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PERSPECTIVE

The new mutation theory of phenotypic evolution


Masatoshi Nei*
Institute of Molecular Evolutionary Genetics and Department of Biology, Pennsylvania State University, 328 Mueller Laboratory,
University Park, PA 16802

Edited by Daniel L. Hartl, Harvard University, Cambridge, MA, and approved June 13, 2007 (received for review April 16, 2007)

Recent studies of developmental biology have shown that the genes controlling phenotypic characters expressed in the early stage
of development are highly conserved and that recent evolutionary changes have occurred primarily in the characters expressed in
later stages of development. Even the genes controlling the latter characters are generally conserved, but there is a large component
of neutral or nearly neutral genetic variation within and between closely related species. Phenotypic evolution occurs primarily by
mutation of genes that interact with one another in the developmental process. The enormous amount of phenotypic diversity
among different phyla or classes of organisms is a product of accumulation of novel mutations and their conservation that have
facilitated adaptation to different environments. Novel mutations may be incorporated into the genome by natural selection (elimi-
nation of preexisting genotypes) or by random processes such as genetic and genomic drift. However, once the mutations are incor-
porated into the genome, they may generate developmental constraints that will affect the future direction of phenotypic evolution.
It appears that the driving force of phenotypic evolution is mutation, and natural selection is of secondary importance.

F
or the last six decades, the domi- occur at the molecular level, but be- families (26, 27). Many multigene fami-
nant theory of evolution has cause they do not affect phenotypic lies are of ancient origin and are shared
been neo-Darwinism, which was characters, they are of little interest to by animals, plants, and fungi. Good ex-
developed by the three founders evolutionists. In this respect, it is inter- amples are homeobox genes that encode
of theoretical population genetics, esting to note that even Kimura (15), transcription factors controlling various
Fisher (1), Wright (2), and Haldane (3), protagonist of the neutral theory of aspects of morphogenesis. The genomes
and was later supported by various evo- molecular evolution, believed in neo- of animals and plants contain a large
lutionists (4–9). Neo-Darwinism asserts Darwinism with respect to phenotypic superfamily of homeobox genes, with
that natural selection is the driving force evolution. By contrast, Nei (17, 24, 25) ⬎200 genes in the human and ⬇80
of evolution, and mutation merely pro- argued that because phenotypic charac- genes in the flowering plant Arabidopsis
vides raw genetic materials with which ters are ultimately controlled by DNA thaliana. Animal homeobox genes can
natural selection produces novel charac- sequences, both molecular and pheno- be divided into at least 49 families (28,
ters. This view is based on the argument typic evolution must occur in similar 29). The most well studied is the HOX
that natural selection enhances the fre- ways. He also suggested that a consider- gene family that controls the anterior–
quencies of advantageous alleles at able portion of morphological evolution posterior segmentation of the animal
many loci and makes it easy to recom- is caused by neutral or nearly neutral body. The homeodomains encoded by
bine them into a single individual and mutations, and the driving force of evo- orthologous and paralogous HOX genes
produce a novel character, especially in lution is mutation at both molecular and from different animals are known to
the presence of gene interaction (1–3). phenotypic levels. However, the evi- have the same or very similar amino
By following this principle, evolutionary dence for supporting this argument was acid sequences (28, 30, 31). In general,
biologists have developed various theo- rather weak. the transcription factor genes involved
ries of natural selection to explain the In recent years substantial progress in the early stages of development are
evolution of sex (9), formation of new has occurred in the study of the molecu- highly conserved (26). This suggests that
species (10), development of social life lar basis of phenotypic evolution, so that the early stages of development are con-
in insects (11), evolution of altruism we can examine the relative importance trolled by the same or similar sets of
(12), etc. In these studies, it is custom- of mutation and selection in detail. In genes in many different phyla or classes
ary to assume that there is a sufficient this article, I will first consider pheno- of organisms.
amount of genetic variation within pop- typic evolution controlled by multigene The highly conserved genes stay in
ulations, and therefore what is necessary families, because there is a large amount the genome not because of a low muta-
is to study how natural selection pro- of interesting data, and the interpreta- tion rate but because of a high degree
duces complex characters or complex tion of new findings in this area is rela- of purifying selection. The degree of
ways of life. tively simple. I will then discuss the purifying selection can be measured by
In the last four decades, the study of evolutionary changes of protein-coding comparing the number of synonymous
molecular evolution has shown that a and regulatory regions of genes in rela- nucleotide substitutions per synonymous
majority of amino acid substitutions in tion to phenotypic evolution and their site (dS) and the number of nonsynony-
proteins are neutral or nearly neutral implications for the general theory of mous substitutions per nonsynonymous
and that only a minority of the substitu- evolution.
tions change protein function (13–18). It
has also been shown that the major fac- Multigene Families and Author contributions: M.N. wrote the paper.
tor of evolution at the molecular level is Phenotypic Evolution The author declares no conflict of interest.
mutation, including gene duplication Conservative and Divergent Evolution. This article is a PNAS Direct Submission.
and other genetic changes (15–17). Recent genomic studies of model organ- Abbreviations: GRN, gene regulatory network; MC1R,
However, most evolutionists still believe isms have made it clear that the ge- melanocortin-1 receptor; OR, olfactory receptor.
in neo-Darwinism with respect to phe- nomes of eukaryotes contain a large *E-mail: nxm2@psu.edu.
notypic evolution and are not interested number of multigene families and that This article contains supporting information online at
in neutral evolution (19–22). Mayr (23) most physiological and morphological www.pnas.org/cgi/content/full/0703349104/DC1.
stated that neutral mutations apparently characters are controlled by multigene © 2007 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703349104 PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 兩 12235–12242


Table 1. Numbers of member genes of several homeobox gene families in animal species
Invertebrates Vertebrates

Gene family Caenorhabditis elegans Fruitfly Tunicate Puffer fish Zebrafish Frog Mouse Rat Human

NKX5 1 1 1 4 3 3 2 2 2
DLX 1 1 3 8 5 6 7 6 6
CDX 1 1 2 2 3 3 3 2 3
HOX 6 8 10 45 40 35 39 39 39
PAX 2 8 4 9 7 3 4 4 4
POU 3 5 2 16 13 15 14 13 16
LIM 7 7 7 21 14 12 12 11 12

Table is modified from Nam and Nei (29).

site (dN) under the assumption that dS the organism or the character involved. cause they are equipped with trichro-
represents the number of neutral mu- For example, the number of gene copies matic color vision (35). For this reason,
tations. In the presence of purifying in the HOX gene family is only 8 in they appear to have smaller numbers of
selection, nonsynonymous nucleotide fruitflies but 39 in mammals (Table 1). OR genes. However, dogs and cows,
substitutions resulting in amino acid This increase is understandable, because which have large numbers of functional
changes may be eliminated. We there- vertebrates need more homeobox genes OR genes, also possess large numbers of
fore expect that dN is smaller than dS, to develop complex morphological pseudogenes. In rats, it is known that
and the extent of purifying selection can characters. A large-scale study of this even if up to 80% of glomeruli in the
be measured by 1 ⫺ dN/dS. When I ap- problem was conducted for 1,219 super- olfactory bulb are removed (OR genes
plied this equation to the concatenated families of genes from 38 eukaryotic knocked out), the individual still can
nucleotide sequences (2,340 codons) of species, and it was shown that the num- live a normal life in the laboratory con-
the homeoboxes of the 39 pairs of hu- ber of genes within each superfamily is dition. Furthermore, Shepherd (36)
man and mouse HOX genes, I obtained generally correlated with the number of pointed out the importance of process-
1 ⫺ 0.001/0.313 ⫽ 0.997. This suggests cell types of the organism (26). ing of odor distinction in the brain, stat-
that 99.7% of nonsynonymous mutations The increase of gene number is, of ing that although humans have a smaller
are eliminated by purifying selection in course, generally caused by gene dupli- number of OR genes, the proportion of
homeobox regions. cation, but gene number sometimes de- brain concerned with olfaction is appar-
However, most proteins are not as creases by gene deletion. Therefore, ently greater in humans than in mice. If
conserved as HOX homeodomains, and multigene families are generally subject we consider these factors, variation in
the average dN/dS ratio obtained from to birth-and-death evolution (27, 33). In the number of functional OR genes
1,000 randomly chosen human and multigene families controlling physiolog- among different species may not be di-
mouse genes is ⬇0.15 (18). This means ical characters, variation in the number rectly related to the ability of olfaction
that, on average, ⬇85% of nonsynony- of gene copies among different species required. This is particularly so in the
mous nucleotide mutations are deleteri- can be enormous. One of the most con- presence of a large number of pseudo-
ous, and only 15% are fixed in the spicuous is the variation of olfactory genes.
population. Many genes that are in- receptor (OR) genes among vertebrate Great variation in the number of gene
volved in various physiological functions species (Table 2). In this gene family, copies among vertebrate species is also
of adult individuals usually evolve with a the number of functional OR genes is observed with pheromone receptor,
higher rate of nonsynonymous substitu- ⬎1,000 in mice but 396 in humans. taste receptor, and Ig genes (Table 2).
tion than HOX genes. Examples are im- Interestingly, humans have more pseu- The reason for this variation is not al-
mune systems genes such as Ig and dogenes than mice, the proportion of ways clear. However, it appears that the
MHC genes, which are for protecting pseudogenes being ⬇55% in humans number of gene copies in these gene
the host from parasites (viruses, bacte- and 24% in mice. Dogs, which are sup- families was originally determined by
ria, etc.). These genes tend to evolve posed to have a good sense of smell, their functional requirement, but after
faster to avoid the attack from ever have 811 functional genes and 289 pseu- the copy number reached a required
changing parasites. However, the rate of dogenes. However, the most notable level, the number has fluctuated by ran-
nucleotide substitution in these genes is organism in this respect is the chicken, dom duplication and deletion of genes.
still much lower than that of pseudo- which has only 82 functional genes but We may call this event random genomic
genes, which is often regarded as the 478 pseudogenes. drift, in analogy with random genetic
neutral substitution rate (17). Despite Why do the numbers of functional drift of allele frequencies in population
this conservative nature of amino acid genes and pseudogenes vary so much genetics. This random genomic drift is
substitution, multigene families may among vertebrate species? The obvious apparently an important factor for the
evolve relatively fast because of the factor would be the requirement for a evolution of phenotypic characters. If
rapid change of the number of member species to adapt to a particular environ- the number of gene copies increases or
genes. mental condition. For most vertebrate decreases by chance for a group of indi-
species, detection of millions of different viduals, these individuals may be able to
Evolutionary Change of the Number of Gene odorants is crucial for their survival. adapt to a new environment. Genomic
Copies. The number of genes contained Yet, animals living in different environ- drift is not just confined to sensory re-
in a genome is not necessarily correlated ments require different types and num- ceptor or immune systems genes but
with the complexity of the organism in bers of olfactory receptors (34). In some appears to be an important evolutionary
eukaryotes (32). However, the number animals such as birds and primates, ol- mechanism for many multigene families.
of gene copies in a gene family tends to faction appears to be less important It is known that human populations har-
increase with increasing complexity of than in other terrestrial vertebrates be- bor extensive polymorphisms of copy

12236 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703349104 Nei


Table 2. Numbers of functional genes and pseudogenes in the sensory receptor and other multigene families of vertebrates
Olfactory Phermone Taste
receptor* receptor† receptor‡ Ig§

Vertebrate OR V1R T2R VH V␭ V␬

Human 396 (425)¶ 5 (115)储 25 (11) 44 (60) 31 (38) 35 (43)


Mouse 1,035 (356) 187 (121) 34 (7) 97 (65) 3 (0) 80 (78)
Dog 811 (289) 8 (33) 14 (4) 43 (37) 52 (64) 16 (9)
Cow 970 (1,159) 40 (45) 11 (13) 11 (6) 28 (30) 9 (13)
Opossum 1,188 (304) 98 (30) 25 (8) 26 (6) 48 (6) 66 (49)
Chicken 82 (476) 0 (0) 3 (0) 1 (58) 1 (25) 0 (0)
Xenopus 410 (478) 21 (2) 48 (5) 39 (41) 6 (1) 71 (18)
Zebrafish 102 (35) 2 (0) 4 (0) 37 (10) 33 (9)**

*Y. Niimura and M. Nei (ref. 113 and unpublished work).


†Shi and Zhang (114).
‡Shi and Zhang (115).
§S. Das and M. Nei, unpublished work.
¶The numbers in parentheses indicate pseudogenes.
储The V1R intact genes in humans are likely to be nonfunctional (112).

**It is unclear whether these genes belong to the V␭ or V␬ genes family. Here, VH, V␭, and V␬ stand for Ig heavy-chain variable, ␭-chain variable, and ␬-chain
variable region genes, respectively.

number of multigene families (37–39) ters should be studied by taking into cated molecular and cellular processes
and that many of these polymorphisms account this gene interaction. If a char- carried out by a large number of genes.
do not seem to affect the fitness of indi- acter is controlled by a large number of In this section, we consider the roles of
viduals even when they are caused by interacting genes, it is possible that the mutation and selection in the evolution
duplication of a genomic region contain- genetic networks involved are robust of physiological characters.
ing ⬇30 genes, as in the case of Ig vari- and resistant to the effects of deleteri-
able region (V␬) genes in humans (40). ous mutations (49). At the same time, Changes in the Protein-Coding Regions of
In plants, there is evidence that the the effects of advantageous mutations Genes. The study of molecular evolution
types and numbers of genes in a genome also may not be manifested significantly started with interspecific comparison of
are reshuffled extensively when in a genetic network with many differ- protein molecules concerned with vari-
polyploidization followed by diploidiza- ent developmental pathways. If this is ous physiological functions (e.g., hemo-
tion occurs (41). the case, a large proportion of muta- globin, cytochrome c, and insulin). This
tions may evolve in a more or less neu- type of study soon revealed that most
Multiple Signal Pathways and Genetic Net- tral fashion. amino acid substitutions occurring in
works. So far, we have considered only structural proteins are more or less neu-
DNA sequence conservation and Evolution of Physiological Characters tral (16), and the functional change of
genomic drift of multigene families. In Strictly speaking, the principle of evolu- proteins is caused primarily by amino
general, a large number of different tion of physiological characters cannot acid substitutions occurring in the active
genes are involved in the development be distinguished from that of morpho- sites of proteins [supporting information
of phenotypic characters, and changes in logical characters, because the formation (SI) Table 3]. This is a general principle
the coordination of temporal and spatial of morphological characters depends on of evolution of proteins controlling
expression of these genes in the devel- various physiological processes in devel- physiological characters (15, 17, 18). Be-
opmental process play important roles opment, and the function of physiologi- cause recent papers on this subject have
in evolution. There are usually several cal characters depends on the anatomy been reviewed by Nei (18), I shall not
signaling pathways for producing the or morphology of the organism. How- repeat the review here. The only com-
same end character, and complex gene ever, it is convenient to treat the evolu- ment I would like to make is that Kimu-
interaction occurs as a form of gene tion of physiological and morphological ra’s (14) definition of neutral mutations
regulatory networks (42–44). The num- characters separately, because the (2Ns ⬍ 1, where N is the effective pop-
ber of genes involved in these signaling former characters are concerned primar- ulation size, and s is the selection coeffi-
pathways or genetic networks generally ily with adult life, and the latter are cient for the mutant allele) is too strict
increases as the phenotypic character products of morphogenesis in the devel- to deal with long-term evolution, and,
involved becomes more complex, and opmental stage. For example, the trans- therefore, a more realistic definition
this increase in gene number is ulti- portation of oxygen from the lungs to based on functional change of genes by
mately caused by gene duplication (45). various tissues in vertebrates is carried Nei (18) will be used in this paper. (He
For this reason, gene duplication is the out primarily by hemoglobin and myo- also proposed a more reasonable form
fundamental process of generating com- globin. Therefore, by examining the of statistical definition of neutrality,
plex organisms (26, 46–48). molecular structures and expression pat- which is given by s公2N ⬍ 1 for a rea-
In the past, it has been customary to terns of these proteins from different sonably large N or approximately
treat each gene as a unit of evolution in organisms, one can study the mechanism ⫺0.001 ⱕ s ⱕ 0.001 for N ⬇ 106.)
population genetics. In reality, however, of evolution of oxygen transportation.
a large number of genes interact with By contrast, to understand the evolution Changes in the Regulatory Regions of
one another temporally or spatially in of morphological characters, one must Genes. However, the evolution of physio-
the developmental process, and there- study the evolutionary change of mor- logical characters is also affected by
fore the evolution of phenotypic charac- phogenesis, which depends on compli- mutational changes of the regulatory

Nei PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 兩 12237


regions of genes that include promoters between species in the regulatory re- receptor (MC1R) and Agouti (61, 62).
and enhancers surrounding the coding gions are generally in conformity with The wild-type coat color of jaguars of
regions of genes. The ␤ globin gene the pattern of neutral evolution (54–58). the cat family is reddish or yellowish
family in humans is known to consist of In this connection, I want to empha- and is determined by phaeomelanin.
a cluster of duplicate genes ␧, ␥A, ␥G, ␦, size that any mutation would never be However, there are mutant genotypes
and ␤ (50). The gene ␧ is expressed in strictly neutral, because its function de- with black coat color. This color is dom-
the early embryonic stage, ␥A and ␥G pends on other genes and environmental inant to the wild type and is caused by
are expressed in fetal liver, and ␦ and ␤ conditions. In this sense, any mutation can deletion of several nucleotides as well as
are expressed in adult individuals. The only be nearly neutral as was conceived by amino acid substitutions in the MC1R
expression of these genes is controlled early molecular evolutionists (18). and Agouti genes (63). Jaguars live in
by the locus control region (LCR) that the jungles of Central and South Amer-
exists in an upstream region of the gene Evolution of Morphological Characters ica, and the selective advantage or dis-
cluster. The molecular components of Any specific morphological characters or advantage of the black form over the
this LCR interact with the regulatory organs such as animal eyes, hearts, and wild type is unclear (62). It is possible
region of each globin gene and deter- limbs and plant flowers, etc. are prod- that the mutant black form has spread
mine the successive activation and sup- ucts of complex processes of temporal through the population largely by ge-
pression of expression of ␤-family genes and spatial expression of many interact- netic drift. Note that small selective ad-
in development (50). A similar LCR is ing genes in development. Developmen- vantage or disadvantage is easily
believed to control the expression of tal biologists often study and compare swamped by the fluctuation of progeny
HOX genes (51) and the expression of the developmental processes of distantly size (18).
olfactory receptor genes (52). related organisms such as humans, ze- However, there are cases in which coat
How these complex systems of gene brafish, sea urchins, and fruitflies. These color is clearly related to the adaptation
expression evolved is unclear. However, studies show that each organism is of organisms. In the Pinacate region of
the regulatory region of each gene must uniquely adapted to its environmental southwest Arizona, the rock pocket
have changed gradually as the number condition or lifestyle, and, therefore, mouse, Chaetodipus intermedius, inhabits
of duplicate genes in the cluster in- natural selection appears to have played both dark and sandy rocky areas of the
creased. In fact, the nucleotide sequence important roles in producing morpho- region. Dark areas have been formed
of a cis-regulatory element is not fixed logical characters (32, 42). However, to by laval flow from a volcanic eruption
but changes in the evolutionary process, understand the mechanism of evolution that occurred ⬎1 million years ago
although it is generally quite conserved. of morphological characters, one should (Mya). Rock pocket mice are generally
The amino acid sequence of the DNA- study the differences in morphogenesis light-colored, but in the laval areas
binding region of a transcription factor of closely related species or polymorphic dark-colored individuals are observed.
also appears to change with time (53). individuals within species. In this case, Nachman et al. (64) showed that there are
These changes in the regulatory ele- the number of genes involved is likely to four amino acid differences in MC1R be-
ments and the DNA-binding regions of be small, so that it would be easier to tween dark-colored and light-colored
transcription factors must be responsible understand the evolutionary process of individuals in this region. Because dark-
for the evolutionary change of gene ex- morphological characters. colored mice were derived from light-
pression pattern and, consequently, the colored mice by mutation, the former
evolutionary change of physiological Changes in the Protein-Coding Regions of were apparently adapted to the dark envi-
characters. Genes. To explain the conspicuous mor- ronment to avoid the attack from preda-
If this is the case, one would expect phological difference between humans tors such as birds and large mammals.
that physiological characters are gener- and chimpanzees despite a small degree Similar adaptation to new environments
ally conserved in the evolutionary pro- of amino acid differences, King and Wil- caused by a single amino acid substitution
cess. Theoretically, when changes in son (59) suggested that morphological in MC1R has been reported in the beach
internal or external environments occur, evolution occurs by mutations of regula- mouse, Peromyscus polionatus, in Florida
they may change relatively quickly be- tory genes rather than structural genes. (65). These examples suggest that new
cause of the mutations occurring at the This view has been accepted by many mutations are responsible for adaptation
cis-regulatory elements and the DNA- developmental biologists (44, 60). By (preadaptation) to new environments and
binding regions of transcription factors. contrast, Nei (ref. 17, chapter 14) pro- they have spread through the population
However, for cis-elements to bind tran- posed that morphological evolution by natural selection (elimination of previ-
scription factors properly, they must occurs by a small proportion of major ous genotypes). The importance of
coevolve with a delicate balance. There- effect mutations whether they are struc- changes of protein sequences has also
fore, the evolution of physiological char- tural or regulatory (major gene effect been reported for the HOX genes deter-
acters is expected to be a slow process. hypothesis). It is still premature to con- mining body segmentation of insects (66,
Of course, it is possible that the nucleo- clude which hypothesis is right, but 67), the Vrs1 transcription factor control-
tide sequences of the regulatory region there are increasing data indicating the ling the six-rowed spike in barley (68), and
outside the cis-elements evolve in a neu- importance of structural gene mutations others (SI Table 3).
tral fashion. However, because the DNA in morphological evolution. These examples show that morpholog-
sequences in cis-elements are generally One of the commonly observed mor- ical characters can be changed by a few
conserved, the average rate of nucleo- phological variations within and between amino acid substitutions, but it should
tide substitution of the entire regulatory related species is that of pigmentation be noted that, as in the case of physio-
region is expected to be lower than the of the hair, skin, and eyes of mammals logical characters, most amino acid
rate of synonymous substitution in the and birds (SI Table 3). Many mamma- substitutions do not affect them appre-
coding regions but higher than the rate lian polymorphisms of black coat color ciably. In the case of MC1R, there are
of nonsynonymous substitution. This (caused by the pigment eumelanin) and 63 aa differences (of 315 shared sites
expectation has been borne out by ac- reddish or yellowish color (caused by compared) between wild-type mice and
tual data for a large number of genes, the pigment phaeomelanin) are con- wild-type rock pocket mice, but the two
and the sequence variations within and trolled by proteins called melanocortin-1 species have essentially the same coat

12238 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703349104 Nei


Fig. 1. Nucleotide sequences of the cis-elements for five bicoid (bcd), three hunchback (hb), and one giant (gt) transcription factors in the regulatory region
of the even skipped (eve) enhancer 2 gene in six Drosophila species. N/A, no homologous sequence identified. –, nucleotide deletion. mel, D. melanogaster; sim,
D. simulans; yak, D. yakuba; ere, D. erecta; pse, D. pseudoobscura; pic, D. picticornis. Adapted from Ludwig et al. (74). The phylogenetic relationships of these
species are shown at the left-hand side of the diagram.

color, indicating that only a few specific tions appear to have occurred, because systems using relatively closely related
mutations can change coat color. This there is continuous variation in the beak species. The Drosophila homeotic gene
observation supports the major gene shape and the expression levels of even-skipped (eve) is known to pro-
effect hypothesis (17). BMP4 and calmodulin among different duce seven transverse stripes along the
species of Darwin’s finches. anterior–posterior axis of the early
Changes in the Regulatory Regions of Another example of rapid morpholog- embryo. Expression of each of these
Genes. Generally speaking, the genetic ical evolution by regulatory mutations is stripes is regulated by ⬎12 cis-elements
basis of morphological evolution is more that of freshwater stickleback fish living in the enhancer (activator and repres-
complicated than that of physiological in lakes near the northern Atlantic and sor) region. Ludwig, Patel, and Kreit-
characters. Darwin’s finches, consisting Pacific. They were apparently derived man (74) studied the tripe 2 enhancers
of 14 species, in the Galapagos Islands from the oceanic marine sticklebacks of the eve gene from six different Dro-
are often used as a textbook example of ⬇12,000 years ago when glaciation sophila species and showed that the cis-
adaptive radiation of morphological started to retreat. Marine sticklebacks elements of this gene are generally
characters. One character that has been have relatively long pelvic (rear) fins, highly conserved, but a few of them
studied well is the beak shape of the but the fins are almost absent or sub- were absent in some species (Fig. 1).
birds living on different islands. Several stantially reduced in freshwater stickle- Furthermore, the number of nucleotide
species of the finches eat insects and backs. It has been shown that the differences in each element increased as
flowers of cactuses, whereas some oth- presence of pelvic fins is associated with the genomic divergence between species
ers feed on seeds dropped on the a high level of expression of transcrip- increased. Nevertheless, when the ge-
ground. Cactus finches generally have tion factor gene, Pitx1, in the pelvic re- netic constructs of enhancers and coding
long and pointed beaks, whereas ground gion of the embryo (72). By contrast, regions from different species were ex-
finches have broad and thick beaks used freshwater sticklebacks showed no or amined, all of them showed essentially
for crushing seeds. Abzhanov et al. (69) low levels of expression of the gene. the same tripe 2 expression. A similar
found that there is a high correlation Study of the PITX1 proteins from ma- but more complicated evolutionary
between the extent of beak breadth and rine and freshwater sticklebacks showed change of the regulatory region of a
the expression level of bone morpho- that there were no amino acid differ- gene resulting in the same phenotype
genic protein, BMP4, in the frontal part ences between them. From these has been reported with respect to the
of beak in the embryonic stage. Later, observations, it was concluded that the mating type MATa and MAT␣ genes in
they searched for other genes affecting formation of pelvic fins is initiated by the ascomycete yeast lineages (75).
the beak shape and showed that calmod- the expression of Pitx1, and the evolu- These results indicate that most nucle-
ulin (CaM), a protein involved in medi- tionary change of the regulatory region otide substitutions in the regulatory re-
ating calcium signaling, is expressed at of the Pitx1 gene is responsible for the gion evolve in a more or less neutral
higher levels in the long and pointed reduction of pelvic fins. There are many fashion, similar to those in the protein-
beak of cactus finches than in the more other examples of cis-regulatory muta- coding region. It is therefore possible
broad beaks of ground finches (70). tions that have generated morphological that the evolutionary change of gene
Therefore, it appears that the breadth changes (73). Therefore, this form of regulation is also controlled by major
and length of finch’s beaks are con- mutation seems to play important roles gene mutations.
trolled primarily by the expression levels in phenotypic evolution. Many develop-
of genes Bmp4 and CaM, respectively. mental biologists seem to believe that Polymorphism in cis-Regulatory Regions and
Darwin’s finches are believed to have cis-regulatory mutations are more im- Gene Expression Level. Mendelian geneti-
originated from the finches in South or portant than the mutations in coding cists have established that quantitative
Central America ⬇2 Mya (71) through a regions of genes, because new morpho- characters are controlled by a large
bottleneck of population size. It is there- logical characters are often associated number of genes (76, 77). This can be
fore likely that the beak shape of the with changes in the expression level of explained partly by the presence of a
finches evolved by new regulatory muta- genes rather than changes in the amino high degree of protein polymorphism
tions and natural selection that occurred acid sequences encoded. observed by electrophoresis at many loci
during the last 2 million years. In this A number of authors have studied the (24, 78). Recent studies have also shown
case, many different regulatory muta- evolutionary change of gene regulatory that there is a large amount of variation

Nei PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 兩 12239


in the level of gene expression among the enormous amount of phenotypic by chance. The observation that the pat-
different alleles at a locus (79–83). In diversity. For example, sea urchin and tern of variation of gene expression lev-
humans, it has been reported that, in starfish, which belong to different els within and between closely related
63% of the genes examined, the expres- classes of the phylum Echinodermata species is consistent with that of neutral
sion level of one allele is 2-fold or more and diverged ⬎540 Mya, show strikingly evolution supports this idea. However,
greater than that of another allele (81). different morphologies, and they are once these mutations are incorporated
These allelic differences of gene ex- apparently well adapted to different life- into the genome, they may generate new
pression level as well as the amino acid styles in different environments. How- developmental constraints that affect
differences detected by protein electro- ever, studies of the early stage of the future direction of phenotypic evolu-
phoresis are likely to influence pheno- embryonic development have shown that tion. This is particularly so when the
typic characters. Probably for this sea urchin and starfish have similar environmental condition changes. Large
reason, most quantitative characters in morphologies and developmental pat- random phenotypic evolution may also
outbreeding populations contain a large terns, and there is a common form of be generated when geological changes
amount of genetic variation, and artifi- gene regulatory network (GRN) consist- such as mass extinction and continental
cial selection is almost always effective ing of approximately six transcription drift occur.
(5). Previously, many neo-Darwinians factor genes (94). This basic core of
claimed that the genetic variation within GRN is specific for the early develop- Prospective and Retrospective Views
species is maintained primarily by bal- ment of echinoderms and has not of Evolution
ancing selection such as overdominant changed for the last 540 million years. The teleological view of evolution has
selection (5, 8). If this is the case, the However, as the development proceeds, been out of fashion for more than a
extent of nonsynonymous nucleotide the GRN in each of the two species century. Yet, human minds appear to be
diversity is expected to be higher than expands into a more complex form in- susceptible to this view consciously or
that of synonymous diversity as in the cluding a large number of genes for unconsciously. In the evolutionary litera-
case of MHC loci (84). In reality, how- transcription factors, signaling proteins, ture, it is not uncommon to see such
ever, this type of genetic variation has and structural proteins. In this process phrases as ‘‘making of Homo sapiens’’
been observed in only a small propor- of expansion of GRN, different genes and ‘‘faster evolution of humans than
tion of genes in diploid organisms (85). are added in the two species so that other primate species.’’ Using these
It is generally more difficult to study their GRNs are gradually differentiated. phrases, investigators often discuss the
the roles of mutation and natural selec- This gradual differentiation of GRNs is evolution of complex organisms without
tion in phenotypic evolution than in responsible for the formation of the considering the fact that many closely
protein evolution, because phenotypic very different morphologies of sea ur- related species have become extinct in
characters are usually controlled by chin and starfish. The basic core of the past. For example, humans appear
many genes and affected by environ- GRN is highly conserved, and any sig- to have evolved a higher level of pheno-
mental factors. Khaitovich et al. (86–88) nificant change of the core results in typic complexity than chimpanzees after
studied the evolutionary divergence of deformation of the organism. This is their divergence ⬇6 Mya. We are there-
gene expression levels of ⬎10,000 genes also true with the GRNs operating in fore tempted to believe that the genes
in several primate species using the mi- successive developmental stages, but the controlling phenotypic characters have
croarray technique and showed that the extent of developmental constraint grad- been subjected to positive Darwinian
extent of evolutionary divergence in- ually becomes weaker as the develop- selection more often in the human lin-
creased roughly in proportion to the ment proceeds. eage than in the chimpanzee (98).
time of divergence between species. This property appears to apply to many However, if we consider evolution as
They also showed that the extent of different animal phyla, and new species in a forward process, this view becomes
gene expression divergence is generally each phylum are generated by mutational dubious. Fig. 2 shows a schematic repre-
higher for the genes whose intraspecific change of GRNs in the final or near-final sentation of the evolution of humans
variation is high than for the genes stages of development (94, 95). In fact, and chimpanzees. Let us imagine that
whose intraspecific variation is low. the evolution of eye spots in the wings of we can go back to the time when the
From these observations, Khaitovich et some butterflies or the evolutionary two populations leading to humans and
al. concluded that the evolutionary di- changes of the number and form of body chimpanzees diverged ⬇6 Mya and are
vergence in gene expression level has segments in insects and vertebrates have asked whether one can predict which of
occurred in a more or less neutral fash- occurred by modification of GRNs in late the two populations is destined to pro-
ion. In genes from tissues such as the stages of development (43, 44, 96). In this duce humans later. Most evolutionists
testes, there were some deviations from view, the evolution of phenotypic diversity would say ‘‘it is impossible.’’ This would
the above general pattern, possibly be- of different phyla has occurred by a con- be true even if the two populations are
cause of positive selection, but they tinuous process of novel mutations and genetically differentiated to a consider-
were rare. Similar results have been ob- elimination of preexisting less-fit geno- able extent. In other words, although we
served in fruitflies (88, 89), yeasts (90), types. Evolutionists have proposed various know that evolution occurs by mutation
fish (91), and others. Although the au- mechanisms by which evolution can occur and natural selection, we cannot predict
thors of these studies did not necessarily so fast that enormous amounts of pheno- the outcome of evolution. Evolution oc-
support neutral evolution, the results typic diversity among organisms can be curs without purpose, and therefore it is
indicate that mutation is the driving explained (1, 2, 97). In reality, evolution is intrinsically unpredictable.
force in gene expression evolution. Es- an intrinsically slow process, and the cur- By contrast, if we study human
sentially the same evolutionary pattern rent phenotypic diversity has been gener- evolution retrospectively by using the
has been observed with protein varia- ated only because there has been a long knowledge of current humans and chim-
tion detected by electrophoresis (92, 93). evolutionary time, ⬎3 billion years. panzees, we can always make a sensible
Theoretically, the evolutionary change story of evolution, although the story
Evolution of Phenotypic Diversity. If we of phenotypic characters can be gener- will be somewhat teleological because it
compare different phyla or classes of ated by neutral or nearly neutral muta- is based on the final products of evolu-
organisms, we are deeply impressed with tions that may be fixed in the population tion. One such story would be that the

12240 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703349104 Nei


human evolution (62), but it should be states ‘‘Natural selection can account for
true with the chimpanzee lineage as both slight and great differences among
well. The cause of extinction of many species, and adaptations are traits that
lineages is not well understood, but part have been shaped by natural selection.’’
of the reason must be the random Although this type of statement is quite
change of genetic materials (genomic common in the evolutionary literature,
drift) and extrinsic environmental it is obvious that any advantageous ge-
changes. If the H. sapiens lineage had notype is produced by mutation includ-
become extinct and another lineage of ing all kinds of genetic changes. Natural
Homo erectus had survived, this world selection occurs as a consequence of
would have been quite different. This mutational production of different geno-
indicates that evolution is opportunistic types, and therefore it is not the funda-
at the species level too. mental cause of evolution.
Most molecular evolutionists are well
Discussion and Perspectives aware of the importance of mutation in
In this article, I have examined various protein evolution. Yet, many investigators
types of molecular data concerning the are trying to identify even the slightest
evolution of phenotypic characters from trace of natural selection using various
the point of view of the selectionism/ statistical methods (99–102). Using these
mutationism controversy. The main con- methods, a number of authors have re-
clusions are as follows. (i) A multigene ported that a substantial proportion of
Fig. 2. Schematic representation of human and family concerned with basic develop- amino acid substitutions are caused by
chimpanzee evolution. The branches of the human mental processes (e.g., HOX genes) is positive Darwinian selection (103–106).
and chimpanzee lineages are the species or sub- generally highly conserved, but the num- However, the statistical methods used are
species that have become extinct in the past. The ber of gene copies involved tends to in- based on many assumptions, which are
retrospective view of evolution is depicted by the crease with increasing complexity of the not necessarily satisfied with actual data
smooth lines aiming at the current morphologies
organism or the character. (ii) When a (18, 107, 108). Furthermore, their esti-
of the two species. It is assumed that the morphol-
ogy of chimpanzees is similar to that of the com-
physiological character is controlled by a mates of selection coefficients are often of
mon ancestor of the two species, whereas the gene family, the number of gene copies the order of 10⫺6 (100, 106) and are un-
human morphology has changed substantially. In may vary extensively among different likely to affect gene function (18). Note
the prospective view, the future evolution is un- organisms, and there are many pseudo- also that although these authors empha-
predictable, and therefore the evolutionary pro- genes in the genome. There are also sized natural selection, they are actually
cess might have been deviated considerably from extensive polymorphisms of copy num- estimating the proportion of mutations
the smooth lines. ber within species. This high degree of that are adaptive.
copy number variation is caused by Historically, the word mutationism
genomic drift as well as by environmen- was used to refer to William Bateson’s
population leading to the human lineage tal factors. (iii) The evolutionary change saltationism or similar ideas, in which
moved to a new habitat, whereas the of physiological and morphological char- natural selection plays little role. Later
chimpanzee lineage stayed in the origi- acters occurs by mutational changes of Morgan (109) presented a more reason-
nal place. For this reason, many new the protein-coding and regulatory re- able form of mutationism taking into
adaptive mutations may have been fixed gions of genes. The genes controlling account the role of natural selection.
in the human lineage and these muta- the characters expressed in the early His view was abstract and based on a
tions led to the evolution of current H. stage of development are highly con- few lines of speculative arguments.
sapiens. However, there is no reason to served, and evolutionary changes occur However, recent molecular studies of
believe that a smaller number of adap- primarily in the characters expressed in phenotypic evolution support the basic
tive mutations have been fixed in the later stages of development. At the nu- ideas of his view and have extended it to
chimpanzee lineage than in the human. cleotide level, the driving force of phe- a more comprehensive view presented in
The chimpanzee lineage also may have notypic evolution is mutation, and there this article. If the new form of mutation
enhanced the adaptability to its own is a significant component of neutral or theory described here is right, even in
habitat, of which the climate and eco- nearly neutral changes. (iv) The pro- its crudest form, more emphasis should
logical community surely changed over spective view of evolution suggests that be given on the roles of mutation in the
geological time. In fact, this view is sup- evolution occurs without purpose by study of evolution. Neo-Darwinians de-
ported by microarray studies of gene mutation and adaptation to new envi- veloped an impressive set of selection
expression levels between humans and ronmental conditions, and therefore it is theories concerning the evolution of sex
chimpanzees (88). In this case, the types intrinsically unpredictable. (110), altruism (12), new species (10),
of mutations fixed in the two lineages As mentioned in the introduction, a and others, without considering mu-
would be different, but if the morpho- majority of current evolutionists believe tations that affect the characters
logical differences are generated by a in neo-Darwinism. In one of the most involved such as male and female repro-
relatively small number of ‘‘major effect popular textbooks on evolution, Fu- ductive organs. These theories should be
mutations’’ (17), it would be difficult to tuyma (ref. 20, p. 10) states that evolu- reexamined by studying the molecular
detect them by standard statistical meth- tionary change is a population process basis of physiological and morphological
ods. Experimental studies would be in which one genotype replaces other components of the characters involved.
necessary. ones, and for this process to occur, mu- It is also important to clarify the mecha-
Note also that any extant species rep- tation is quite ineffective because of its nism of formation of novel characters by
resents only one evolutionary lineage low rate of occurrence, whereas even mutation whether selection is involved
surviving among many that appeared the slightest intensity of natural selec- or not. A group of molecular biologists
but became extinct in the past (Fig. 2). tion can bring about substantial change are already working in this direction
This is well documented in the case of in a realistic amount of time. He also (111), but participation of population

Nei PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 兩 12241


biologists and genomic scientists in this I thank E. Holmes, J. Klein, Z. Lin, K. Ma- ments. This study was supported by
enterprise would speed up our under- kova, J. Nam, N. Nikolaidis, Y. Suzuki, N. National Institutes of Health Grant
standing of phenotypic evolution. Takahata, and J. Zhang for valuable com- GM020293.

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12242 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703349104 Nei

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