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. 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- F 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) Ͼ200 genes in the human and Ϸ80 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: [email protected]. 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.org͞cgi͞doi͞10.1073͞pnas.0703349104 PNAS ͉ July 24, 2007 ͉ vol. 104 ͉ no. 30 ͉ 12235–12242 Downloaded by guest on September 26, 2021 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 Ϫ 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 Ϫ 0.001/0.313 ϭ 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 Ϸ0.15 (18).