Phylogenetic evidence for parallel adaptive origins of digestive RNases in Asian and African leaf monkeys: a response to Xu et al. (2009).

Identification of parallel amino acid substitutions accompanying parallel phenotypic evolution is of considerable interest to molecular evolutionists because such parallel substitutions are likely to be adaptive and functionally important (Stewart et al., 1987; Yokoyama and Yokoyama, 1990; Zhang and Kumar, 1997). In 2006, I reported that the gene encoding pancreatic ribonuclease (RNase1) was duplicated independently in Asian and African colobine monkeys (Zhang, 2006). Statistical analyses of DNA sequences, functional assays of reconstructed ancestral proteins, and site-directed mutagenesis showed that the new genes acquired enhanced digestive efficiencies through three parallel amino acid replacements driven by positive selection. They also lost a non-digestive function independently, under a relaxed selective constraint. In a recent Short Communication, Xu and colleagues suggested that the independent duplications that I reported were actually only one duplication event and that the adaptive parallel substitutions I described were no longer existent or were explainable by hypermutations at CpG sites (Xu et al., 2009). Xu et al.’s claims were not supported by available evidence. Below, I provide a detailed response and offer additional phylogenetic evidence for parallel adaptive evolution of digestive RNases in Asian and African colobines. In my original analysis (Zhang, 2006), the RNase gene tree was reconstructed using both the coding region and flanking noncoding regions, totaling 1954 nucleotides. The tree was reconstructed with maximum-likelihood, neighbor-joining, and maximum-parsimony methods, and all methods supported the same tree with high bootstrap values (>98% for every node by every method). In the reanalysis, however, Xu et al. reconstructed the gene tree using only 450 nucleotides of the coding region. Their tree (Fig. 2 in Xu et al. (2009)) is less reliable than my tree (Fig. 2 in Zhang (2006)) for five reasons. First, it is well-known that molecular phylogenies are generally more reliable when they are based on many nucleotides than based on few nucleotides. Xu et al.’s tree was based on a much smaller number of nucleotide sites than was my tree. As a result, the bootstrap values in their tree are low, as they admitted (Xu et al., 2009). For example, the clade of all RNase1B sequences has a bootstrap value of only 75% in their Fig. 2A and 61% in their Fig. 2B. The statistical support for their tree topology is not significantly greater than for mine even when only the coding region is considered. Second, the topology of their tree is not consistent with their hypothesis of one duplication event in the common ancestor of colobines. If their hypothesis were correct, one should observe that (i) all RNase1 sequences of colobine monkeys form a monophyletic group and that (ii) the phylogenetic relationships among colobine RNase1 genes are identical to the relationships