þÿ

Inverse relationship between genetic diversity and epigenetic complexity

Shi Huang Ph.D.

The Burnham Institute for Medical Research

10901 North Torrey Pines Roads

La Jolla, CA 92037

shuang@burnham.org

Tel: 1-858-646-3120

Fax: 1-858-646-3192

Key words: Genetic equidistance result, evolution, molecular clock, maximum genetic diversity

hypothesis, epigenetic complexity, Neo-Darwinian hypothesis

Running title: The maximum genetic diversity hypothesis

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Abstract

Early studies of molecular evolution revealed a correlation between genetic distance and

time of species divergence. This observation provoked the molecular clock hypothesis and in

turn the `Neutral Theory', which however remains an incomplete explanation since it predicts a

constant mutation rate per generation whereas empirical evidence suggests a constant rate per

year. Data inconsistent with the molecular clock hypothesis have steadily accumulated in

recent years that show no correlation between genetic distance and time of divergence. It has

therefore become a challenge to find a testable idea that can reconcile the seemingly conflicting

data sets. Here, an inverse relationship between genetic diversity and epigenetic complexity

was deduced from a simple intuition in building complex systems. Genetic diversity, i.e., genetic

distance or dissimilarity in DNA or protein sequences between individuals or species, is

restricted by the complexity of epigenetic programs. This inverse relationship logically deduces

the maximum genetic diversity hypothesis, which suggests that macroevolution from simple to

complex organisms involves a punctuational increase in epigenetic complexity that in turn

causes a punctuational loss in genetic diversity. The hypothesis explains a diverse set of

biological phenomena, including both for and against the correlation between genetic distance

and time of divergence.

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The only real valuable thing is intuition......The whole thing of science is nothing more

than a refinement of everyday thinking.

- Albert Einstein

Introduction:

It is remarkable that the human mind is able to comprehend nature. The scientific

understanding of nature is largely based on mathematics. Since mathematics is premised on

axioms or self-evident intuitions, it can be easily inferred that intuition is the ultimate foundation

of science. The relationship between intuition and a natural phenomenon is sometimes indirect

or follows the hierarchy from intuition to mathematics, to physics, to chemistry, and to biology.

But it can also be direct, for example, Newton's three laws of motion were originally postulated

as `axioms'. Intuition may directly impact the science of biology without going through the

bridge of mathematics, or chemistry, or physics, although such an intuition-based law of biology

has yet to be uncovered. An intuition-based theory is true on its own logical coherence (like a

mathematical proof) and does not in principle need validation from empirical data. In contrast,

no amount of experimental data could prove a provisional theory that is based on empirical

observations.

The molecular clock hypothesis is an essential part of the modern evolution theory. The

hypothesis was triggered by the empirical observation of a correlation between genetic distance

as measured by DNA or protein sequence dissimilarity and time of species divergence as

inferred from fossil records. Two kinds of sequence alignment can be made using the same set

of sequence data. The first aligns a recently evolved organism such as a mammal against

those that evolved earlier such as amphibians and fishes. The second aligns an outgroup

organism such as fishes against those sister species that appeared later such as amphibians

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and mammals. The first alignment indicates a linear correlation between genetic distance and

time of divergence, implying indirectly a constant mutation rate among different species. The

second alignment shows the genetic equidistance result where sister species are approximately

equidistant to the outgroup. This directly triggered the idea of constant mutation rate among

different species. Since both alignments use the same sequence data set, either alone is

sufficient to reveal any information on genetic distance. But the data that most directly and

obviously triggered the interpretation of constant mutation rate is the genetic equidistance result.

The molecular clock hypothesis was first informally proposed in 1962 based largely on

data from the first alignment (Zuckerkandl and Pauling, 1962). Margoliash in 1963 performed

both alignments and made a formal statement of the molecular clock after noticing the genetic

equidistance result (Margoliash, 1963). "It appears that the number of residue differences

between cytochrome c of any two species is mostly conditioned by the time elapsed since the

lines of evolution leading to these two species originally diverged. If this is correct, the

cytochrome c of all mammals should be equally different from the cytochrome c of all birds.

Since fish diverges from the main stem of vertebrate evolution earlier than ether birds or

mammals, the cytochrome c of both mammals and birds should be equally different from the

cytochrome c of fish. Similarly, all vertebrate cytochrome c should be equally different from the

yeast protein."

The molecular clock hypothesis asserts that the rate of amino acid or nucleotide

substitution is approximately constant per year over evolutionary time and among different

species. Two different species are thought to gradually accumulate mutations over time since

their most recent common ancestor. Their genetic distance in ancient times is thought to be

smaller than their distance today. None of these assertions are based on intuitions or could be

considered as self-evident. Nor do they have direct experimental support. They are all ad hoc

interpretations of the genetic equidistance result.

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The empirical observation of an apparently constant mutation rate has provoked the

`Neutral Theory'. But this theory is now widely acknowledged to be an incomplete explanation.

For example, Ayala noted: "The theoretical foundation originally proposed for the clock, namely

the neutrality theory of molecular evolution, is untenable. The vagaries of molecular rates of

evolution have contributed much to invalidating the theory."(Ayala, 1999). Pulquerio and

Nichols noted: "The `Neutral Theory' is not a complete explanation, however. For example, it

predicts a constant substitution rate per generation, whereas empirical evidence suggests

something closer to a constant rate per year." (Pulquerio and Nichols, 2007).

The constant mutation rate interpretation of the genetic equidistance result represents

an over-interpretation of the actual result, since the result shows merely the outcome of

evolution and says nothing about the past mutation process. In fact, the equidistance result has

been found to be independent of mutation rate variations (Huang, 2008a). Violation of rate

constancy does not mean violation of the equidistance result and the equidistance result does

not necessarily mean rate constancy. The constant mutation rate interpretation of the

equidistance result is a non-testable tautology and is not a real scientific explanation of the

equidistance result (Huang, 2008a).

The common practice of relative rate tests that often interprets small deviations from an

exact equidistance as being statistically significant is in fact flawed as it does not consider

sampling variations (Huang, 2008a). It also overlooks the striking fact that the deviations are

rarely large. If the real phenomenon here is non-equidistance with equidistance being

coincidental, one would expect to see much larger variations in distance. Thus, the data shows

that the real phenomenon here is equidistance while the small deviations from exact

equidistance are coincidental and non-significant sampling variations.

Although there clearly exists a correlation between genetic distance and time of

divergence, such correlation is not universal and is often violated as more data became known

in recent years. Numerous studies based on extant organisms have questioned the constancy

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of mutation rate (Ayala, 1999; Ho and Larson, 2006; Pulquerio and Nichols, 2007). A study of

DNA and protein sequences of ancient fossils challenged a fundamental premise of the modern

evolution theory (Huang, 2008b). It shows that genetic distance had not always increased with

time in the past history of life on Earth. Another study showed that the genetic distance among

flowering plants is much greater than that among mammals, even though flowering plants have

evolved for similar amount of time as mammals (Huang, 2008a). The genetic distance between

two subpopulations of medaka fish that had diverged for ~ 4 million years is 3-fold greater than

that between two different primate species (humans and chimpanzees) that had diverged for 5-7

million years (Kasahara et al., 2007). The genetic distance measured on genealogical

timescales (< 1 million years) is often an order of magnitude greater than that on geological

timescales (> 1 million years) (Ho and Larson, 2006), suggesting that genetic distance

measured in evolutionary time is independent of actual mutation rate measured in real time.

The molecular clock hypothesis was originally an ad hoc idea triggered by the genetic

equidistance result and remains unsupported by any other independent facts despite the effort

of the past 45 years. While it may explain the correlation between genetic distance and time of

divergence, it clearly cannot explain the frequent factual violations of the correlation. A new and

more complete idea is needed that must be able to reconcile the seemingly conflicting data sets.

Here, a simple intuition in building complex systems was used to derive a novel principle of

biology, the inverse relationship between genetic diversity and epigenetic complexity. This

principle or its logical deduction, the maximum genetic diversity hypothesis, was found to

explain a large set of biological data, including both for and against the correlation between

genetic distance and time of divergence.

An intuition in building complex systems/machines

It is a self-evident intuition that simpler systems/machines can tolerate more

variations/choices in building blocks. The more complex the system, the more restriction would

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be placed on the choice of building blocks. A one-story house can be build by all varieties of

bricks but only the stronger ones among them can qualify for a 100-story building because the

weaker ones cannot withstand the weight of a 100-story building. The number of choices of

different materials for constructing a toy bicycle is much greater than that for a space shuttle.

Inverse relationship between genetic diversity and epigenetic complexity

The building blocks for biological organisms are DNAs. The complexity of organisms is

reflected by the ways a set of DNAs is used to make a cell or an organism with multiple distinct

cell types. The more the cell types, the more the number of ways of using the same set of

DNAs, and the more complex the organism. Phenotypes are determined by the primary

sequence of DNAs or genotypes as well as by the ways by which DNAs are used or expressed,

often termed epigenotypes or epigenetic programs. Each cell type represents a distinct

epigenetic program of the same genotype. Cell types with distinct functions differ only in

epigenotypes but not in genotypes (a small number of special cell types such as antibody

producing cells are exceptions).

From the self-evident intuition of building complex machines, it is easy to deduce an

equivalent principle in constructing biological organisms. Thus, simpler organisms with low

epigenetic complexity can tolerate more variations in DNAs or have higher genetic diversity.

Genetic diversity is defined here as genetic distance or dissimilarity in DNA or protein

sequences between different individuals or species. Simple organisms are built more by the

primary function of a gene rather than by a specific expression pattern of the gene. A gene may

only have one expression pattern in simple organisms and many variants of the gene may be

able to fit within that one expression pattern. In contrast, when an organism is built by multiple

distinct gene expression patterns or cell types, the variation in gene sequence would be

necessarily restricted.

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The reason is easy to understand. If cell type A is determined by expression pattern X

and cell type B by pattern Y of the same gene, a mutational variant of the gene must be

compatible with both expression pattern X and pattern Y. Such multilevel compatibility reduces

the number of variants of the gene that can meet the multiple requirements. If ten mutational

variants can fit with expression pattern X, then may be only three of the ten would fit with both

patterns X and Y. The more expression patterns or cell types or functional pathways/networks a

gene is involved with, the more restriction would be placed on the number of variants of the

gene. Genetic diversity is restricted by epigenetic complexity and vice versa. It is impossible to

build complex epigenetic programs if the DNAs are constantly changing. To compensate for the

loss in the range of genetic diversity, complex organisms use different epigenetic programming

of the same gene set, in addition to mutation, to adapt to environments and to evolve new

phenotypes. Fish and human share nearly identical gene sets and the evolution from fish to

human is in a large part a process of epigenetic programming, analogous to writing distinct

books with the same set of vocabulary.

Complex organisms and epigenetic programs

Epigenetic programs are not only inherited during mitotic cell division but are also

transmitted through the germline to the next generation (Cropley et al., 2006; Hitchins et al.,

2007). They control both expression levels of genes and the specific combination of co-

expressed genes within a specific cell type. The epigenetic programs are here broadly defined,

including both the primary epigenetic proteins as well as those secondary or tertiary proteins

that could regulate the primary proteins. The number of human genes is only about 1.6 fold

more than that of a fruit fly and about the same as the mouse or fish. However, the number of

certain enzymes responsible for epigenetic gene organization, the PRDM subfamily of histone

methyltransferases, increases dramatically during metazoan evolution: 0 in bacteria, yeasts,

and plants; 2 in worms, 3 in insects; 7 in sea urchins, 15 in fishes, 16 in rodents, and 17 in

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primates (Fumasoni et al., 2007; Huang, 2002). This faster pace of expansion of certain

epigenetic enzymes, relative to the pace for the genome, in complex metazoan indicates a

correlation between complex epigenetic programs and complex organisms.

Complex organisms are here defined as those that have complex epigenetic programs.

Whether an organism is more complex than another organism can be roughly estimated based

on a comparison of the number of genes involved in epigenetic programs. This is informative to

differentiate unicellular organisms: yeasts have more epigenetic enzymes than bacteria and are

therefore more complex; yeasts have several histone acetylases and SET domain histone

methyltransferases while bacteria have none. Based on the number of the PRDM family of

epigenetic enzymes, it is also easy to conclude that vertebrates are more complex in epigenetic

programs than invertebrates or that primates are more complex than rodents or fishes.

When the numbers of epigenetic enzymes are similar for some multicellular organisms,

then the number of tissue or cell types is a good measure of epigenetic complexity since each

tissue or cell type is representative of a distinct epigenetic program or gene expression pattern.

The more tissue types an organism has, the more the number of distinct epigenetic programs

and hence the more complex the epigenetic program. The exact number of tissue types for any

complex organism remains unknown, largely because there are many more neuronal cell types

than we can presently recognize (Stevens, 1998). But this may not prevent one from drawing

the conclusion that organisms that appeared early in evolution generally have less number of

cell types than their descendant but distinctly different organisms that appeared later.

The number of neuronal cell types likely represents a major proportion of the total

number of cell types in a complex animal. Also, epigenetic programs may control the complex

interaction and organization of these neuronal cell types that manifest as intelligent brain

functions. Thus, organisms with complex and intelligent brains are likely to contain more cell

types or more complex interaction and organization of neuronal cell types. It is therefore easy to

infer that the first primate has more cell types or complex organizations than the first mammal

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which has more cell types or complex organizations than the first vertebrate. Also, animals that

go through complex and prolonged developmental process contain more complex epigenetic

programs since the development from a fertilized egg to an adult organism is largely an

epigenetic process. The same tissue type often exhibits different expression patterns or

epigenetic programs at different stages of development.

Organisms with the most complex and advanced brain (but not necessarily the largest in

volume) are necessarily more complex in epigenetic programs or have more varieties of

neuronal cell types and more complex interactions. Humans obviously have more distinct cell

types and more complex neuronal interactions, thanks to our complex brain, than any other

species that ever lived and are necessarily the most complex and diversified in epigenetic

programs. Human brain shows dramatically more methylated DNAs than chimpanzees (Enard

et al., 2004).

Epigenetic restriction of genetic diversity

Research on epigenetic programs is still at its infancy. Based on the limited knowledge

of today, we can still envision several ways by which epigenetic programs may restrict genetic

diversity. First, most genes are needed for the proper functioning of multiple fetal and adult

tissues. A germline mutation in these genes needs to be compatible with multiple tissue types.

Thus, the number of viable mutant variants is limited by the number of tissue types with which

the gene is involved.

Second, some genes are only expressed in one tissue type, such as hemoglobin in red

blood cells. These genes however still exhibit different expression patterns at different time

points during development. The gene expression pattern of fetal red blood cells is different from

adult red blood cells. So these genes still need to be compatible with several different

developmental gene expression patterns. Furthermore, they need to be repressed in most cell

types during development and during normal adult life. They need to be packaged into a

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chromatin state that silences gene expression. Some mutant variants may interfere with such

chromatin mediated repression and would be negatively selected.

Third, some genes are expressed in only one cell type but the function of the gene is

needed for most cell types of an organism. The function of hemoglobin is needed for the oxygen

supply of every cell type. Also, many house keeping genes such as actin are needed for most

cell types. Such general function of a protein like hemoglobin and actin may be fine-tuned for

the need of multiple tissues. A house keeping gene may also exhibit new functions or

connections with new networks in complex organisms that are absent in simple organisms, such

as the apoptosis function of cytochrome c. Also, for a complex organism to evolve a new cell

type, it is necessary to keep the house keeping genes unchanged so that new cell types can

evolve with the least amount of unnecessary disruption to existing cell types. It may not matter

much as to which specific version of a house keeping gene is used but it is important to stick

with one once it is selected by an organism.

Fourth, the coding region of every gene in complex organisms encodes not only amino

acids but also epigenetic information such as the nucleosome code (Segal et al., 2006). A

nucleosome code allows the nucleosome to locate in the right position in the genome. A silent

mutation may nevertheless affect the nucleosome code and alters the chromatin packaging

state of the gene, which may affect either gene repression or activation.

Fifth, complex organisms can eliminate reproductive cells carrying severe mutations

(Fan et al., 2008). Also, embryos of complex organisms may die before birth if they did not

develop properly due to mutations.

Sixth, epigenetic enzymes execute a senescence response to oncogenic mutations, thus

nullifying the harmful effects of such mutations (Braig et al., 2005).

Finally, the non-coding and non-expressed regions of the genome are nevertheless

packaged into chromatin and encode the nucleosome code and other information necessary for

gene expression and organization, and are therefore not free from epigenetic restrictions. Many

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epigenetic proteins interact with the genome in a sequence specific fashion such as the PRDM

family that contains DNA-binding zinc-finger motifs (Huang, 2002). Even when an epigenetic

enzyme has no intrinsic DNA binding property, it nevertheless interacts with a DNA binding

transcription factor and therefore requires a specific DNA motif to function as either coactivators

or corepressors (Rosenfeld et al., 2006).

The maximum genetic diversity (MGD) hypothesis

The inverse relationship between genetic diversity and epigenetic complexity is logically

and self-evidently true on its own, just like the original intuition that triggered it. It in turn

logically deduces what may be termed the maximum genetic diversity (MGD) hypothesis. The

hypothesis has three themes. First, empirical facts of evolution show both macroevolution and

microevolution (Figure 1). Macroevolution involves major advances in epigenetic complexity.

The overall direction towards higher complexity however does not necessarily exclude

occasionally going in the opposite direction. An organism is more complex if it has a higher

degree of epigenetic complexity as indicated by its number of cell types or its number of

epigenetic enzymes. Unlike macroevolution, microevolution is a gradual process of

accumulating mutations due to either drift or selection as described by a watered down version

of the molecular clock hypothesis or the `Neutral Theory' and the Neo-Darwinian selection

hypothesis. It may also involve some low degree of stochastic epigenetic reprogramming

without a significant net change in epigenetic complexity.

Second, complex organisms are constructed more by epigenetic programs relative to

simple organisms and are in turn inherently less tolerant of mutations. The maximum genetic

diversity allowed for a complex organism is smaller than that allowed for a simple organism.

The notion that genetic distance is roughly a function of time and mutation rates only applies to

diverging organisms of similar complexity over short time scales prior to reaching the maximum

cap. Most of the shared residues between two species are due to shared functions and

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epigenetic complexity. A small fraction of the shared residues may be due to common

adaptation to a common environmental selection that may vary from time to time (Figure 2). For

distinctly different kinds of organisms, their genetic distance is independent of mutation rates

and time but is determined by the maximum genetic diversity of the simpler organism. The

gradual increase in epigenetic complexity with time during macroevolution of distinct organisms

results in the linear correlation between maximum genetic distance and time of species

divergence. Such a correlation holds only for macroevolution and is not related to mutation

rates. It is fundamentally different from the correlation between genetic distance (prior to

reaching maximum) and time of divergence during microevolution in short time scales. Actual

mutation rates are usually fast enough for maximum genetic distance to be reachable in

evolutionary time.

Finally, while both micro- and macro-evolution involve gradual accumulation of mutations

and minor variations in epigenetic complexity, macroevolution from simple to complex

organisms is associated with a punctuational increase in epigenetic complexity and in turn a

punctuational loss in genetic diversity (Figure 2 and 3). From a common ancestor, the genetic

distance between two splitting descendants may gradually increase with time until reaching a

maximum level. This maximum genetic distance will stay roughly unchanged with time

thereafter (Figure 3). Mutations still occur but only affect saturated sites or sites that suffer

repeated hits. For microevolution, no major changes in epigenetic complexity will take place in

either of the two splitting species. For macroevolution, one of the two splitting organisms will

undergo a sudden increase in epigenetic complexity. This may take place soon after the two

splitting organisms have reached their maximum genetic distance. The sudden increase in

epigenetic complexity may be a response to the inadequacy of mutation alone in adapting to

new environmental challenges. This punctuational jump in epigenetic complexity forces the

genetic diversity of the new species to be lower than its sister species that remains largely

unchanged in epigenetic complexity. This in turn causes the genetic distance between the new

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species and its simpler sister species to be strictly determined by the maximum genetic diversity

of the sister species.

The maximum genetic diversity hypothesis explains numerous facts

A large number of well established but puzzling observations can now be easily

explained by the MGD hypothesis and a selected few are shown in the following to further

illustrate the hypothesis. In addition, a few novel facts have been uncovered that would

represent confirmations of the predictions of the hypothesis. None of these observations are

needed to invoke the hypothesis in the first place, since the hypothesis was deduced from

intuition. Therefore, all of them can be considered as independent lines of evidence in support

of the hypothesis.

1. Relationship between genetic diversity and time of origin. It is well established that

genetic diversity within a biological kind of old lineage is greater than that within a biological kind

of young lineage (Figure 4A). The genetic diversity of bacteria is greater than eukaryotes

(Ciccarelli et al., 2006). The fact that simple organisms with inherently high-level tolerance of

genetic diversity evolved earlier in history generates the apparent correlation between the time

of origin and genetic diversity (Figure 4A). But an equally valid relationship is between the time

of origin and the epigenetic complexity of the organism (Figure 4B). If epigenetic complexity

sets up a maximum cap on genetic diversity and if simple organisms appeared earlier than

complex organisms, then the apparent correlation between time of origin and genetic diversity

can be explained as an epiphenomenon of epigenetic complexity that is largely independent of

mutation rates, generation times, and population size.

2. The MGD hypothesis predicts the genetic equidistance result. The maximum diversity

allowed for an organism X is the same as the maximum genetic distance between X and all

descendants of X. The equidistance from X shared by all different descendants of X is strictly

determined by the epigenetic constraints imposed on X but is not linked to the more severe

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epigenetic constraints imposed on the descendants of X. This notion is illustrated by a

hypothetical case as shown in Table 1. If fish is allowed a maximum diversity of 60% difference

in a hypothetical protein sequence of 10 amino acids as shown in Table 1, then fish 1 would

differ from a maximum diverged fish 2 in 6 of the 10 amino acid positions. All evolutionary

descendants of fish, whether a different subspecies of fish or an amphibian or a human, would

all have the same maximum genetic distance with an extant fish that is equivalent to the

maximum diversity of 60% allowed for fish (Table 1).

If amphibian is allowed a maximum diversity of 40% difference, which is lower than fish

because amphibian is more complex, all evolutionary descendants of amphibian, whether a

different subspecies of amphibian or a mouse or a human, would all have the same genetic

distance from an extant amphibian that is equivalent to the maximum diversity of 40% allowed

for amphibian (Table 1). But the epigenetic constraint on amphibian has no effect on the

distance between amphibian and fish, which is strictly a result of the epigenetic constraint on

fish. The epigenetic constraint on amphibian only affects or determines the equidistance to

amphibian shared by all different descendants of amphibian. All fish descendants that do not

look like fish can be viewed as maximum diverged fishes and should show approximately the

same maximum distance with an extant fish that is the same as the maximum diversity allowed

for fishes.

It is well known that sequence regions conserved in simple organisms are often also

conserved in complex organisms. Sequence regions not conserved in complex organisms are

also often not conserved in simple organisms. This explains the fact as illustrated in Table 1

that a comparison of fish (with a hypothetical maximum diversity of 60%) and human (with a

hypothetical maximum diversity of 10%) should result in a dissimilarity of 60% equaling the

maximum diversity of fish, rather than 70%.

This notion that the maximum genetic diversity of a simple kind of organism determines

and is about the same as the maximum distance between the simple organism and the later

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appearing complex organisms can be illustrated by the example of cytochrome c. The

maximum diversity in this protein sequence is about 70% difference within bacteria, for

example, between Bordetella parapertussis and Paracoccus Versutus. The maximum distance

between bacteria and mammals is about 65% difference, such as between Bordetella

parapertussis and Pan troglodytes (chimpanzees). Within fungi, the maximum diversity is about

40% difference, for example, between Aspergillus oryzae and

Yarrowia lipolytica. The

maximum distance between fungi and mammals is about 43% difference, such as between

Aspergillus oryzae and Pan troglodytes. Within arthropods, the maximum diversity is about 24%

difference, for example, between Drosophila melanogaster and Tigriopus californicus. The

maximum distance between arthropods and mammals is about 25% difference, such as

between Drosophila melanogaster and Pan troglodytes.

3. The relationship between time and genetic distance in microevolution is different from

that in macroevolution. Most genes (about 90%) have been found to behave consistently as

good clocks in macroevolution, and show the same pattern as originally found for cytochrome c

(Fitch and Margoliash, 1967; Margoliash, 1963): human is more related to primates, less to

rodents, still less to birds, still less to frogs, and still less to fish (e.g., see Table 2). However,

despite their consistent pattern in macroevolution, many genes give erratic or contradictory

results when the timing of split in microevolution is measured. For example, pufferfish (Takifugu

rubripes) and zebrafish (Danio rerio) are believed to have diverged not more than 140-200

MyBP (million years before present) based on the first fossil evidence of teleostei in the early

Cretaceous period (Powers, 1991). If the situation between the two fishes is similar to what one

originally found for cytochrome c in macroevolution, one would expect 90% of all genes to show

more identity between the fishes than between human and bird since the time of divergence for

human and bird is much earlier (310 MyBP).

In a survey of 40 randomly picked genes, I found 36 (90%) that show the expected

macroevolution pattern where human is more related to bird, less to frog, and still less to fish. In

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contrast, only 19 (48%) show more identity between the two fishes than between human and

bird. Depending on which gene is used as clock, the time of divergence between the two fishes

would vary from 91 to 420 million years (Table 2). In fact, I employed the molecular clock

method to derive an average time of divergence using these 40 genes by calibrating against the

fossil divergence time between human and bird (310 MyBP). However, I obtained an obviously

incorrect time (417+/-172 MyBP) that is more than two fold greater than the actual time as

indicated by the fossil record. As a positive control to show that my method is similar to those of

others, I derived a mean time of divergence between human and amphibians and found it to be

similar to that obtained by others (Kumar and Hedges, 1998).

Apparently, some of the subspecies split or microevolution is not equivalent to the

changes in macroevolution, but the Neo-Darwinian hypothesis treats them the same. In

contrast, the MGD hypothesis considers them to be very different in evolutionary dynamics. So,

clocks derived from macroevolution should not be expected to work also for microevolution.

Genetic distance between two distinct species of macroevolution always reflects the maximum

genetic distance. However, genetic distance between two similar species that have diverged

more recently would gradually increase as a function of time before they reach the maximum

(Figure 2). Different genes would diverge according to different mutation rates. If the time is not

enough for all genes to reach the maximum diversity level, some genes may reach a diversity

level closer to the maximum than some other genes. The genes in fish are allowed a maximum

diversity level greater than genes in birds and humans. So if some genes reached a diversity

level closer to the maximum, they would put the time of split between the two fishes earlier than

that between birds and humans. But some other genes may only reach a certain diversity level

much lower than the maximum because of slower rate of mutations and insufficient time. These

genes would put the time of split between the fishes later than that between birds and humans.

4. Radiation of mammals and the Cambrian explosion. The two main areas of

disagreement between molecular clocks and the animal fossil record concern the radiation of

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mammal orders around the Cretaceous-Tertiary boundary (65 MyBP) and animal phyla at the

Cambrian explosion 520 MyBP (Hedges, 2002). In each case, molecular clocks show much

deeper divergence. The MGD hypothesis suggests that the rates of change in genetic distance

for macroevolution are determined by epigenetic complexity. They tend to be slower than the

actual mutation rates. If these slower rates are used to date microevolution in the horizontal

direction, we would expect to see a deeper time of divergence than the actual time, as we have

already seen above for the two fishes. Some mammalian radiation events involve varieties

within a kind, such as the split between mouse and rat, and may represent microevolution in the

horizontal direction. They may accumulate mutations faster than the slower rate observed for

macroevolution. This would cause the time of split between mouse and rat to be older than the

actual time: 23-41 million years from the molecular clock estimate versus 10-12 million years

from the fossil record (Hedges, 2002). Some mammalian radiation events may involve a major

change in kinds and represent macroevolution, such as the split between primates and rodents.

They may involve a higher than average rate of change in epigenetic complexity and in turn in

maximum genetic distance. In this case, if the average rate of change in epigenetic complexity

is applied, it would give a deeper time of divergence than the actual time.

The rate of change in epigenetic programs between phyla may be much greater than

that between different species within one phyla. For example, vertebrates have a much greater

number of PRDM epigenetic enzymes than arthropods (Huang, 2002). But the number of

PRDM genes among different species of vertebrates is similar. The rate of change in epigenetic

programs in macroevolution within the vertebrate phyla may be slower than that between phyla

or between arthropods and vertebrates. So when the slow rate estimated from speciation events

within one phyla, that of vertebrate, is used to calibrate the time of phyla divergence between

arthropods and vertebrates, the time would be estimated to be deeper than the actual time

(1000 MyBP versus 520 MyBP) (Hedges, 2002).

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5. Simpler organisms show higher genetic diversity than complex organisms after

evolving for the same amount of time. The MGD hypothesis predicts that simple organisms

should show higher genetic diversity than complex organisms after the same amount of time of

evolution. Indeed, flowering plants have much greater genetic diversity than mammals even

though they have both coevolved for similar amount of time (Huang, 2008a). Flowering plants

are less complex in epigenetic programs and have zero PRDM family of epigenetic enzymes

while mammals have 16 to 17. It is also obvious that flowering plants have less number of cell

types than mammals. The genetic diversity of flowering plants after less than 125 million years

of evolution is about equivalent to that reached by vertebrates after 450 million years of

evolution. So the hypothesis explains equally well both data for and against the correlation

between genetic diversity and time of divergence.

6. Direct evidence of maximum genetic diversity. The MGD hypothesis predicts that the

genetic distance between some ancient species of similar kind or epigenetic complexity may

have reached a maximum cap long before present. I tested this prediction for the fungi kingdom.

The baker's yeast Saccharomyces cerevisiae belongs to the Ascomycota phylum, the

Saccharomycotina subphylum, the Saccharomycetes class, the Saccharomycetales order, the

Saccharomycetaceae family, and the Saccharomyces genus. A large number of observations

have established the well-known top-down direction of evolution where the major pulse of

divergence of phyla occurs before subphyla or classes, classes before that of order, orders

before that of families, and families before that of genera. If many fungi may share similar

epigenetic complexity, the MGD hypothesis predicts that, if time is long enough for genetic

distance to reach the cap, the maximum genetic distance between two fungi genera of the same

family should be similar to that between two fungi families, or orders, or phyla. In contrast, the

molecular clock hypothesis predicts that the genetic distance between two fungi genera of the

same family should be smaller than that between families, still smaller than that between orders,

still smaller than that between classes or subphyla, and still smaller than that between phyla.

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I randomly picked three proteins for analysis, Pin1, CytC (cytochrome c), and CMD

(Calmodulin). As shown in Table 3, the protein sequence identity in Pin1 between two distant

genera (S. cerevisiae/Saccharomyces and D. hansenii/Debaryomyces) of the same family is

44%, which is about the same as that between two families of the same order (39% between S.

cerevisiae/Saccharomycetaceae and Y. lipolytica/Dipodascaceae), or about the same as that

between two subphyla of the same phylum (42% between S. cerevisiae/ Saccharomycotina and

G. zeae PH-1/Pezizomycotina), or about the same as that between two phyla of the same

kingdom (41% between S. cerevisiae/Ascomycota and C. Neo-formans/Basidiomycota). For the

protein CytC, the identity between two distant genera (78% identity) seems to be larger than

that between two distant families (73% identity) which is still larger than that between two distant

subphyla (67% identity) which is still larger than that between two distant phyla (60% identity)

(Table 3). This pattern is consistent with the top down direction of evolution and suggests that

the time may not yet be long enough for the genetic distance in CytC among the presently

sequenced fungi taxa to reach the maximum cap, consistent with the known slow mutation rate

of the CytC protein. For the protein CMD, genetic distance between taxa above the family level

appears to have reached a maximum at 56-60% identity. These data show that there is a

maximum cap on genetic distance at some faster mutating loci like Pin1 and CMD between two

species of similar kind in the fungi kingdom. The cap may be gradually reached by gradual

accumulation of mutations within a certain amount of time.

I also found direct evidence of maximum cap in fishes. Zebrafish (D. rerio) and

pufferfish (T. nigroviridis) diverged not more than 140-200 MyBP ago as mentioned above. If

they diverged by the gradual model and if time is long enough for at least some genes to reach

the maximum genetic distance, the MGD hypothesis predicts that some genes would show a

genetic distance between the two fishes that is similar to the maximum genetic diversity allowed

for fishes. The maximum genetic diversity of fishes is of course roughly the same as the genetic

distance between fishes and a distinct fish descendant such as a mammal. I examined a large

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number of chromatin modifying enzymes and found 13 out of 32 with a distance between the

fishes to be the same or slightly greater than the distance between a fish and a mammal (Table

4). The SET domain family of histone lysine methyltransferases (KMTs) is specifically more

enriched with genes that evolved fast with 6 out of 9 genes analyzed reaching maximum cap in

the fishes. This feature of the KMT family is significantly different from a slowly evolving family

such as ribosomal proteins with only 2 of 12 proteins analyzed reaching maximum cap in the

fishes (P< 0.05, Fisher's exact test, two tailed). Not a single gene was found to have

significantly greater distance between the two fishes than between fish and mammal, indicating

clearly the existence of a cap on genetic distance.

7. Actual mutation rate in real time is faster than that calculated from phylogenetic

analysis. It is well known that mutation rate from pedigree analysis on genealogical timescales

is often an order of magnitude or more greater than mutation rate from phylogenetic analysis

over geological time (Ho and Larson, 2006). Thus, phylogenetic diversity or distance over

geological time is uncoupled from actual mutation rate observed on genealogical timescales. It

suggests that actual mutation rates are often fast enough for most organisms to reach a

maximum cap in genetic distance over geological timescale. Indeed, if actual mutation rates are

slower than those from phylogenetic analysis, it would falsify the MGD hypothesis.

The phylogenetic diversity or distance reflects the maximum diversity allowed for an

organism. Some of the variants at a particular time period accumulated as a result of random

mutations may not persist long over geological time and may have to be replaced by another set

of variants at a later time period (Figure 2). Maximum genetic distance between two species

would stay constant over time while the same genetic distance may be maintained by different

sets of variants at different times (Figure 2). A set of variants best suited for life at one time may

not be the best at a different time and would have to be replaced.

8. Stasis and punctuation in the fossil record. The MGD hypothesis suggests that

morphological phenotypes for complex organisms are better correlated with epigenotypes.

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Advances in epigenotypes in macroevolution occur largely via punctuation (Figure 2). Such

punctuation events are followed by stasis in epigenotypes in microevolution. Thus the

hypothesis predicts both stasis and punctuation at the level of epigenotypes and in turn at

morphological levels. Consistently, the fossil record shows both stasis and punctuation at

morphological levels (Gould and Eldredge, 1993).

9. Cancer as a disease of both genetics and epigenetics. The MGD hypothesis predicts

that high epigenetic complexity has a way of limiting the incidence of mutations. A relaxation in

epigenetic control may be expected to allow more mutations to occur. Indeed, human cancer

provides a good illustration of this prediction. Mutations are common in cancer. Epigenetic

programs are often deregulated in cancer and methylation deficiency is a hallmark of cancer

(Feinberg and Tycko, 2004; Huang, 2002). Loss of epigenetic control as indicated by loss of

DNA methylation occurs during aging and precedes mutations in cancer (Suzuki et al., 2006). A

rate-limiting step in carcinogenesis by major environmental factors such as nutrient-imbalanced

diet is the deregulation of an epigenetic enzyme RIZ1/PRDM2 (Zhou et al., 2008). In addition,

the hypothesis predicts that high genetic diversity or too many mutations would interfere with

epigenetic programming. Indeed, too many mutations, either germ line or somatic, are well

known to cause cancer, which is essentially a disease where the normal epigenetic programs

have been replaced by a cancer specific program. Thus, the hypothesis unifies cancer genetics

and epigenetics and explains why cancer appears to be a disease of both genetic mutations

and epigenetic anomalies.

10. Copy number variations of the genome. Advances in epigenetic complexity may

involve changes that affect large regions of the genome, such as amplification or deletion of

long stretches of DNA. Thus, such copy number changes may be expected to be a common

behavior of the genome just like point mutations are. Indeed, copy number variations are

observed to be common in the human genome (Redon et al., 2006). Within a specific level of

epigenetic complexity, a certain range of neutral and random copy number changes are allowed

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that may affect slightly epigenetic programs, just like a certain range of random point mutations

are allowed. Relaxation of epigenetic programs is expected to allow more abnormal copy

number changes to occur. Indeed, cancer is commonly caused by loss of epigenetic control

and often exhibits aneuploidy and amplifications or deletions of long stretch of DNA.

11. Genetic diseases. The prevalence of genetic or familial diseases in humans

indicates plainly that a large portion of genetic diversity, i.e. those represented by those disease

mutations, cannot become a part of the normal range of genetic diversity among humans. Most

genetic diseases affect only a tiny population of humans. Just imagine how much more

diversified the human race would be if all those rare disease mutations would become fixed in

the whole population. If mutations in the retinoblastoma gene, a cell cycle regulator important

for many different cell types, do not cause cancer in the retina of children, the diversity in the

retinoblastoma gene locus would be greatly expanded. The fact of rare disease mutations in

humans is sufficient to prove the hypothesis that there is an upper limit to the amount of genetic

diversity in an organism. The fact that those rare disease mutations are mostly tissue specific is

consistent with the notion that the upper limit is set up by the complexity of epigenetic programs.

If humans lack the retina cell type or the retina specific epigenetic program, most of the

mutations in the retinoblastoma gene would have been tolerated as normal variations and the

genetic diversity of humans would have been in turn expanded. Also, numerous disease alleles

in humans correspond to normal alleles in rhesus macaques (Gibbs et al., 2007). Thus, many

alleles or mutant variants that can be tolerated in a less complex organism in fact cause

diseases in humans.

12. Anomalies of the genetic equidistance result. A small number of genes show

anomalies and are routinely excluded from phylogenetic analysis based on the molecular clock

hypothesis. An example is the mitochondrial protein ND6. My analysis showed that all

vertebrates ND6 proteins are equidistant to the outgroup sea urchin but fishes ND6 proteins are

closer to frogs than to mammals. The molecular clock hypothesis has no explanation for such a

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gene that shows both equidistance as well as non-equidistance. However, the MGD hypothesis

easily explains it. Some of the shared sequences are due to common environmental selections

(Figure 2). Fishes may have more in common with frogs than with mammals in their adaptation

strategies for the ND6 protein.

13. Inverse correlation between genome size and genetic diversity. Large size

genomes (measured here as number of genes) require more complex epigenetic regulation

than small genomes and are expected to show less genetic diversity. Indeed, there is a strong

inverse correlation between genome size and genetic diversity (Ciccarelli et al., 2006). Genetic

diversity is more responsive to changes in genome size in bacteria than in eukaryotes,

indicating that genetic diversity is restricted more by epigenetic complexity than by genome size

in eukaryotes.

In microbes, there is an inverse relationship between genome size and mutation rate per

base pair per replication (Drake et al., 1998). In four metazoans analyzed, the mutation rate per

base pair per replication is lowest for humans, higher for mice, and still higher for drosophila or

worm. These data are expected from the MGD hypothesis.

14. No bacterium lineage could be identified as the closest relative of eukaryotes.

Based on the overall trend in evolution from simple to complex organisms and the earliest fossil

evidence of life on Earth, it is almost certain that bacteria were the ancestors of the eukaryotes.

However, the MGD hypothesis predicts that no single bacterium lineage could be identified

among bacteria as the closest relative of eukaryotes. Such a lineage, if indeed exists, would

have long reached maximum diversity and would show equidistance to eukaryotes as other

bacteria. In contrast, if there is no maximum cap on diversity or if time is not long enough yet

and if the Neo-Darwinian hypothesis is true, one should be able to identify the bacterium lineage

that is closer to eukaryotes than most other bacteria. But extensive studies show that no such

bacterium lineage can be identified. Recent data show that the identification of archaea as

closer to eukaryotes is only true for some class of genes such as those involved in translation

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(Pennisi, 1998). For many other genes, archaea are in fact more distant to eukaryotes than

eubacteria. The overall pattern of genetic similarity suggests that common selection and

coincidence may account for most of the sequence identities between eukaryotes and bacteria.

The closer relationship between a bacterium species and eukaryotes in some genes but

not others has been commonly interpreted to mean horizontal gene transfer, even though there

is little independent evidence for it. It is more likely however that the closer relationship are

fortuitous due simply to the fact that bacteria have much greater genetic diversity and some

gene variants of bacteria would by chance resemble an eukaryotic version. If one compares a

gene from a mammalian species against orthologous genes of all species of bacteria in the

Genbank, one would find that the degree of similarity would vary to a great extant (e.g., for

GLUD1, the identity between human and all bacteria ranges from 30% to 50%). In contrast, if

one compares a gene from an individual bacterium species against all vertebrate species in the

Genbank, one would find that the degree of similarity falls within a very narrow range (e.g., for

GLUD1, the identity between the bacterium Pedobacter sp. BAL39 and all vertebrates ranges

from 47% to 53%). Vertebrates have lower genetic diversity and there is much less probability

for a variant of vertebrates to be much more closely related by chance than other variants to an

individual variant of bacteria.

There are data against the idea of horizontal gene transfer. If a gene was transferred

from a prokaryotic lineage into the vertebrate lineage, this likely occurred within the past 400 to

500 million years, after most of the major prokaryotic phyla were established. Therefore, any

transferred gene should be more closely related to its donor lineage than to any other

prokaryotic lineage, which would be detectable in phylogenetic trees. However, it was found

that most of the genes shared between vertebrates and bacteria did not show patterns

consistent with bacterial to vertebrate gene transfer (Salzberg et al., 2001).

15. Ubiquitously expressed genes have lower genetic diversity than tissue specific

genes. The MGD hypothesis predicts that ubiquitously expressed genes have lower diversity

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than tissue specific genes due to selection against mutations that cannot fit with multiple cell

types. Indeed, an analysis of 2400 genes between human and rodent found that ubiquitously

expressed proteins have average genetic distances between human and rodent that were

threefold lower than those of tissue-specific genes (Duret and Mouchiroud, 2000).

16. Stability of epigenetic programs. It is well known that artificial selection or breeding

of animals can only generate varieties of the same type but never of a different type. This fact

plainly indicates that genetic variation within an organism is not without a limit. The epigenetic

program that allows a genome to manifest a dog phenotype also prevents the same genome

from randomly drifting into something that is not allowed by the epigenetic program. Indeed,

random drifting is far more likely to give rise to cancer rather than a novel functional organ. If

genotypes can be rather unstable or easily influenced by random mutations, the epigenotypes

are relatively much more stable. Indeed, when cultured for up to ten years, hundreds of cell

divisions later, Drosophila wing disc cells can still give rise to adult wing structures (Hadorn,

1967). The stability of epigenotypes is also indicated by the stasis and extinction phenomena in

the fossil record. If environment becomes unsuitable for survival, a species would more often

than not go extinction rather than change itself in its basic epigenetic programs. In today's

world, all we observe is extinction of species rather than drastic transformation of species.

A specific epigenetic program allows a certain degree of variation in genotypes and in

turn a certain range of adaptive capability in response to environmental changes. When the

environmental changes exceed the adaptive capability allowed within a specific epigenetic

program, the organism would simply go extinct rather than change. Change is not without a

limit. Change is not the only feature of evolution. Equally important and obvious as change is

the opposite of change. If constant random change within a limited range in genotypes is a

hallmark of evolution, then long period of stasis and stability in epigenotypes followed by short

period of punctuational advance in epigenotypes is an equally important hallmark of evolution.

Indeed, the genetic code is the optimal code for error minimization or for minimizing the effects

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of random changes; it is the most stable of all possible codes and is optimal for stability rather

than for random changes (Freeland et al., 2000).

17. Low genetic diversity of chromosome X. Comparisons of genomes have shown a

lower rate of sequence divergence on chromosome X than the autosomes for many species

(Patterson et al., 2006). Chromosome X undergoes X inactivation in females, which is an

epigenetic event. Thus, genes located on X encounter more epigenetic restrictions than genes

on autosomes and are therefore expected from the hypothesis to be less tolerant of mutations.

This explanation is far more reasonable than the suggestion of interbreeding between humans

and chimpanzees (Patterson et al., 2006).

18. Human has the lowest genetic diversity. The genetic diversity within chimpanzees

is two to three times greater than within humans, even though both species are thought to have

evolved for the same amount of time since their most recent common ancestor (Becquet et al.,

2007). The striking fact that human shows the lowest DNA diversity among all species has

commonly been explained by the bottleneck hypothesis: most human populations are thought to

go extinct at one time in history except one small population that survived to produce the six

billion people living today. But there is no independent evidence of such near extinction event

and there is little hope of ever uncovering such evidence. There are also lines of evidence

against the bottleneck hypothesis (Li and Sadler, 1991; Xiong et al., 1991). In contrast, the

homogeneity of human DNA can be easily explained by the MGD hypothesis. The organism

with the most complex and diverse epigenetic programs or the most number of cell types is

necessarily supposed to have the lowest diversity in DNA.

Neanderthals appeared earlier than modern humans and have slightly larger brains. It is

unclear whether Neanderthals may be less intelligent or have less complex brain than modern

humans, which may explain their mysterious extinction. The MGD hypothesis predicts that

Neanderthals are less complex in epigenetic programs and have a less complex brain than

modern humans because Neanderthals appear to exhibit more DNA diversity (Krings et al.,

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2000; Orlando et al., 2006). This prediction is obviously consistent with the fact that it is modern

humans rather than the Neanderthals that dominate the Earth today. It is also consistent with

the evolution trend that less complex organisms appeared earlier in history.

19. Mammals are more distant to snakes than to birds. Mammals and reptiles

(including birds) were separated ~310 MyBP. Thus, all the reptiles (including birds) should be

equidistant to a mammal if the constant mutation rate idea is true. But the MGD hypothesis

predicts that simpler reptiles such as snakes, which lost limbs, should have higher genetic

diversity and hence be more distant to a mammal than complex reptiles such as birds. A

random sampling of five proteins indeed shows that snakes are more distant to humans than

birds are (Hemoglobin alpha chain, albumin, cytochrome b, PDGF, and ND6).

20. More recently evolved complex brachiopods are closer to mammals. The inarticulate

brachiopod genus Lingula (order Linguilida) is the oldest, relatively evolutionarily unchanged

animal known. The oldest Lingula fossils are found in Lower Cambrian rocks dating to roughly

550 MyBP. Terebratulids are modern articulate brachiopods and appeared later in evolution

around ~430 MyBP. The molecular clock hypothesis predicts that mammals should be

equidistant to Lingula and terebratulids. But the MGD hypothesis predicts that mammals should

be closer to terebratulids given that they evolved later and should have lower genetic diversity.

Indeed, a random sampling of several proteins showed that mammals are closer to terebratulids

than to Lingula (Cox1, Cox2, Cox3, ND1, and COB). Also, terebratulids are closer to mammals

than to a fellow brachiopod Lingula.

In contrast to the brachiopods, complex plants (flowering plants) that appeared later in

evolution and simpler plants (mosses) that appeared earlier are about equidistant to mammals

in several randomly analyzed genes (EF1a, Adh1a, EIF2b, Pin1, PP1, RPC1, and Cox1). The

identity between flowering plants and mosses are much greater than between mammals and

mosses, in contrast to brachopods where the distance between mammals and Lingula is similar

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to that between terebratulids and Lingula. Thus, plants have evolved plant-specific conserved

domains since separating from mammals but before divergence of mosses and flowering plants.

21. The genetic diversity of tuatara. The tuatara of New Zealand is a living fossil reptile

and has very slow metabolic and growth rates, long generation times and slow rates of

reproduction. Contrary to expectations from Neo-Darwinian theory, tuatara has high `mutation

rates', significantly higher than those of mammals measured in real time by the same method of

using mitochondrial D-loop DNA sequences from fossils of ~10,000 years old (Hay et al., 2008).

However, this result is to be expected from the MGD hypothesis since reptiles should have

higher maximum genetic diversity than mammals. If ~10,000 years is sufficient for reptiles and

mammals to reach maximum cap in genetic diversity in the D-loop region, then the reptiles

would show higher genetic diversity, resulting in the appearance of a higher `mutation rate'. But

in reality, tuatara can have slower mutation rate but still show higher genetic diversity than a

mammal if time is long enough for tuatara to reach the maximum cap or to be close to the cap.

22. Evidence from fossil DNA and protein sequences. Finally, the MGD hypothesis

explains the recent results that ancient fossil specimens are more distant to an outgroup than

extant sister species are and that ancient fossil specimens have greater genetic distance than

extant sister species (Huang, 2008b). The Neo-Darwinian gradual mutation hypothesis predicts

that ancient specimens of extinct species cannot be more distant to an outgroup than extant

sister species are (Figure 5A). Also, two distinct ancient specimens from different era cannot be

more distant than their extant sister species are. But the MGD hypothesis predicts the exact

opposite (Figure 5B). The recent analysis of fossil DNA and protein sequences fully conforms

to the predictions of the MGD hypothesis.

Implications for molecular phylogeny

Molecular phylogeny analysis aims to classify the time of divergence between

morphologically similar species that either do not have fossil records or cannot be clearly

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distinguished by fossil records. A mutation rate is usually calibrated using fossil records of

vertebrate macroevolution. The most commonly used calibration date is the divergence time of

310 million years between birds and mammals. However, as discussed here, the `mutation rate'

deduced from macroevolution is not the real mutation rate as it is known in real time

measurements. It therefore cannot be used to time microevolution of species that have

diverged only recently and have not yet reached a maximum cap in genetic diversity. Such

microevolution reflects the real mutation rate and should be timed using a mutation rate that is

measured by real time analysis such as pedigree analysis. For example, to date the divergence

of pufferfish and zebrafish, one should only use genes that have not reached a maximum

diversity. Thus, cytochrome c may be used but most KMTs should not be used. However, the

mutation rate of cytochrome c should be deduced not from divergence of birds and mammals

but from pedigree analysis of living pufferfish and zebrafish.

Based on mutation rates derived from pedigree analysis of the mitochondrial D-loop

region, the human race is estimated to be only ~ 6500 years old (Parsons et al., 1997).

However, what the result means is uncertain since the maximum genetic diversity of the

mitochondrial D-loop region is unknown for humans. If the genetic diversity has not yet reached

a cap within 6500 years, then we may conclude that the human race is indeed 6500 years old.

On the other hand, if the cap has been reached in 6500 years, then we will not be able to

discern the real age of human race using the mitochondrial D-loop DNA. The age could be

much older while the diversity of the D-loop DNA would no longer increase with time after

reaching the cap. Given that the oldest fossil of modern humans is much older than 6500 years

(about 30,000 to 65,000 years old), it is likely that the maximum diversity of the mitochondrial D-

loop can be reached in ~ 6500 years. Thus, if the fossil record is true, the maximum distance in

the D-loop that we observe today within the human race would in fact represent the maximum

allowable for the human organism.

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Conclusions:

The inverse relationship between genetic diversity and epigenetic complexity is self-

evident. It does not need independent validation of empirical facts, just like the intuition that a

complex system is more selective in building materials than a simple system. Nonetheless, it or

its necessary logical deduction, the maximum genetic diversity hypothesis, has found support in

numerous facts and has yet to meet a factual contradiction, as would be expected for any self-

evident logical truth. It explains more facts than does the molecular clock hypothesis and

represents the first testable or scientific explanation of the genetic equidistance result. Many

data that were simply ignored before can now be understood. Most of the existing literature of

molecular evolution would need to be re-interpreted in light of the new hypothesis. The

molecular clock hypothesis and the Neutral Theory cannot account for macroevolution and are

only relevant to some microevolution events over short timescales. The inference of divergence

time based on sequence identity is still practically useful in some cases. But a distinction must

be made between divergence that has reached a maximum and divergence that has not.

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Acknowledgments:

This work was supported by NIH (RO1 CA 105347). I thank Drs. Phil Skell and Klara

Briknarova for critical reading of the manuscript.

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Table 1. Genetic equidistance explained by the maximum genetic diversity hypothesis.

A hypothetical protein sequence of 10 amino acids is listed for each organism. Conserved

positions are represented by numbers. Positions that change from time to time are represented

by X. The hypothetical maximum diversity allowed for fish is 60%, for amphibian 40%, and for

human 10%. The maximum diversity of 60% for fish necessarily determines that all

descendants of fish, whether amphibian or human, would all have the same maximum distance

of 60% with fish that is identical to the maximum diversity allowed for fish.

Species

Sequence

Fish 1

0123xxxxxx

Fish 2

012326xxxx

Fish 3

012326xxxx

Amphibian 1

012334xxxx

Amphibian 2

01233424xx

Amphibian 3

01233424xx

Human 1

012334315x

Human 2

012334315x

Human 3

012334315x

Maximum diversity (percent difference)

Fish 1 vs. fish 2

Amphibian 1 vs. amphibian 2

Human 1 vs. human 2

Maximum distance (percent difference)

Human vs. amphibian

Human vs. fish

Amphibian vs. fish

Nature Precedings : hdl:10101/npre.2008.1751.1 : Posted 1 Apr 2008

Table 2. Molecular clocks give consistent timing for macroevolution but inconsistent

timing for microevolution. Percent identities between species are listed for four randomly

selected genes. All four genes behave as good clocks in macroevolution from fish (D. rerio) to

frog (X. laevis) to bird (G. gallus) to mouse (M. musculus) to human (H. sapiens), which is

consistent with the timing based on the fossil record as indicated for each divergence. In

contrast, they give wildly contradictory timing when used to time microevolution divergence

between pufferfish and zebrafish. The estimated time varies from 420 to 91 million years

depending on which of the four genes is used as clock. The mutation rate or clock rate of each

gene was derived from plotting the number of amino acid changes between protein sequences

against species age estimated from fossil evidence. MyBP, million years before present. N.A.,

gene sequence not available.

Percent identity

MyBP

Prdm2 BTK CytC GCA1A

H. sapiens v.s. D. rerio

450

H. sapiens v.s. X. laevis

N.A. 85

360

H. sapiens v.s. G. gallus

310

H. sapiens v.s. M. musculus

F. rubripes v.s. D. rerio

420

400

200

Nature Precedings : hdl:10101/npre.2008.1751.1 : Posted 1 Apr 2008

Table 3. Genetic distance among different species of fungi. Three proteins, Pin1, CytC,

and CMD from the baker's yeast were used to BLAST against the fungi database of NCBI .

Percent identities in protein sequence between species of different genus, families, subphyla,

and phyla are listed.

Percent identity

Pin1 CytC Cmd

Between genera within the same family Saccharomycetaceae

S. cerevisiae v.s. D. hansenii/Debaryomyces

S. cerevisiae v.s. E. gossypii/Eremothecium

S. cerevisiae v.s. K. lactis/Kluyveromyces

Between families within the same order Saccharomycetales

S. cerevisiae vs Y. lipolytica/Dipodascaceae

S. cerevisiae v.s. C. albicans/mitosporic Saccharomycetaceae

Between subphyla within the same phylum Ascomycota

S. cerevisiae v.s. G. zeae PH-1/Pezizomycotina

S. cerevisiae v.s. S. pombe/Schizosaccharomycetes

Between phyla within the same kingdom Fungi

S. cerevisiae vs. R. oryzae/Zygomycota

S. cerevisiae vs. C. Neo-formans/Basidiomycota

S. cerevisiae vs. C. cinerea/Basidiomycota

S. cerevisiae vs. U. maydis/Basidiomycota

S. cerevisiae vs. B. emersonii/Chytridiomycota

Nature Precedings : hdl:10101/npre.2008.1751.1 : Posted 1 Apr 2008

Table 4. The genetic distance between two fishes in many chromatin modifying enzymes

is similar to that between a fish and a mammal. The percent identity between zebrafish (D.

rerio) and pufferfish (T. nigroviridis), human (H. sapiens), or mouse (M. musculus) is shown for

a number of chromatin modifying epigenetic enzymes. Genes are considered as having

reached maximum distance in fishes if the distance between the two fishes is equal or slightly

greater than between a fish and a mammal.

(% identity)

D. rerio vs.

T. nigroviridis H. sapiens

M. musculus

Genes reached maximum distance
Suv39H1/KMT1A

Smyd2/KMT3C

SET7/9/KMT7

PRDM11

PRDM4

PRDM15

PRMT4

Lsd1/KDM1

Jarid1b/KDM5b

MYST1/KAT8

SIRT5

HDAC1

HDAC4

Genes not yet reached maximum distance
Suv4-20H1/KMT5B

EZH2/KMT6

PRDM2/KMT8

PRMT6

PRMT7

PRMT5

PRMT8

Jmjd2b/KDM4b

HAT1/KAT1

PCAF/KAT2B

CBP/KAT3A

MYST2/KAT7

Clock/KAT13D

SIRT3

SIRT4

SIRT6

SIRT7

HDAC3

HDAC8

Nature Precedings : hdl:10101/npre.2008.1751.1 : Posted 1 Apr 2008

Figure legends:

Figure 1. Macroevolution and microevolution. The vertical direction is macroevolution and

involves major changes in epigenetic complexity over time. The horizontal direction is

microevolution and involves changes in varieties within a specific level of epigenetic complexity.

The estimated number of species for each kind of organisms is indicated in parentheses. Time

is not to scale and in the direction from past to future.

Figure 2. Genetic distance between two splitting organisms at various times during

macroevolution. A 10 amino acid peptide with amino acids represented by numbers is shown

to illustrate the dissimilarity or genetic distance between the species at various times during

evolution. X represents amino acid positions that may change from time to time. A fraction of

these X residues may be shared in different organisms due to common external environments

that may differ from time to time. The ancestor organism A0 gives rise to two descendant

lineages that gradually accumulate genetic distance until reaching a maximum at time T1. At

this time, a punctuational jump in epigenetic complexity occurs in one of the lineages generating

B1. The descendant organism A1 remains phenotypically similar to the ancestor A0. The

lineage leading to B1 is phenotypically similar to A1 prior to the punctuational jump at time T1.

The epigenetic jump in B1 reduces the genetic diversity of B1, as indicated by the reduction in

the number of X positions.

Figure 3. The Neo-Darwinian hypothesis versus the maximum genetic diversity

hypothesis. (A) The Neo-Darwinian model of microevolution and macroevolution. Genetic

distance increases with time with no maximum cap. Fish and amphibians are used as

examples. The transition from fish to amphibian is indicated by the dashed line. The starting

point of the dashed line represents the time when amphibian epigenotype or phenotype first

Nature Precedings : hdl:10101/npre.2008.1751.1 : Posted 1 Apr 2008

became obviously distinct from that of fish. (B) Model of microevolution and macroevolution

based on the MGD hypothesis.

Figure 4. Inverse relationship between genetic diversity and epigenetic complexity. (A)

Maximum genetic diversity within each type of organisms in cytochrome c correlates with the

time since the first appearance of each type. The percent amino acid change in cytochrome c

within each type of organism was obtained by BLAST against protein database at the National

Center for Biotechnology Information. Similar data was first reported in the 1960s (Fitch and

Margoliash, 1967; Margoliash, 1963). (B) High epigenetic complexity as measured by the

number of cell types per organism inversely correlates with genetic diversity and the time since

the first appearance of each organism. The first eukaryote is more complex than bacteria in

having more cellular compartments and more epigenetic enzymes. The number of cell types is

estimated based on the complexity of the nervous systems to be relatively the most in the first

primate, less in the first mammals, and still less in the first vertebrate. The figure is meant to

show this relative trend but does not intend to show the precise number of cell types.

Figure 5. Genetic distance between organisms at various times during macroevolution.

A. Genetic distance according to the Neo-Darwinian gradual mutation hypothesis. B. Genetic

distance according to the MGD hypothesis. A 10 amino acid peptide with amino acids

represented by numbers is shown to illustrate the dissimilarity or genetic distance between the

species at various times during evolution. X represents amino acid positions that may change

from time to time. A fraction of these X residues may be shared in different organisms due to

common external environments that may differ from time to time.

Nature Precedings : hdl:10101/npre.2008.1751.1 : Posted 1 Apr 2008