Convergent Evolution in Rod Vision Genes Figure 2. Convergent evolution of the SAG gene in bats based on a tree derived from the Bayesian analysis of nucleotide sequences. Numbers and symbols above the branches are the positions and amino acid replacements. Sequences at internal nodes were reconstructed by the Maximum Likelihood method in PAML. doi:10.1371/journal.pone.0034564.g002 for positive selection was not detected on either of these two branches, instead we found that the sequences of these three vision genes had undergone parallel sequence evolution. The failure to detect positive selection may reflect the difficulty of obtaining statistically significant evidence by these methods despite the presence of positively selected amino acid substitutions [39]. Non-neutral convergent evolution of morphological characters should cause bias in phylogenetic inference [40,41,42]. Phyloge- netic analyses of Prestin [3,4,43], CRX (this study) and RH1 [5] using protein sequences or nonsynonymous sites fail to recover the expected species tree due to convergent evolution. However, we failed to detect evidence for convergence in the SAG gene using phylogenetic approaches, and this difference is likely due to the number of changes that occurred in the sequences. Many amino acid substitutions occurred in the SAG sequences on both branches of the Old World fruit bats and sheath-tailed bats, thus the single parallel amino acid site did not result in a ‘‘false’’ tree, as the larger number of other changes overwhelmed this signal, and instead support a the species phylogeny consistent with that generated by other types of data. Convergent evolution, however, was detected in the SAG sequences when ancestral sequences were reconstructed for the internal nodes. Vision plays a basic role in the survival of most animals. Bats are nocturnal, however Old World fruit bats, and sheath-tailed bats may use eyesight to navigate and, compared with other insect- feeding bats that rely on echolocation, have larger eyes. Our study showed, in addition to our previous finding of the RH1 gene [5], that the genes CRX and SAG have undergone parallel sequence evolution in two divergent lineages of bats with larger eyes (Pteropodidae and Emballonuroidea). These parallel changes in these three genes in two branches of bat phylogeny likely result from the common selection for amino acid-altering mutations [3] that are beneficial for dim light vision. The finding of multiple convergences in the network of genes essential for rod vision in bats reflect the complex mechanisms that drove the adaptation to dim light environments during the successful radiation of the second most diverse order of mammals as they exploited the aerial nocturnal niche. Similarly, recent studies have shown that at least two genes, through adaptive evolution, contributed to the evolution of echolocation [3,43,44,45]. Our study demonstrates that greater attention should be focused on the molecular evolution of pathways and networks for a better understanding of phenotypic convergence. Materials and Methods Ethics Statement All research involving animals used in this study followed the guidelines of the by-laws on experimentation on animals, and was approved by the Ethics and Experimental Animal Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ_YP201002). Source of data and primary treatments CRX and SAG gene sequences of the little brown bat (Myotis lucifugus), flying fox (Pteropus vampyrus), cow and dog were downloaded from the Ensembl database. Sequences of these genes were aligned using CLUSTALX 1.81 [46]. Gene-specific primers were designed based on conserved regions. Fresh eye tissue was available for Old-World bat species, thus RNA was isolated, PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34564