J Mol Evol (1994) 39:160-173 Journal of Molecular Evolution © Springer-Verlag New YorkInc. 1994 Analysis of Directional Mutation Pressure and Nucleotide Content in Mitochondrial Cytochrome b Genes Lars S. Jermiin, Dan Graur,* Roger M. Lowe, Ross H. Crozier School of Genetics and Human Variation, La Trobe University, Bundoora, Victoria 3083, Australia Received and accepted: 23 February 1994 Abstract. We present a new approach for analyzing directional mutation pressure and nucleotide content in protein-coding genes. Directional mutation pressure, the heterogeneity in the likelihood of different nucleo- tide substitutions, is used to explain the increasing or de- creasing guanine-cytosine content (GC%) in DNA and is represented by ~D, in agreement with Sueoka (1962, Proc Natl Acad Sci USA 48:582-592). The new method uses simulation to facilitate identification of significant A + T or G + C pressure as well as the comparison of directional mutation pressure among genes, even when they are translated by different genetic codes. We use the method to analyze the evolution of directional mutation pressure and nucleotide content of mitochondrial cy- tochrome b genes. Results from a survey of 110 taxa in- dicate that the cytochrome b genes of most taxa are subjected to significant directional mutation pressure and that the gene is subject to A + T pressure in most cases. Only in the anseriform bird Cairina moschata is the cytochrome b gene subject to significant G + C pres- sure. The GC% at nonsynonymous codon sites decreases proportionately with increasing A + T pressure, and with a slope less than one, indicating a presence of se- lective constraints. The cytochrome b genes of insects, nematodes, and eumycotes are subject to extreme A + T pressures (#D = 0.123, 0.224, and 0.130) and, in par- allel, the GC% of the nonsynonymous codon sites has decreased from about 0.44 in organisms that are not subjected to A + T or G + C pressure to about 0.332, * Permanent address: Department of Zoology, George S. Wise Fac- ulty of Life Science, Tel Aviv University, Ramat Aviv 69978, Israel Correspondence to: L.S. Jermiin 0.323, and 0.367, respectively. The distribution of taxa according to the GC% at nonsynonymous codon sites and directional mutation pressure supports the notion that variation in these parameters is a phylogenetic com- ponent. Key words: Directional mutation pressure -- A + T pressure -- G + C pressure -- Synonymous codon sites -- Nonsynonymous codon sites -- Mitochondrial cy- tochrome b gene -- Metazoa -- Magnoliophyta -- Chlorophyta -- Eumycota Introduction More than 30 years age Freese (1962) and Sueoka (1962) formulated a quantitative theory of directional muta- tion pressure and used it to explain wide interspecific (Lee et al. 1956; Belozersky and Spirin 1958) and nar- row intragenomic (Sueoka 1959; Sueoka et al. 1959; Rolfe and Meselson 1959) heterogeneities in the base composition of bacterial DNA. The theory is based on the assumption that all nucleotide substitutions in DNA are not equiprobable, but can have an overall direc- tionality toward higher or lower GC%. The theory pre- dicts that the GC% is determined by selective con- straints and two antagonistic mutation rates u and v; u being the mutation rate from an A:T or a T:A nucleotide pair (cx pairs) to a G:C or a C:G nucleotide pair (y pairs), with v being the reverse mutation rate. Mathematically, directional mutation pressure is defined as occurring when gi) v~ 0.5, where FtD = v/(v + u), and is equal to the GC% in those nucleotide sites that are selectively neutral and in equilibrium (Sueoka 1962, 1988, 1992).