**3. The diversity**

are endowed with the ability of *self-organization* and that this capability has evolved from

This obscure stage has possibly involved *abiotic molecules* slowly evolving towards selfreplicating forms (Paragraph 1) in a kind of *chemical evolution* (Martin and Russell, 2003) that goes beyond this discussion. Self-replicating means keep track of itself and preserving memory of the experiment, but the question is, obviously, about the nature of the first self-replicating molecule; however, since replication is accomplished in modern cells through the cooperative action of *proteins* and *nucleic acids*, there is a general agreement on their essential contribution to the development and maintenance of any living organism. Hence at the origin of the cellular organization are elementary properties that, through self-replication, are transmitted to the

The appearance of the *prokaryotes* dating back approximately 3,500 million years ago represents the first remarkable result of this process, but is just a step towards the refinement of life. A real breakthrough in cellular organization is the advent of the *eukaryotes* where elementary properties are re-arranged to a higher level of complexity. But, how was it possible? Nobody knows, however this seems to be related to the endosymbiosis, an evolved form of phagocy‐ tosis that consists in using the competence of other organisms, instead of the energy. From this peculiar type of *symbiosis* (Mereschkowsky, 1926) derive certain essential properties of the cellular organization, such as *cooperation*, development of specific skills or *competence*,

The appearance of multicellular organisms as occurred fairly rapidly in the experiment of life must be seen as an evolutionary stage that does not necessarily involve a significant increase in the complexity of the genetic program (Prochnik et al., 2010); this process has been repro‐

Furthermore, since the evolution of multicellularity has not resulted in the replacement of the *prokaryotic prototype* that is still alive, for example, in *modern bacteria*, it seems logical to assume that this stage represents a necessity in evolution (Furusawa and Kaneko, 2000) for *some* living organismsexpeciallywhenexposedtohighlyselectiveenvironmentalconditions.Theseextreme conditionsoneartharepossiblyresponsibleforthe*evolutionarypeaks*recordedafterlongperiods of stasis (Eldredge et al., 2005) and thus for the evolution of multicellularity. At this regard it is noteworthythatarchaeaandbacteria,inspiteoftheirearlyevolution,exibitaverysmallnumber of species (about5,000)if comparedwithmulticellularorganisms,andareassociatedwithahigh levelofresistanceinallecosystems(Staley,2006).Therefore,itisplausibletoassumethatbacteria are a *source of backup* capable of restarting the experiment of life on earth even after catastroph‐

ic climate change or, possibly, in other places in the universe (Wickramasinghe, 2004).

*isms*(Furusawa and Kaneko, 2000).

Returning to the metaphor of the *tree of life*, having a common trunk or origin means to share, at least, some features and/or functions, and in multicellularity some properties of unicellular organisms are reallocated on a larger scale with the evolution of *cellular differentiation* and *specialization*. This new kind of cooperation establishes *functional hierarchies* and leads from the development of *finely detailed pattern* up to the evolution of fully developed *complex organ‐*

*complexity increase*, development of *interaction patterns* up to *multicellularity*.

duced recently in vitro by using an eukariotic model (Ratcliff et al., 2012).

elementary properties of chemical compounds essential to kick off life on earth.

offspring.

648 Regenerative Medicine and Tissue Engineering

The *diversity* in biology is "...the variety and variability among living organisms and the ecological complexes in which they occur." (Assessment., 1987). The diversity can also be calculated as the *number* of different items and their *relative frequency*; these items are organized at many levels, ranging from entire ecosystems to the chemical structures that are the molecular basis of heredity (Paragraph 2).

As we have seen before, the diversity originated very early in the tree of life with a very rapid expansion characterized by the evolution of an extraordinary variety of living organisms in a relatively short time followed by a long lasting substantial stasis (Eldredge et al., 2005). This expansion phase is possibly the second *obscure stage* in the experiment of life, after the initial one (Figure 3), since it is really hard to imagine a cause-effect relationship during those phases beyond the theories of transformation-evolution (Figura 4).

Schematic representation of the origin of life and diversity. The upper part is a speculation and represent an intelligent creator above the experiment of life in the universe. The intermediate part is a representation of the hypothesis that life on Earth has an extraterrestrial origin thanks to space vectors such as meteorites or asteroids. The lower part re‐ calls the second obscure stage [generation machine] and the evolutionary theory explaining the origin of diversity.

The road to the diversity is marked by milestones including the transition from *duplication* to *reproduction* and this innovation is believed to coincide with the evolution of the *eukaryotic* cell that adopts the *meiosis*. This peculiar form of cell division produces greater variability contri‐ buting to increase the diversity of living organisms.

But writing of diversity in biology can be complicated, precisely because of different points of view covering the whole scenario; indeed, from a purely *descriptive* point of view, there are both visible and invisible differences. Focusing on a *population* represented by all the living organisms that belong to the same species and live in the same geographical area, since individuals are not identical, some visible differences are expected and these differences are even more noticeable when taking into account *different populations* or species.

one (Figure 3), since it is really hard to imagine a cause-effect relationship during those phases

Schematic representation of the origin of life and diversity. The upper part is a speculation and represent an intelligent creator above the experiment of life in the universe. The intermediate part is a representation of the hypothesis that life on Earth has an extraterrestrial origin thanks to space vectors such as meteorites or asteroids. The lower part re‐ calls the second obscure stage [generation machine] and the evolutionary theory explaining the origin of diversity.

The road to the diversity is marked by milestones including the transition from *duplication* to *reproduction* and this innovation is believed to coincide with the evolution of the *eukaryotic* cell that adopts the *meiosis*. This peculiar form of cell division produces greater variability contri‐

**Figure 4.** On the origin of life

buting to increase the diversity of living organisms.

beyond the theories of transformation-evolution (Figura 4).

650 Regenerative Medicine and Tissue Engineering

Nonetheless the visible differences give way to *basic similarities* when are taken into consider‐ ation the internal features, invisible at naked eyes. These common features include the general *molecular structures* and principles that are the basis of *biochemical functions* in all living organisms and clearly demonstrate the concept of *branching of the tree of life* starting from a *common ancestor*.

Thus the biological life is characterized by a *partial and transient independence* as the result of the dynamic interaction between the environment and the biochemical function of some *common macromolecules* such as lipids, nucleic acids, namely DNA and ribonucleic acid (RNA), proteins and carbohydrates reallocated on a larger scale with the evolution of *cellular differen‐ tiation* and *specialization*.

The *nucleic acids* of all organisms are constituted by the same set of nucleotides and the proteins blocks are almost made by the same amminoacids. Moreover, several *cellular events* share the same mechanisms and machinery; events like mitosis, DNA duplication, protein syntesis are indeed based on the same molecular steps and the molecular structures involved are the same both in prokayotes and eukaryotes, such as histones for chromatin packing, polymerase for DNA duplication, ribosomes for protein syntesis and so on.

From a molecular point of view, only the analysis of structures, such as the analysis of how many and which amino acids compose a protein with a similar function in two different species, can state the diversity, assess the *phylogenetic proximity* between species and outline the branches of the tree of life (Figure 5).

Lookingnowatthe*geneticprogram*thatdeterminestheshapeandthefunctionoflivingorganisms, it is represented by different *gene sequences* that, because of *diploidy*, in humans consists of two or more copies of genes called *alleles*. Thus the *gene pool* of a population is made by the totally of alleles,their *combination* or *aplotype* andtheir*relativefrequencies* among the individuals.Thus the *genetic diversity* describes the existence of many different versions of the same individual, a *differentphenotype*istheresultofageneticvariationinaspecificenvironmentandiscalled*variant*. As stated before, the *genetic diversity* among individuals in a given population and even more between species, increases the chances of survival in case of highly selective environmental conditions;thus *evolution*takesplacewhenthereare *any*changes inthegeneticpoolthatincrease the adaptation process (Paragraph 1). There are few processes that can lead to *new genetic combinations* and the most relevant for this discussion are *mutation*, *recombination* and *natural selection*, therefore we do not take into account the effects of *migration* into a population from another one, with different gene frequencies, as a *source of variation*.

Essentially these processes can be *random*, meaning that they take place independently of the needs of the organism, *probabilistic* and *directional*; mutations are random, recombination is

The picture shows the estimated geological age of the last common ancestor of each pair of specified animals. Each time estimate is based on comparisons of amino acid sequences of orthologous proteins; more time had a pair of ani‐ mals to evolve independently, smaller is the percentage of amino acids remaining identical. The final estimates and the time scale has been calibrated to match the fossil evidence that the last common ancestor of mammals and birds lived 310 million years ago. Numbers in the top bar gives data on sequence divergence for a particular protein chosen arbitrarily, i.e the α-chain of hemoglobin. The clear irregularities in growing divergence with increasing time reflect the randomness of the evolutionary process and, probably, the action of natural selection, which drives particularly rapid changes in some organisms subjected to special physiological requirements.

**Figure 5.** Phylogenetic proximity

probabilistic, depending on the distance between genes (Russell, 1998), and natural selection is directional since it follows a direction which favors the survival of the fittest organism.

Both mutation and recombination define the *allelic variability*, which determines a difference between the genotypes of individuals; this variability will be further shaped by the environ‐ ment, that ultimately determine the presence of different *phenotypes*, based on instructions dictated by the *genotype* (Russell, 1998). Mutations, as source of variation, are quite limited since their rates are very low. On the other side, recombination is a primary source of variation; most of the attention of evolutionary geneticists has focused on the extensive *genetic recombi‐ nation* that takes place during meiosis and, in particular, during pairing of sisters chromatides and its significance as a generator of genetic diversity in organisms with sexual reproduction (Charlesworth, 1988; Edwards, 2000).

In organisms with asexual reproduction, such as bacteria, recombination is very limited since mutations are the only source of gene combinations, thus, asexual organisms may evolve more slowly, under natural selection, than sexual ones (Griffiths et al., 1999). Although the mutations play a more limited role as a source of variability in multicellular organisms with sexual reproduction, together with the effects of gene recombination, they can still be transmitted to offspring only by the *germ cells*.

In multicellular eukaryotes these cells are the connection between the different generations since they are the only ones that undergo *meiosis*, as well as *mitosis*, in contrast to *somatic cells*, practically all the others, that divide only by mitosis. In organisms that have evolved forms of *sexual reproduction*, the life cycles start through the union of two germ cells, male and female, and their nuclear fusion originates the *zygote*. Several reasons support the evolutionary necessity and the benefits of sexual reproduction in multicellular organisms (Roze, 2012; Wong and Wessel, 2006) and their prevalence in animals (Engelstadter, 2008).

Thus the zygote is a *diploid cell* resulting from the mating of two *haploid cells*, that gives rise, after three cell divisions, to the first *stem cells*. These cells are the only ones capable of differ‐ entiating into all cell types that make up a multicellular organism. This extreme versatility is known as *potency*, it is maximal during the pre-natal life in the *embryonic stem cells*, and progressively decreases in post-natal life remaining confined within the *adult stem cells*.

With regard to the definition given at the beginning of the paragraph, it should be remarked that the *biodiversity* implies the overall biological variability, including both genetic and environmental features. Environmental changes, both cyclic and irregular ones, may be a serious challenge for the experiment of life causing usually a *switch* in the phenotype. There are two opposite types of switching of the phenotype: the *reactive type*, which occurs as a direct response to an external cause detected by a *sensory mechanism,* such as a receptor, and the *stochastic type*, which occurs without the mediation of a sensory mechanism (Kussell and Leibler, 2005).

The *phenotypic diversity* is generated by *phenotype switching* caused mainly by stochastic mechanisms; thus the *population diversity* and the coexistence of subjects differently adapted to a certain environment, can be a way to respond to *irregular environmental changes* (Soll and Kraft, 1988; Perez-Martin et al., 1999; Bayliss et al., 2001; Lachke et al., 2002; Bonifield and Hughes, 2003; Kearns et al., 2004; Balaban et al., 2004).
