Codon-based tests of positive selection, branch lengths, and the evolution of mammalian immune system genes

Abstract

Using basic probability theory, we show that there is a substantial likelihood that even in the presence of strong purifying selection, there will be a number of codons in which the number of synonymous nucleotide substitutions per site (d (S)) exceeds the number of non-synonymous nucleotide substitutions per site (d (N)). In an empirical study, we examined the numbers of synonymous (b (S)) and non-synonymous substitutions (b (N)) along branches of the phylogenies of 69 single-copy orthologous genes from seven species of mammals. A pattern of b (N) > b (S) was most commonly seen in the shortest branches of the tree and was associated with a high coefficient of variation in both b (N) and b (S), suggesting that high stochastic error in b (N) and b (S) on short branches, rather than positive Darwinian selection, is the explanation of most cases where b (N) is greater than b (S) on a given branch. The branch-site method of Zhang et al. (Zhang, Nielsen, Yang, Mol Biol Evol, 22:2472-2479, 2005) identified 117 codons on 35 branches as "positively selected," but a majority of these codons lacked synonymous substitutions, while in the others, synonymous and non-synonymous differences per site occurred in approximately equal frequencies. Thus, it was impossible to rule out the hypothesis that chance variation in the pattern of mutation across sites, rather than positive selection, accounted for the observed pattern. Our results showed that b (N)/b (S) was consistently elevated in immune system genes, but neither the search for branches with b (N) > b (S) nor the branch-site method revealed this trend.

Fig. 1

NJ tree of BRCA2 amino acid sequences illustrating the numbering scheme for branches used in this paper

Fig. 2

Probability of a codon with no synonymous differences and one or more non-synonymous differences, as a function of p_S. Curves are plotted for three different p_N/p_S ratios: 0.1, 0.2, and 0.5. A Poisson distribution is assumed and the expected numbers of synonymous and non-synonymous sites per codon (0.745 and 2.255, respectively) are based on the data of Hughes and Friedman (2005)

Fig. 3

a Numbers of individual branches (in trees for individual genes) with b_N>b_S, illustrated for each branch of the tree. b Percent of branches with b_N>b_S plotted against mean b_S (r_S=−0.817; P=0.002); the points corresponding to each branch of the tree are numbered as in Fig. 1. c Plot of coefficients of variation (CV) in b_N (solid diamonds; r_S=−0.773; P=0.005) and b_S (open circles; r_S=−0.745; P=0.008) plotted against mean b_S for each branch of the tree

Fig. 4

Median values of a b_S; b b_N; and c b_N/b_S for individual branches categorized according to the function presence (Im+) or absence (Im−) of immune system function and the presence (Sig+) or absence (Sig−) of signaling function. In each case, there was a significance difference among the four categories (Kruskal–Wallis test; P<0.001). Individual comparisons (Dunn 1964) between the Im+/Sig− category and other categories: *P<0.05; **P<0.01; ***P<0.001. Numbers of individual branches in each category are indicated in a and c

Fig. 5

Plot of the ration of mean b_N in immune system genes to mean b_N in other genes vs. mean b_S (r_S=0.745; P=0.008); the points corresponding to each branch of the tree is numbered as in Fig. 1

Fig. 6

Histograms of p_S (cross-hatched bars) and p_N (solid bars) between 117 codons identified as positively selected by the branch-site method and their immediate ancestors (reconstructed by maximum parsimony)