miller 1998). The pattern of a tree is realized when genes are transmitted virtually unchanged from one generation to the next, i.e., when descent is vertical. When genes are swapped horizontally, instead, they become part of many branches simultaneously and the resulting pattern is no longer a tree but a web (Doolittle 1999; Doolittle and Bapteste 2007).

The phylogenetic record, in other words, has been heavily blurred by horizontal gene transfer and the three primary kingdoms emerge clearly only from molecules that have largely avoided that process, i.e., from molecules—like the ribosomal RNAs—that have been highly conserved in evolution. The result is that the blurring of the phylogenetic trees caused by horizontal gene transfer reopened the discussion about the nature of the first cells.

According to Carl Woese, as we have seen, the common ancestor gave origin to three primary kingdoms, but according to other authors it gave origin only to two primary kingdoms—Archaea and Bacteria—and the eukaryotes appeared at a later stage as a result of an extraordinary cell fusion between an archaeon and a bacterium.

This last hypothesis has been expressed in various ways. According to William Martin and Miklòs Müller, the original cell fusion took place between a methanogenic archaeon and the alfa-proteobacterium whose descendants evolved into mitochondria (Martin and Müller 1998). According to Lòpez-Garcia and Moreira (1999) the eukaryotes emerged from the fusion of a methanogenic archaeon with a myxobacterium.

In both cases, the fundamental assumption is that the eukaryotic cell is a chimera that originated from the fusion of an archaeon with a bacterium in an extraordinary episode of transmutation that Franklin Harold described as “effectively a miracle” (Harold 2014, p.124).

Today, the idea that eukaryotes arose from a fusion of two prokaryotic cells has become the dominant theory in this field, and yet, as we will see, there is no compelling evidence in its favor.

8 Two or Three Primary Kingdoms?

The phylogenetic trees obtained from individual organic molecules have produced discordant results, but a much more powerful approach to phylogeny was realized when it became possible to study not only individual molecules but entire genomes (Koonin 2003; Snel et al. 2005; Simonson et al. 2005; Jun et al. 2010). One of the most important results of this extended technology was the discovery that all modern eukaryotes belong to five or six major groups that radiated from what has been called the last eukaryotic common ancestor (LECA) (Baldauf 2003; Adl et al. 2005; Keeling et al. 2005; Koonin 2012).

This tells us that there have been two major events in the evolution of the cells. The first was the appearance of the Last Universal Common Ancestor (LUCA) the population that evolved the genetic code; the other was the appearance of the Last Eukaryotic Common Ancestor (LECA) the population from which all modern eukaryotes have descended.

The common ancestor of all cells appeared around 3.5 billion years ago, whereas the common ancestor of all eukaryotes arrived 2 billion years later, around 1.5 billion years ago (Harold 2014).

Another outstanding result of genome phylogeny was the discovery that the eukaryotic cells received genes from both Archaea and Bacteria. More precisely, they received about 20% of their genes from Bacteria and about 10% from Archaea while the remaining 70% are exclusively found in eukaryotes and are referred to as eukaryotic signature genes (Lane 2015).

The fact that 70% of the eukaryotic genes do not exist in bacteria suggests that the common ancestor gave origin to three primary kingdoms, as suggested by Woese, and it has been pointed out that this conclusion is fully compatible with the experimental evidence (Kurland et al. 2006).

Despite this, however, today the most popular theory in this field claims that the common ancestor gave origin only to two primary kingdoms, Archaea and Bacteria. The supporters of this theory have proposed that the exclusively eukaryotic genes in the beginning were bacterial genes that later became transformed beyond recognition. In reality, we have no evidence of this transmutation of genes, but the theoretical possibility that it could have taken place has apparently been enough for the supporters of the idea that the bacteria have been the sole first cells that evolved from the common ancestor (the bacterial theory of life).

In addition to the evidence provided by the exclusively eukaryotic genes, however, there are at least other two arguments that should be taken into account.

  1. 1.

    The idea that the first eukaryotes originated by a fusion of two prokaryotes does not seem to be compatible with the fact that eukaryotes are consumers whereas prokaryotes are producers of organic matter. How could the fusion of two producer-cells give origin to a consumer-cell?

  2. 2.

    The idea that the first eukaryotes originated by a fusion of two prokaryotes does not seem to be compatible with the fact that eukaryotes had the ability to generate new codes whereas prokaryotes have lost that ability. How could the fusion of two cells that could not generate new codes give origin to a cell that had the ability to generate new codes?

Today the idea that the last common ancestor gave origin to two bacterial primary kingdoms is the dominant view, but it is an experimental fact that 70% of the eukaryotic genes do not exist in bacteria, and in my opinion Woese’s proposal of three primary kingdoms is still the best explanation of that fact.

9 The Problem of Complexity

The fossil record has revealed the presence of fossilized bacteria in Precambrian rocks, and has shown that the stromatolites built by cyanobacteria between 2 and 3 billion years ago are virtually identical to those built by their modern descendants (Barghoorn and Tyler 1965; Knoll 2003). The bacteria, in other words, appeared very early in the history of life and have conserved their size, their shapes and the number of their components—in short their complexity—ever since. This point has been beautifully illustrated by Nick Lane: “… the bacteria and archaea have barely changed in 4 billion years of evolution. There have been massive environmental upheavals in that time. The rise of oxygen in the air and oceans transformed environmental opportunities, but the bacteria remained unchanged. Glaciations on a global scale (snowball earths) must have pushed ecosystems to the brink of collapse, yet bacteria remained unchanged….Nothing is more conservative than a bacterium” (Lane 2015, p. 158).

The eukaryotes, on the other hand, did the very opposite. They repeatedly increased the complexity of their cells and eventually broke the cellular barrier and gave origin to plants and animals. This gives us a major problem: why have the prokaryotes not increased their complexity throughout the history of life while the eukaryotes have become increasingly more complex?

An unexpected solution to this problem has come from the discovery that the prokaryotes became increasingly committed to fast replication and adopted a streamlining strategy in order to achieve this goal, a strategy that prevented them from evolving new codes. Let us illustrate this point with two examples.

The descendants of the common ancestor that adopted a streamlining strategy abolished all the intermediate steps between transcription and translation and the result was that the transcription of the genes was immediately followed by their translation into proteins. The other descendants maintained a physical separation between transcription and translation and this allowed them to gradually introduce in between the operations of splicing. The prokaryotes, in other words, could not evolve a splicing code because they had abolished the separation between transcription and translation that is the very precondition of splicing.

A second example comes from the histone code. In order to maximize the replication of the genes it was necessary to remove any surrounding material from the genes, and this is why the streamlining strategy produced genes with no protein wrapping around them. The ancestral systems that did not follow that strategy, on the other hand, continued to carry genes surrounded by positively charged molecules and eventually some of these molecules evolved into histones. The potential to evolve the histone code, in other words, survived only in the descendants of the common ancestor that did not adopt the streamlining strategy of the bacteria.

We have in this way a solution to the problem of complexity: the cells that adopted a streamlining strategy lost the potential to evolve new codes and have conserved the same complexity ever since; the cells that did not adopt a streamlining strategy maintained the potential to evolve new codes and gave origin to increasingly complex systems (Barbieri 2017). The complexity of a living system, in other words, is closely related to the number of its codes.