**6. Conclusions and discussion**

18 Gene Duplication

range of *1/2 < S < 1,* and the probability *Pdn* is within the range of *2nQnrn > Pdn > Qnrn*. If the homologous chromosomes are randomly partitioned into the children regardless of carrying a new gene or not, *rn* takes the value of *(1/2)n* and the above relation of *Pdn* becomes *Qn > Pdn > Qn/2n*. Moreover, the value of *Qn* becomes smaller for the larger value of *n*, and the probability *Pdn* becomes lower as the number of new genes assembled by hybridization is increased. The lower probability means the longer time or more generations for a new style organism carrying more kinds of new genes to appear. Thus, the diploid organism has a chance to acquire many kinds of new genes by hybridization, but it takes a longer time to

Moreover, the process to establish the homozygote is further continued after the new style organism carrying *n* kinds of new genes heterogeneously is generated with the probability *Pdn*. Although it is laborious to follow this process completely, the essence of this process can be elucidated by investigating the ratio of children that receive these new genes homogeneously and heterogeneously from the mating between the organisms each carrying *n* kinds of new genes heterogeneously. If the chromosomes in each homologous pair are randomly partitioned into the children regardless of carrying a new gene or not, the ratio of children receiving *(n-k)* kinds of new genes is calculated to be *nCk3n-k/4n* with the normalization factor *4n*, where *k* takes a value ranging from zero to *n*. This indicates that more than half of the children receive all new genes *(k = 0)* for *n = 1*, *2*. If the one or two new genes exhibit an excellent character, therefore, the descendants increase their fraction monotonously as a new style of organisms. However, the ratio of children receiving a full set of new genes becomes smaller for a larger value of *n*. In the case of *n = 5*, for example, the ratio of the children that receive five kinds of new genes *(k = 0)* decreases to *(3/4)5*, while other five types of children each appear with the ratio of *(3/4)4/4* by receiving four kinds of new genes *(k = 1)* in different ways. When a biologically meaningful character is expressed by five kinds of new genes, therefore, only *(3/4)5* of the children succeed in expressing this character but other five types of children are reserved as those carrying 'hidden genes' for producing other characters by further hybridization with other types of variants. Such divergence of characters becomes more outstanding when a larger number of new genes are required for the expression of a character. This divergent property in the process to establish many kinds of new genes as the homozygote explains the explosive divergence of body plans that has occasionally occurred in diploid organisms, because the cell differentiation is a representative character expressed by many kinds of genes and its hierarchical evolution constructs body plans, as will be discussed in the next section. Until the new style organisms are established as the homozygote, the mating between the variants of heterozygote also regenerates the original style of organisms. The phenomenon called the "reversion" or "atavism" in classical biology may be the vestige of this evolutionary process to establish the

If the influence of transposons is explicitly considered, it makes the above process more complicated in such a way that duplicated genes are separately transferred to different kinds of chromosomes. When various origins of duplicated genes or new genes are concentrated on one chromosome, however, the descendants received such a chromosome may be extinct due to the incompatibility of this chromosome with its partner chromosome not carrying any new gene. Thus, many kinds of new genes for expressing a new character may be scattered over different kinds of chromosomes in survivors just like the result of the

realize this chance.

homozygote.

present model scheme.

The variants, which experienced gene duplication, first decline to be minor members in a population by the load of carrying extra gene(s), but some of them revives as a new style of organisms by the generation of new gene(s) from the counterpart of duplicated genes. After the new gene(s) appear, the new style organisms increase their fraction being further elaborated by Darwinian evolution. This course of the large-scale evolution is essentially the same in any type of organisms, and this is a necessary condition for the new style of organisms and the original style of organisms to be able to coexist utilizing different material and energy sources or to live in separate areas, showing a striking contrast to the survival of the fittest in Darwinian evolution. This evolutionary pattern also gives an explanation to the punctuated mode of evolution, which has been proposed from paleontology against the gradual accumulation of variants in Darwinian evolution (Eldredge & Gould, 1972).

However, the detailed processes of this large-scale evolution are different depending on the types of genome constitution and transmission. The monoploid organism is suitable to generate one new gene step by step testing its biological function, but hardly generates many kinds of new genes simultaneously. The lower eukaryote, whose genome consists of the plural number of chromosomes, resolves this difficulty to produce a new style of organisms receiving many kinds of new genes by the conjugation of variants carrying different origins of new genes. The diploid organism can also produce a new character responsible for multiple kinds of new genes by the successive hybridization of different variants but its conservative property requires the succeeding process to establish the homozygote of these genes. This process becomes longer for a larger number of new genes to be established. During this long process, the further hybridization with other variants also occurs, occasionally yielding the explosive divergence of new characters depending on the combinatorial sets of new genes. This conclusion of the present study explains the recently revealed evolutionary patterns of prokaryotes and eukaryotes to a great extent, getting an insight into the problems how and why the monoploid eukaryotes have evolved to the diploid eukaryotes.

According to the analyses of base-pair changes in ribosomal RNAs, the main lineages of present-day prokaryotes diverged *3.0x109* years ago, developing various chemical syntheses, *O2*-releasing photosynthesis and *O2* respiration, respectively (Otsuka et al., 1999), after the earlier divergence of archaebacteria, eubacteria and eukaryotes (Sugaya & Otsuka, 2002). Several stages from simple electron transport pathways to *O2* respiration and *O2*-releasing photosynthesis are still observed in the present-day eubacteria and the elongation of the pathways has taken place stepwise by gene duplication, as can be traced from the amino acid sequence similarities between their component proteins and the ubiquitous permeases (Otsuka, 2002; Otsuka & Kawai, 2006), although such similarity search of amino acid sequences is not systematically carried out yet for chemical syntheses. However, the excellent abilities of O2 respiration and O2-releasing photosynthesis cannot be fully exhibited in the simple cell structure of prokaryotes (Otsuka, 2005), and the genome size of the eubacteria having these abilities is also limited to the order of *106 bp* compactly encoding *3,000* ~ *4,000* genes like the other prokaryotes (Wheeler et al., 2004).

On the other hand, the eukaryotes have experienced much more evolutionary events until some of them establish the diploid state. The ancestral eukaryote probably became the predator of eubacteria by developing the intracellular structure, endocytosis and exocytosis

A Theoretical Scheme

occurred about *3.8x108* years ago.

characters for survival.

of the Large-Scale Evolution by Generating New Genes from Gene Duplication 21

them, the green plants, which have also acquired the chloroplasts as the endosymbionts of photosynthetic eubacteria independently of sea algae, provide a representative example of the above five stages of evolution from the monoploid organisms to the diploid organisms. The *Cojugatae* such as *Roya* and *Spirogyra* are at the stage (a), the *Chara* of *Charophyta* is at the stage (b), the *Bryophyta* is at the stage (c), where the fertilized egg on female gametophyte grows into sporangium, the *Pterophyta* is at the stage (d), and the seed plants are at the stage (e). According to the recent analysis of neutral nucleotide base substitutions in *rbcL* genes on the chloroplast genomes (Kawai & Otsuka, 2004), the divergence of *Charophyta* and *Bryophyta* occurred more than *109* years ago, the divergence of *Bryophyta* and *Pterophyta* occurred around *4.7x108* years ago, and the divergence of *Pterophyta* and seed plants

The molecular mechanism underlying the cell differentiation is not fully clarified yet, but it is probably based on a set of receptors, the corresponding ligands, signal transduction proteins, transcriptional regulators as well as the proteins exhibiting the respective cell-type specific functions. Moreover, the amino acid sequences of these proteins under the control of signal transduction network become longer by the attachment of special amino acid residue repeats such as serines and threonines. Thus, the assembly of so many kinds of large genes into a genome must have first progressed under the scheme of the conjugation of monoploid eukaryotes with the plural number of chromosomes. After a set of genes responsible for cell differentiation are established in the monoploid state, the increase in the repertoire of the respective members would have occurred relatively easily. In particular, a small number of nucleotide base substitutions could bring about the expansion of such protein families as transcriptional regulators, receptors and kinases associated with the signal transduction network, although these kinds of proteins have their origins at the stage of unicellular eukaryotes. The increase in acquired energy by the cell differentiation in the monoploid state makes it possible to realize the cell differentiation in the diploid state. The example of green plants suggests that the cell differentiation in the diploid state has started from the zygote and gradually spread to form other organs of diploid cells, resulting in the alternation of the monoploid generation and the diploid generation. The diploid state is suitable to protect the differentiated cells from the point mutations, as will be discussed in the last part of this section, but it takes a longer time or many generations to establish a set of many genes for advancing the further cell differentiation in the diploid state as the homozygote. Although this is the barrier lying between the stage (d) and the stage (e), the diploid organisms having gone over this barrier receive a good chance to produce various combinatorial sets of new genes leading to the explosive divergence of morphological characters. Such explosive divergence has the merit of testing simultaneously various

Although any example of animals at the stages (b) and (c) is hardly found at the present time, the *Cnidaria* still alternates the monoploid generation and the diploid one. The divergence of *Cnidaria* and the common ancestor of other animals occurred immediately after the animal-plant divergence (Otsuka & Sugaya, 2003). The famous explosion of body plans giving rise to *Annelida, Mollusca, Arthropoda, Echinodermata* and *Chordata*, which is first found by the fossil record of Ediacara and Avalon faunas (Mathews & Missarzhersky, 1975; Rozanov & Zhuravlev, 1992) and of Cambrian Burgess Shale (Gould, 1989) and then estimated to have occurred successively during the period of 9~6x108 years ago by the analysis of neutral nucleotide base substitutions (Otsuka & Sugaya, 2003), is probably based on the evolutionary scheme of diploid organisms described in sections 5, because these

as well as the signal transduction network. Such cell structure would have been suitable to acquire the mitochondria as the endosymbionts of O2-respiratory eubacteria, which is estimated to have occurred *2.0x109* years ago (Margulis, 1981; Yang et al., 1985; Otsuka et al., 1999). Under the supply of abundant ATP molecules efficiently synthesized by the mitochondria, the yeast *Sacchromyces serevisiae*, which appeared *1.8 x109* years ago (Otsuka et al., 1997), has expanded its genome to *1.2x107 bp* encoding *6, 300* genes (Wheeler et al., 2004) and can take the diploid state under nutrient conditions, although it usually takes the monoploid state. The enlarged genome consisting of the plural number of chromosomes also requires the special apparatus for the faithful segregation of sister DNAs upon cell division, in contrast to the prokaryotes where the membrane attachment mechanism of DNA only operates (Jacob et al., 1963; Ogden et al., 1988; de Boer, 1993). Multiple kinds of gene products such as the primitive spindle pole and kinetochore and polar microtubules are already present in the yeast (Alberts et al., 1994) while several bundles of microtubules only pass through tunnels in the typical *Dinoflagellates* (Kubai, 1975; Hearth, 1980; Wise, 1988). Thus, the components of this auxiliary apparatus for cell division may have evolved step by step at the stage of unicellular eukaryotes. Although the molecular mechanism underlying the switching from the monoploid to diploid states and vice versa is not fully clarified yet, the example of yeast indicates that this mechanism itself has also evolved at the stage of unicellular eukaryotes.

However, the evolution from the monoploid eukaryote to the diploid eukaryote has taken place considerably gradually via several stages. This is reasonable because the diploid state is an extreme case of gene duplication. If the genome size jumps from *N* to *2N*, this means the increase in the stored energy and systematization from *Es(N,SN)* and *SN* to *Es(2N, S2N)* and *S2N*. Thus, the acquired energy must be also increased to maintain the biological activity. As indicated already (Otsuka, 2008), this increase in acquired energy is possibly attained by the cooperative action of differentiated cells. However, the evolution of cell differentiation cannot occur suddenly. On this problem, the present result throws light, in the point that the conjugation of lower eukaryotes with the plural number of chromosomes is suitable to assemble many kinds of new genes necessary for cell differentiation. In fact, the recently revealed phylogeny of eukaryotes strongly suggests at least the following five stages in the evolution from the monoploid to diploid eukaryotes. (a) First, the monoploid eukaryote evolves the conjugation to exchange the homologous chromosomes. (b) Second, this eukaryote then develops multicellularity and cell differentiation in the monoploid state by assembling many kinds of new genes. (c) Third, the cell differentiation also advances to the cells in the diploid state. (d) Fourth, the eukaryote evolves to alternate the monoploid generation and the diploid generation. (e) Finally, the eukaryote evolves to the diploid organism with the higher hierarchy of cell differentiation.

As far as the present knowledge of the phylogeny of eukaryotes and their genome constitution (Otsuka et al., 1997) is concerned, the first lineages having evolved multicellularity and cell differentiation are some of the fungi that appear after yeast and the sea algae, which have further acquired photosynthetic eubacteria as the endosymbionts in the lineage of fungi (Van den Eynde et al., 1988). However, the most advanced one of them still remains at the stage (d), alternating the monoploid generation and the diploid generation. Apart from the lineages of fungi and algae, the evolution of advancing the cell differentiation to the diploid state has taken place in the animals and the green plants, whose divergence is estimated to have occurred *1.2x109* years ago (Dickerson, 1971). Among

as well as the signal transduction network. Such cell structure would have been suitable to acquire the mitochondria as the endosymbionts of O2-respiratory eubacteria, which is estimated to have occurred *2.0x109* years ago (Margulis, 1981; Yang et al., 1985; Otsuka et al., 1999). Under the supply of abundant ATP molecules efficiently synthesized by the mitochondria, the yeast *Sacchromyces serevisiae*, which appeared *1.8 x109* years ago (Otsuka et al., 1997), has expanded its genome to *1.2x107 bp* encoding *6, 300* genes (Wheeler et al., 2004) and can take the diploid state under nutrient conditions, although it usually takes the monoploid state. The enlarged genome consisting of the plural number of chromosomes also requires the special apparatus for the faithful segregation of sister DNAs upon cell division, in contrast to the prokaryotes where the membrane attachment mechanism of DNA only operates (Jacob et al., 1963; Ogden et al., 1988; de Boer, 1993). Multiple kinds of gene products such as the primitive spindle pole and kinetochore and polar microtubules are already present in the yeast (Alberts et al., 1994) while several bundles of microtubules only pass through tunnels in the typical *Dinoflagellates* (Kubai, 1975; Hearth, 1980; Wise, 1988). Thus, the components of this auxiliary apparatus for cell division may have evolved step by step at the stage of unicellular eukaryotes. Although the molecular mechanism underlying the switching from the monoploid to diploid states and vice versa is not fully clarified yet, the example of yeast indicates that this mechanism itself has also evolved at the

However, the evolution from the monoploid eukaryote to the diploid eukaryote has taken place considerably gradually via several stages. This is reasonable because the diploid state is an extreme case of gene duplication. If the genome size jumps from *N* to *2N*, this means the increase in the stored energy and systematization from *Es(N,SN)* and *SN* to *Es(2N, S2N)* and *S2N*. Thus, the acquired energy must be also increased to maintain the biological activity. As indicated already (Otsuka, 2008), this increase in acquired energy is possibly attained by the cooperative action of differentiated cells. However, the evolution of cell differentiation cannot occur suddenly. On this problem, the present result throws light, in the point that the conjugation of lower eukaryotes with the plural number of chromosomes is suitable to assemble many kinds of new genes necessary for cell differentiation. In fact, the recently revealed phylogeny of eukaryotes strongly suggests at least the following five stages in the evolution from the monoploid to diploid eukaryotes. (a) First, the monoploid eukaryote evolves the conjugation to exchange the homologous chromosomes. (b) Second, this eukaryote then develops multicellularity and cell differentiation in the monoploid state by assembling many kinds of new genes. (c) Third, the cell differentiation also advances to the cells in the diploid state. (d) Fourth, the eukaryote evolves to alternate the monoploid generation and the diploid generation. (e) Finally, the eukaryote evolves to the diploid

As far as the present knowledge of the phylogeny of eukaryotes and their genome constitution (Otsuka et al., 1997) is concerned, the first lineages having evolved multicellularity and cell differentiation are some of the fungi that appear after yeast and the sea algae, which have further acquired photosynthetic eubacteria as the endosymbionts in the lineage of fungi (Van den Eynde et al., 1988). However, the most advanced one of them still remains at the stage (d), alternating the monoploid generation and the diploid generation. Apart from the lineages of fungi and algae, the evolution of advancing the cell differentiation to the diploid state has taken place in the animals and the green plants, whose divergence is estimated to have occurred *1.2x109* years ago (Dickerson, 1971). Among

stage of unicellular eukaryotes.

organism with the higher hierarchy of cell differentiation.

them, the green plants, which have also acquired the chloroplasts as the endosymbionts of photosynthetic eubacteria independently of sea algae, provide a representative example of the above five stages of evolution from the monoploid organisms to the diploid organisms. The *Cojugatae* such as *Roya* and *Spirogyra* are at the stage (a), the *Chara* of *Charophyta* is at the stage (b), the *Bryophyta* is at the stage (c), where the fertilized egg on female gametophyte grows into sporangium, the *Pterophyta* is at the stage (d), and the seed plants are at the stage (e). According to the recent analysis of neutral nucleotide base substitutions in *rbcL* genes on the chloroplast genomes (Kawai & Otsuka, 2004), the divergence of *Charophyta* and *Bryophyta* occurred more than *109* years ago, the divergence of *Bryophyta* and *Pterophyta* occurred around *4.7x108* years ago, and the divergence of *Pterophyta* and seed plants occurred about *3.8x108* years ago.

The molecular mechanism underlying the cell differentiation is not fully clarified yet, but it is probably based on a set of receptors, the corresponding ligands, signal transduction proteins, transcriptional regulators as well as the proteins exhibiting the respective cell-type specific functions. Moreover, the amino acid sequences of these proteins under the control of signal transduction network become longer by the attachment of special amino acid residue repeats such as serines and threonines. Thus, the assembly of so many kinds of large genes into a genome must have first progressed under the scheme of the conjugation of monoploid eukaryotes with the plural number of chromosomes. After a set of genes responsible for cell differentiation are established in the monoploid state, the increase in the repertoire of the respective members would have occurred relatively easily. In particular, a small number of nucleotide base substitutions could bring about the expansion of such protein families as transcriptional regulators, receptors and kinases associated with the signal transduction network, although these kinds of proteins have their origins at the stage of unicellular eukaryotes. The increase in acquired energy by the cell differentiation in the monoploid state makes it possible to realize the cell differentiation in the diploid state. The example of green plants suggests that the cell differentiation in the diploid state has started from the zygote and gradually spread to form other organs of diploid cells, resulting in the alternation of the monoploid generation and the diploid generation. The diploid state is suitable to protect the differentiated cells from the point mutations, as will be discussed in the last part of this section, but it takes a longer time or many generations to establish a set of many genes for advancing the further cell differentiation in the diploid state as the homozygote. Although this is the barrier lying between the stage (d) and the stage (e), the diploid organisms having gone over this barrier receive a good chance to produce various combinatorial sets of new genes leading to the explosive divergence of morphological characters. Such explosive divergence has the merit of testing simultaneously various characters for survival.

Although any example of animals at the stages (b) and (c) is hardly found at the present time, the *Cnidaria* still alternates the monoploid generation and the diploid one. The divergence of *Cnidaria* and the common ancestor of other animals occurred immediately after the animal-plant divergence (Otsuka & Sugaya, 2003). The famous explosion of body plans giving rise to *Annelida, Mollusca, Arthropoda, Echinodermata* and *Chordata*, which is first found by the fossil record of Ediacara and Avalon faunas (Mathews & Missarzhersky, 1975; Rozanov & Zhuravlev, 1992) and of Cambrian Burgess Shale (Gould, 1989) and then estimated to have occurred successively during the period of 9~6x108 years ago by the analysis of neutral nucleotide base substitutions (Otsuka & Sugaya, 2003), is probably based on the evolutionary scheme of diploid organisms described in sections 5, because these

A Theoretical Scheme

**7. References** 

D556-D561

New York

New York

of the Large-Scale Evolution by Generating New Genes from Gene Duplication 23

same sequence length of gene duplication, therefore, the reduction factor may take a smaller value in animals than in lower eukaryotes and prokaryotes. Thus, the fraction of variants carrying the 'hidden genes' generated from gene duplication may be high enough to hybridize between them in higher eukaryotes, especially in animals. Such 'hidden genes' belong to the category of 'genetic polymorphism', which has been first proposed by Ford (1965) and is subsequently disclosed by electrophoretic studies, although the 'genetic polymorphism' was only regarded as the result of random fixation

It is still somewhat mysterious that the introns and spacers are more expanded in animal genomes than in the genomes of other eukaryotes. Such expansion can be seen from the ratio of the genome size to the number of encoded genes described above. It is conceivable that the introns are necessary for messenger RNAs to pass through the nuclear membrane and the spacers enhance the crossing over of homologous chromosomes without injuring established genes, but the expansion of introns and spacers in the higher eukaryotes might

Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K. & Watson, J. D. (1994). *Molecular Biology of the Cell.* 3rd Ed. Garland Publishing Inc., New York & London. pp. 941-943 Birney, E. & Other fifty persons (Ensembl 2006). Database Issue. *Nucleic Acids Res.* Vol. 1,

de Boer, R. A. J. ( 1993). Chromosome Segregation and Cytokinesis in Bacteria. *Curr. Opin.* 

Dickerson, R. E. (1971). The Structure of Cytochrome c and the Rate of Evolution. *J. Mol.* 

Dobzhansky, T. (1941). *Genetics and the Origin of Species.* 2nd Ed. Columbia University Press,

Eigen, E. (1971). Selforganization of Matter and Evolution of Biological Macromolecules. *Die* 

Eldredge, N. & Gould, S. J. (1972). Punctuated Equilibria: An Alternative to Phyletic

Fairon-Demaret, M. & Scheckler, E. S. (1987). Typification and Redescription of *Moresnetia* 

Ferris, S. D. & Whitt, G. S. (1979). Evolution of the Differential Regulation of Duplicated

Fisher, R. A. (1930). *The General Theory of Natural Selection.* Oxford Univ. Press, London and

Gould, S. J. ( 1989). *Wonderful Life. The Burgess Shale and the Nature of History.* W. W. Norton

Belgium. *Bull Inst Roy Sci Nat Belg Sci Terre*, Vol. 57, pp. 193-199

Gene after Polyploidization. *J. Mol. Evol.,* Vol. 12, pp. 267-317

Ford, E. B. (1965). *Genetic Polymorphism*. Faber & Faber, London Gilbert, W. (1978). Why Genes in Pieces ? *Nature*, Vol. 271, p. 501

Gradualism. In: *Models in Paleobiology,* T. J. M. Schopf, (Ed.). p. 82, Freeman and

*zalesskyi* Stockmans, 1948, an Early Seed Plant from the Upper Famennian of

Carroll, R. L. (1988). *Vertebrate Paleontology and Evolution.* W. H. Freeman, New York

of selectively neutral or nearly neutral mutations by the neutralist (Kimura, 1977).

imply any other biological role of their nucleotide sequences.

Darwin, C. (1859). *The Origin of Species*. John Murry, London

*Natürwissenshaften,* Vol. 58, pp. 465-523

*Cell Biol*., Vol 5, pp. 232-237

*Evol*., Vol. 1, pp. 26-45

Cooper, San Francisco

& Company Inc., New York

animals show the living style defined as the diploid organism in the present chapter. Such divergence of body plans occasionally occurred afterwards in each of the above phyla. The examples well investigated in paleontology are the divergence of *Placodermi*, cartilaginous fish and bony fish, the divergence of amphibians, reptiles and mammals, and the divergence of dinosaurs and birds, which occurred in the *Chordata* within the recent 4x108 years (Carroll, 1988). The seed plants also show the similar tendency in the successive divergence of *Coniferophyta*, *Anthophyta* and their relatives (Fairon-Demaret & Scheckler, 1987; Rothwell et al., 1989; Rowe, 1992; Stewart & Rothwell, 1993; Kawai & Otsuka, 2004), although many of these seed plants can also self-reproduce by the parthenogenesis and their explosive feature seems mild. Although the explosive divergence of body plans can be also explained by the biological activity expressed in terms of the interaction between differentiated cells (Otsuka, 2008), the present study derives this divergence from the aspect of the generation of new genes from gene duplication in diploid organisms.

The fossil record of these examples indicates that the original style of organisms prospered over a wide region when new styles of organisms diverged, being consistent with the present theory. The prosperity of the original style of organisms means that their biological activity is high, and this is necessary to permit the existence of variants carrying duplicated genes in the population and further to enhance the chance of assembling many kinds of new genes into a genome by hybridization. This is in contrast with Darwinian evolution generating new species adapted to the special environment of a geographically isolated district by accumulating point mutations.

Finally, some discussions will be given to the problem why the cell differentiation has been shifted from the monoploid state to the diploid state. This problem arises from the present result that the diploid organism is not necessarily superior to the monoploid organism with the ability of exchanging homologous chromosomes in assembling many kinds of new genes for cell differentiation. The main reason of this shifting may be the protection of differentiated cells from the point mutations due to the miss in repairing damaged nucleotide bases. First of all, many more genes are needed to develop the higher hierarchy of cell differentiation. In fact, the genome size of higher eukaryotes is expanded to the order of *108 ~ 109 bp*, e. g., *1.2x108 bp* encoding *24,000* genes in *Arabidopsis thaliana*, *1.4x108 bp* encoding *13,000* genes in *Drosophila melanogaster* and *3.1x109 bp* encoding *30,000*  genes in *Homo sapiens* (Wheeler et al., 2004). Second, it takes a longer time, one or more years, to develop the higher hierarchy of cell differentiation to form an adult form in the higher eukaryotes, although the growth rate and the lifetime seem to be further regulated differently depending on species. On the other hand, the mutation rate due to the miss in repair is *10-9* per site per year in eukaryotes as well as in prokaryotes (Kimura, 1980; Otsuka et al., 1997). As the evidence for the above discussion, the males of some species of ants and bees are born by the haploid parthenogenesis, showing that the monoploid state is sufficient for the high hierarchy of cell differentiation during their short lifetime. Although the accuracy in repairing damaged DNAs can be raised by the additional energy for proofreading (Hopfield, 1974), the evolution of organisms has not been directed to use such additional energy. On the contrary, the nucleotide base substitution rate becomes about tenfold faster in animal mitochondrial genome than in the host cell genome, as is used to resolve the phylogeny of recently diverged animals (Hasegawa et al., 1985; Pesole et al., 1999; Otsuka et al., 2001). This faster mutation rate strongly suggests that the energy to proofread the small genome of mitochondria is diminished and instead the saved energy is used to raise the biological activity of the host cell. For the same sequence length of gene duplication, therefore, the reduction factor may take a smaller value in animals than in lower eukaryotes and prokaryotes. Thus, the fraction of variants carrying the 'hidden genes' generated from gene duplication may be high enough to hybridize between them in higher eukaryotes, especially in animals. Such 'hidden genes' belong to the category of 'genetic polymorphism', which has been first proposed by Ford (1965) and is subsequently disclosed by electrophoretic studies, although the 'genetic polymorphism' was only regarded as the result of random fixation of selectively neutral or nearly neutral mutations by the neutralist (Kimura, 1977).

It is still somewhat mysterious that the introns and spacers are more expanded in animal genomes than in the genomes of other eukaryotes. Such expansion can be seen from the ratio of the genome size to the number of encoded genes described above. It is conceivable that the introns are necessary for messenger RNAs to pass through the nuclear membrane and the spacers enhance the crossing over of homologous chromosomes without injuring established genes, but the expansion of introns and spacers in the higher eukaryotes might imply any other biological role of their nucleotide sequences.
