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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
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
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
**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
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
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